Engineering a membrane protein chaperone to ameliorate the proteotoxicity of mutant huntingtin

Engineering a membrane protein chaperone to ameliorate the proteotoxicity of mutant huntingtin | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Engineering a membrane protein chaperone to ameliorate the proteotoxicity of mutant huntingtin Hyunju Cho, Jeonghyun Oh, Christy Catherine, Eun Seon Kim, Kwang Wook Min, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4292547/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Jan, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Toxic protein aggregates are associated with various neurodegenerative diseases, including Huntington’s disease (HD). Since no current treatment delays the progression of HD, we developed a mechanistic approach to preventing mutant huntingtin (mHttex1) aggregation. Here, we engineered the ATP-independent cytosolic chaperone PEX19, which targets peroxisomal membrane proteins to peroxisomes, to remove mHttex1 aggregates. Using yeast toxicity-based screening with a random mutant library, we identified two yeast PEX19 ( sc PEX19) variants and engineered equivalent mutations into human PEX19 ( hs PEX19). These variants prevented mHttex1 aggregation in vitro and in cellular HD models. The mutated hydrophobic residue in the α4 helix of hs PEX19 variants binds to the N17 domain of mHttex1, thereby inhibiting the initial aggregation process. Overexpression of the hs PEX19-FV variant rescues HD-associated phenotypes in primary striatal neurons and in Drosophila . Overall, our data reveal that engineering ATP-independent membrane protein chaperones is a promising therapeutic approach for rational targeting of mHttex1 aggregation in HD. Biological sciences/Biochemistry/Protein folding/Protein aggregation Biological sciences/Biochemistry/Protein folding/Chaperones Biological sciences/Biological techniques/Molecular engineering/Protein design Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Maintaining proper protein homeostasis is essential for healthy cells. However, the cell is under continuous risk from newly synthesized proteins that might expose hydrophobic surfaces in the crowded cellular environment, leading to protein misfolding and aggregation 1 – 4 . To overcome these problems, cells invest in a sophisticated integrative chaperone network that supports accurate de novo protein folding, facilitates refolding of misfolded proteins, and prevents protein aggregation 1 , 2 , 5 , 6 . However, environmental stresses, genetic mutations, and aging can reduce the overall capacity of molecular chaperones, resulting in the accumulation of toxic aggregates and misfolded proteins in cells 7 – 9 . Such aggregates eventually lead to various diseases, including neurodegenerative diseases and type 2 diabetes 10 , 11 . Huntington’s disease (HD) is the most common dominantly inherited neurodegenerative disorder and is caused by the abnormal expansion of CAG (polyQ) repeats in exon 1 of the huntingtin gene (Httex1) 12 , 13 . The length of polyQ repeats in the mutant Httex1 (mHttex1 with > 36 repeats) positively correlates with an increasing propensity to form aggregates and correlates inversely with the age of disease onset 14 , 15 . Aggregation of the polyQ repeat domain is also mediated by its flanking domains, the N-terminal conserved N17 domain and the C-terminal proline-rich domain (PRD). The N17 domain stimulates mHttex1 aggregation, whereas the PRD inhibits it 16 – 19 . Accumulation of mHttex1 aggregates in the nucleus and cytoplasm impairs the proteostasis network and disrupts cellular endomembranes, thus leading to dysregulation of diverse cellular processes including transcription, mitochondrial respiration, ER homeostasis, vesicular trafficking, and axonal transport 20 – 23 . One suggested approach to correcting protein misfolding and removing pathological aggregates involves engineering a molecular chaperone to increase chaperone capacity in affected cells 24 – 26 . Indeed, the yeast AAA + protein disaggregase, Hsp104, has been engineered to rescue the proteotoxicity of TDP43, FUS, and α-synuclein for amyotrophic lateral sclerosis (ALS) and Parkinson’s disease 24 , 27 , 28 . However, most chaperones, including Hsp104, require subunit assembly, oligomerization, co-chaperones, or cofactors, such as ATP and metal ions, for their optimal activities 5 . Thus, engineered chaperones that rely on cellular ATP concentrations and the expression levels of their subunits and co-chaperones 29 may complicate therapeutic applications. PEX19, an ATP-independent cytosolic chaperone, mediates the targeting of peroxisomal membrane proteins (PMPs) during peroxisome biogenesis 30 – 32 . Importantly, PEX19 does not require any co-chaperone or cofactors for its chaperone activity. Therefore, we hypothesized that PEX19 could be readily engineered to provide a robust approach for mitigating mHttex1 proteotoxicity. Here, using yeast toxicity-based screening 33 with a random mutant library, we isolated yeast PEX19 ( sc PEX19) variants that suppress the proteotoxicity of mHttex1 aggregates. Using this information, we engineered the equivalent human PEX19 ( hs PEX19) variants and found that they also potently suppress toxic mHttex1 aggregates. Biochemical assays revealed that the isolated hs PEX19 variants directly bind the hydrophobic side of the amphipathic helix at the N17 domain of mHttex1, thereby preventing mHttex1 aggregation. Overexpression of the hs PEX19 variant further rescued mHttex1-induced neurite degeneration in mouse striatal neurons and improved both the climbing ability and lifespan of flies expressing mHttex1-93Q. Altogether, our study suggests that fine-tuning the sequences of ATP-independent membrane protein chaperones could be a feasible approach to designing therapeutic chaperones for HD and potentially other diseases linked to protein aggregation. Results Engineered sc PEX19 variants suppress the toxicity of mHttex1 in yeast To isolate an sc PEX19 mutant gene that suppresses the cellular toxicity of mHttex1 protein, we used the yeast toxicity-based screening method 33 (Fig. 1 a). Deletion of PRD in Httex1-97Q enhances its polyQ-induced toxicity in yeast 19 . This mutant is more optimal for screening since it results in a larger difference in cell viability compared to expressing non-toxic Httex1-25Q. To this end, we generated yeast strains carrying chromosomally integrated Httex1 genes (Httex1-25QΔP and Httex1-97QΔP), which encode an N-terminal FLAG tag, the first 17 amino acids of Httex1 (N17 domain), 25 or 97 repeats of glutamine, and a C-terminal GFP gene under the control of the galactose-inducible promoter (Fig. 1 a). Expression of the wild-type sc PEX19 did not alter the cellular toxicity of Httex1-97QΔP when compared with the empty vector control (Extended Data Fig. 1 a). We randomly mutated the entire sc PEX19 gene and screened the sc PEX19 plasmid library against Httex1-97QΔP toxicity. Among approximately 90,000 transformants, 21 colonies were able to grow on galactose plates. After assessing the cell viability of those colonies, we found that two sc PEX19 variants, m1 and m2, effectively suppressed the cellular toxicity of Httex1-97QΔP in yeast (Fig. 1 b). The isolated sc PEX19 variants share two common mutation sites, L288F and E292V (Fig. 1 a). Therefore, we hypothesized that the mutation of these two sites accounts for the ability of sc PEX19 variants to rescue Httex1-97QΔP-induced toxicity in yeast. To test this hypothesis, we generated a double mutant sc PEX19-L288F/E292V. The results of the spotting assay showed that sc PEX19-L288F/E292V is sufficient to suppress the cellular toxicity of Httex1-97QΔP (Fig. 1 b). In contrast, coexpression of the single mutants of sc PEX19-L288F or sc PEX19-E292V with Httex1-97QΔP did not restore cell viability (Extended Data Fig. 1 b), suggesting that sc PEX19-L288F/E292V is a minimally mutated suppressor of polyQ-induced toxicity in yeast. In addition, we substituted E292 with other hydrophobic amino acids on the sc PEX19 variant. We found that only sc PEX19-L288F/E292I suppressed Httex1-97QΔP-induced toxicity to the same degree as sc PEX19-L288F/E292V (Fig. 1 c), possibly due to the structural similarity between the valine and isoleucine side chains. Therefore, we identified two sc PEX19 variants, sc PEX19-L288F/E292V ( sc PEX19-FV) and sc PEX19-L288F/E292I ( sc PEX19-FI), that potently suppress polyQ toxicity in yeast. Consistent with the results obtained with the spotting assay, microscopy and Western blot analyses showed that sc PEX19-FV and sc PEX19-FI significantly reduced the aggregation of Httex1-97QΔP proteins compared to sc PEX19-WT (Fig. 1 d-f). Over 50% of Httex1-97QΔP was found in SDS-insoluble aggregates in sc PEX19-WT expressing cells (Fig. 1 e, f). In contrast, overexpression of sc PEX19-FV and sc PEX19-FI drastically reduced the relative amount of SDS-insoluble 97Q aggregate and simultaneously increased SDS-soluble 97Q protein levels (Fig. 1 e, f, and Extended Data Fig. 1 c-d). This enhancement of Httex1-97QΔP solubility by sc PEX19-FV and sc PEX19-FI is not due to different expression levels of PEX19 in the cells (Extended Data Fig. 1 c, e). During protein targeting to peroxisome membranes, farnesylation of the C-terminal cysteine residue in the PEX19-CaaX motif increases the binding affinity of PMPs, effectively preventing PMP aggregation in the cytosol 34 . In addition, the PMP-bound PEX19 is recruited to the peroxisomal membrane by PEX3, which interacts with an N-terminal αa helix in PEX19 35–37 . Noticeably, the levels of farnesylated PEX19 were significantly reduced in sc PEX19-FV and sc PEX19-FI overexpressing cells (Extended Data Fig. 1 c, f). Therefore, we tested whether two major aspects of PEX19-mediated PMP targeting, farnesylation and interaction with PEX3 34–36,38 , are crucial for ameliorating polyQ-induced cellular toxicity in yeast. To this end, we introduced two further mutations, a farnesylation-defective mutation sc PEX19-C339S and a PEX3 binding-defective mutation sc PEX19-ΔN, into sc PEX19-WT and the toxicity-reducing variants (Extended Data Fig. 2 a). The results of a spotting assay showed that coexpression of sc PEX19-FV/C339S, sc PEX19-FV/ΔN, sc PEX19-FI/C339S, and sc PEX19-FI/ΔN with Httex1-97QΔP did not alter cell growth compared to sc PEX19-FV and sc PEX19-FI (Extended Data Fig. 2 b, c). Therefore, both farnesylation and recruitment of PEX19 to the peroxisomal membrane by binding to PEX3 are dispensable for sc PEX19-FV and sc PEX19-FI to suppress the cellular toxicity of Httex1-97QΔP in yeast. hs PEX19 variants suppress mHttex1 aggregation Both mutated residues (L288F/E292V and L288F/E292I) are located in the α4 helix of PEX19 protein (Fig. 2 a, green highlighted box). Sequence alignment analysis showed that these residues are highly conserved from Human (M255/Q259) to Arabidopsis (M202/Q206) (Fig. 2 a). In addition, the M255 residue of hs PEX19 directly interacts with the farnesyl group in its C-terminal end 34 (Fig. 2 b), suggesting that this residue could be important for substrate recognition. Due to their highly homologous sequences, we hypothesized that introducing identical mutations (M255F/Q259V or M255F/Q259I) into hs PEX19 could also enhance suppression of mHttex1 aggregation. To test whether both purified sc PEX19 and hs PEX19 variants directly prevent Httex1-51Q aggregation in vitro , we used the well-established filter trap assay that detects heat-stable, SDS-insoluble aggregates 16 , 39 . In this assay, the N-terminal Httex1-51Q can be exposed by cleaving off a GST-tag using TEV protease, thus initiating polyQ aggregation (Extended Data Fig. 3 a). In the absence of a chaperone, Httex1-51Q readily formed SDS-insoluble aggregates at 3 h (Fig. 2 c, d). In contrast, sc PEX19-FV and sc PEX19-FI effectively suppressed aggregation of the purified Httex1-51Q protein, while sc PEX19-WT was insufficient to prevent Httex1-51Q aggregation (Fig. 2 c). Similar to sc PEX19 variants, hs PEX19 variants effectively prevented aggregation of Httex1-51Q protein in vitro (Fig. 2 d). This enhanced chaperone activity of hs PEX19 variants was not due to different TEV cleavage efficiency caused by their mutations (Extended Data Fig. 3 b). In addition, our negatively stained transmission electron micrograph (TEM) analysis showed that hs PEX19-WT was not sufficient to prevent Httex1-51Q fibril formation (Fig. 2 e). In contrast, hs PEX19-FV completely suppressed the formation of Httex1-51Q fibrils at 15 h (Fig. 2 e). Consistent with the results of the filter trap assay (Extended Data Fig. 3 c), hs PEX19-FI also prevented fibril formation by Httex1-51Q (Fig. 2 e, lower), although in some cases, TEM analysis of hs PEX19-FI revealed both larger Httex1-51Q aggregates and small fibril fragments (Fig. 2 e, upper). Indeed, hs PEX19 variants were unable to redissolve preformed Httex1-51Q aggregates when added at 3 h, suggesting that they do not have a disaggregase activity (Extended Data Fig. 3 d). Therefore, we conclude that hs PEX19 variants function as a holdase that prevents the initial aggregation process of Httex1-51Q. To test whether hs PEX19 variants are also effective in reducing mHttex1 aggregation in a mammalian HD model, we coexpressed hs PEX19 variants with Httex1-19Q-GFP or Httex1-134Q-GFP in HEK293T cells 40 . Overexpression of hs PEX19-FV and hs PEX19-FI at ~ 3-fold over endogenous PEX19 levels strongly prevented the aggregation of Httex1-134Q, as demonstrated by both fluorescence microscopy analysis and the filter trap assay (Fig. 2 f-h and Extended Data Fig. 4 a, c). In contrast, overexpression of hs PEX19-WT reduced the Httex1-134Q aggregates by ~ 50% on average, suggesting that hs PEX19-WT itself exhibits a mild chaperone activity toward polyQ proteins as supported by the in vitro aggregation assay (Fig. 2 g, h). The difference in rescuing effects observed in hs PEX19 variants relative to their wild-type protein was not due to different expression levels of exogenous PEX19 or Httex1-134Q (Extended Data Fig. 4 a-c). Critically, overexpression of hs PEX19 variants did not perturb the peroxisomal localization of the peroxisomal membrane protein PMP70, suggesting that this approach is unlikely to interfere with peroxisome biogenesis (Extended Data Fig. 4 d, e). Therefore, these data demonstrate that the substitution of two conserved residues on the α4 helix of hs PEX19 significantly increases its chaperone activity toward mHttex1. PEX19 variants bind the N17 domain of mHttex1 The N17 domain of Httex1 has an amphipathic helical property, which contributes to the initiation and acceleration of mHttex1 aggregation 16 , 41 (Fig. 3 a). Furthermore, a recent study suggested that structural coupling between the N17 and polyQ repeat domains stabilizes the helical content of Httex1 and accelerates aggregation 42 . Deletion of the N17 domain of Httex1-51Q (Httex1-51Q-ΔN) delays the kinetics of Httex1-51Q aggregation 16 . Given that hs PEX19 variants generate a more hydrophobic environment at their C-terminal domain (CTD) than hs PEX19-WT, we hypothesized that they bind to the hydrophobic amino acids in mHttex1, possibly at the N17 domain of mHttex1. Thus, we tested whether hs PEX19 variants also suppress Httex1-51Q-ΔN aggregation in vitro . In contrast to Httex1-51Q-WT, hs PEX19 variants were unable to suppress the aggregation of Httex1-51Q-ΔN (Fig. 2 d vs 3b). Furthermore, hs PEX19 variants did not suppress the aggregation of another polyQ repeat protein, Ataxin3 (Extended Data Fig. 5 a). Since Ataxin3-78Q has only the polyQ-repeat domain in common with Httex1 43,44 , it is plausible that hs PEX19 variants do not target this polyQ-repeat domain (Extended Data Fig. 5 a). Taken together, these results suggest that the N17 domain could be the primary recognition site of hs PEX19 variants within the Httex1-51Q protein. To check whether the mutated hydrophobic residues at the hs PEX19 variants directly interact with Httex1-51Q, we used the Bpa crosslinking assay that uses a photocrosslinker, p-benzoyl-l-phenylalanine (Bpa) (Fig. 3 c). We site-specifically incorporated Bpa into F255 at the hs PEX19 variants using amber suppression 45 . Suppression of Httex1-51Q aggregation by hs PEX19-FV Bpa and hs PEX19-FI Bpa was also observed at 3 and 6 h, albeit to a lesser extent than the unincorporated hs PEX19 variants (Extended Data Fig. 5 b vs Fig. 2 d). At 3 h incubation, hs PEX19-FVxHttex1-51Q or hs PEX19-FIxHttex1-51Q crosslink at ~ 70 kDa was readily detectable, whereas there was no observed crosslinked band in the presence of Httex1-51Q-ΔN (Fig. 3 d, e). Therefore, these results indicate that the F255 residue in the hs PEX19 variants specifically binds to the N17 domain of Httex1-51Q. We further tested whether the hydrophobic amino acid residues in the N-terminal amphipathic helix of mHttex1 also bind the hs PEX19 variants (Fig. 3 a). To minimize structural perturbation, we incorporated Bpa at the F11 residue on Httex1-51Q among seven hydrophobic amino acid residues (Fig, 3a). Bpa incorporation on Httex1-51Q did not alter the aggregation kinetics (Extended Data Fig. 5 c vs Fig. 2 d). Similar to Httex1-51Q-WT, both hs PEX19-FV and hs PEX19-FI suppressed the aggregation of Httex1-51Q-F11 Bpa more efficiently than hs PEX19-WT (Extended Data Fig. 5 c). In the presence of hs PEX19 variants, two distinct hs PEX19-Httex1-51Q crosslink bands at ~ 70 and ~ 80 kDa were observed (Fig. 3 f, g), suggesting that Httex1-51Q -F11 Bpa binds hs PEX19 variants, possibly with two different conformations. In contrast, hs PEX19-WT resulted in a distinct crosslinked band at ~ 80 kDa and a weak diffuse band at ~ 70 kDa (Fig. 3 f, g). Consistent with its mild chaperone activity, hs PEX19-WT also binds to Httex1-51Q-F11 Bpa , but likely with one dominant conformation (Fig. 3 f, g). These observed differences in the aggregation and Bpa crosslinking assays are not due to different TEV cleavage efficiency (Extended Data Fig. 5 d). Therefore, our data demonstrate that the F11 hydrophobic residue on Httex1-51Q directly interacts with hs PEX19 and consistent with the results in Fig. 2 d, its variants increase this interaction. The α4 helix of hs PEX19 variants serves as a specific binding site for the N17 domain of mHttex1 PEX19 binds to the moderately hydrophobic transmembrane domains (TMDs) of peroxisomal and mitochondrial membrane proteins 30 , 37 , 46 , 47 (Fig. 4 a). In addition, PEX19 interacts with TMDs located in diverse topologies of membrane proteins, multi-spanning PMPs, tail-anchored membrane proteins (TAs), and N-terminal signal-anchored membrane proteins (Fig. 4 a). Since hs PEX19 binds to these moderately hydrophobic TMDs, we hypothesized that hs PEX19 variants might also interact with the isolated N17 domain of Httex1 (Fig. 4 a). To test this, we fused the N17 domain of Httex1 to the N-terminus of the Maltose binding protein (MBP) (Fig. 4 b). The hs PEX19-FV Bpa and hs PEX19-FI Bpa proteins readily crosslinked to the N17-MBP protein, whereas no crosslinked band appeared in the presence of wild-type MBP protein (Fig. 4 c). These results suggest that the N17 domain of Httex1 is a minimum recognition motif for hs PEX19 variants that allows suppression of the mHttex1 aggregation. In contrast to hs PEX19 variants, SGTA, a cytosolic co-chaperone that binds highly hydrophobic TMDs of ER TAs 48 , 49 , was unable to suppress the aggregation of Httex1-51Q (Fig. 4 d). Collectively, our results suggest that mutations on the α4 helix of hs PEX19 enable binding to the relatively low hydrophobic N17 domain of mHttex1. Several studies suggested that the α1 helix of PEX19-CTD serves as a binding site of PMPs 32 , 34 , 50 . Given that the Httex1-51Q binds to the α4 helix of hs PEX19 variants, we checked whether the hs PEX19 variants also interact with a bona fide PEX19 substrate, the peroxisomal TA, PEX26 31 (Fig. 4 e, f). At an approximately 3-fold excess concentration of the endogenous hs PEX19 51 , the amounts of PEX26 loaded onto hs PEX19 variants were comparable to hs PEX19-WT, indicating that these mutations on the α4 helix of hs PEX19 do not largely alter the overall binding capacity of PEX26 (Fig. 4 e, f). In contrast to Httex1-51Q, both hs PEX19-FV Bpa and hs PEX19-FI Bpa did not crosslink to PEX26 (Fig. 4 g-i). These results suggest that F255 and V259/I259 mutations on hs PEX19 could create a specific binding site for the N17 domain of Httex1-51Q, eventually resulting in robust suppression activity of Httex1-51Q aggregation. We tested whether hs PEX19-FV also prevents aggregation of a non-polyQ protein, TDP43, which is associated with another neurodegenerative disease, ALS. To this end, we performed an established in vitro aggregation assay using the purified TDP43-TEV-MBP-His 6 protein 52 . The addition of TEV protease enables initiation of TDP43 aggregation (Extended Data Fig. 6a, black). In contrast to Httex1-51Q aggregation in Fig. 2 d, incubation with hs PEX19-WT or hs PEX19-FV exhibited only a minor delay in TDP43 aggregation kinetics (Extended Data Fig. 6a, blue and red). To further monitor TDP43 aggregation in cells, we generated a stable HEK293 cell line (TDP43-BiFC) that expresses both TDP43-VN and TDP43-VC. Given that phosphorylation and acetylation on TDP43 promote its aggregation 53 – 56 , we used Forskolin as a phosphorylation activator and Apicidin as an acetylation-inducing agent for TDP43 57,58 . Treatment with either Forskolin or Apicidin significantly increased the fluorescence intensities of TDP43-BiFC in the cytosol (Extended Data Fig. 6b, c). Overexpression of hs PEX19-WT or hs PEX19-FV showed at most a minor rescue of Forskolin or Apicidin-induced cytosolic TDP43 aggregation in HEK293 cells (Extended Data Fig. 6d-g). Together with Fig. 2 , we conclude that hs PEX19-FV selectively suppresses the aggregation of mHttex1 in vitro and in mammalian cells. hs PEX19-FV rescues HD-associated phenotypes To test whether hs PEX19-FV protects striatal neurons from mHttex1 proteotoxicity, we coexpressed Httex1-134Q-GFP with hs PEX19-WT or hs PEX19-FV at 7 days in vitro (DIV) in primary striatal neurons (Fig. 5 a). In contrast to Httex1-19Q-GFP- and vector control- coexpressing striatal neurons, at 48 h post-transfection, we observed largely fragmented neurites in the striatal neurons when coexpressed with Httex1-134Q-GFP and vector control, suggesting that mHttex1 induces neuritic degeneration 59 , 60 (Fig. 5 a). Striatal neurons coexpressing Httex1-134Q-GFP and hs PEX19-FV exhibited unfragmented healthy neurites, while partially fragmented neurites were observed in the Httex1-134Q-GFP-and hs PEX19-WT-coexpressing neurons (Fig. 5 a). These results suggest that hs PEX19-FV effectively protects neuritic degeneration in mHttex1-expressing mouse striatal neurons. We next tested whether the hs PEX19-FV variant could rescue HD-associated phenotypes in Drosophila HD models. To this end, we generated transgenic fly lines expressing pACU2 empty vector (vector control), hs PEX19-WT, or hs PEX19-FV and coexpressed Httex1-20Q or Httex1-93Q under the control of Elav-GAL4 (pan-neurons) or D42-GAL4 (motor neurons) drivers (Supplementary Table 1). As a negative control, we used the W 1118 fly line which does not carry a Httex1 transgene. Compared to W 1118 /vector control and Httex1-20Q/vector control flies, motor- or pan-neuronal Httex1-93Q overexpression in Httex1-93Q/vector control flies led to a significant defect in their locomotion capacities (Fig. 5 b, c). In contrast to hs PEX19-WT, hs PEX19-FV expression partially restored the impaired climbing ability of flies overexpressing Httex1-93Q in motor- and pan-neurons. Consistent with these results, the numbers of Httex1-93Q-positive puncta in Httex1-93Q/hsPEX19-FV flies were significantly reduced in both motor- and pan-neurons compared to Httex1-93Q/vector control flies (Extended Data Fig. 7a-f). Furthermore, despite the exclusive cytosolic localization of hs PEX19-FV, Httex1-93Q/hsPEX19-FV flies displayed nuclear-localized soluble Httex1-93Q in both motor- and pan-neurons (Extended Data Fig. 7g, h). Overexpression of hs PEX19-FV significantly increased the lifespan of flies expressing Httex1-93Q, whereas it did not affect the W 1118 and Httex1-20Q flies (Fig. 5 d-f). Taken together, hs PEX19-FV provides effective neuroprotection in both mouse striatal neurons and Httex1-93Q-expressing flies. Discussion Here, we used the yeast toxicity-based screening method to identify two yeast PEX19 variants, sc PEX19-FV (L288F/E292V) and sc PEX19-FI (L288F/E292I), that rescue the toxicity of mHttex1 in yeast. Since the sites of these mutations in the α4 helix of PEX19 are highly conserved, we further generated the human variants hs PEX19-FV (M255F/Q259V) and hs PEX19-FI (M255F/Q259I). We confirmed that hs PEX19 variants effectively suppress mHttex1 aggregation in vivo and in mammalian cells. The mutated phenylalanine residue in the α4 helix of hs PEX19 variants directly interacts with the N17 domain of mHttex1, thereby preventing aggregation of mHttex1. Finally, our results demonstrate that hs PEX19-FV rescues mHttex1-induced neuritic degeneration in primary striatal neurons and HD-associated behavioral deficits and lifespan in the Drosophila HD model. Several chaperones have been identified as mHttex1 aggregation suppressors, which target different domains of mHttex1. Previous studies showed that the TRiC chaperonin and Hsc70 chaperone bind to the N17 domain of mHttex1, thereby preventing mHttex1 aggregation 16 , 61 . In addition, two J-domain proteins (JDPs), DNAJB6 and DNAJB8, and the β subunit of the nascent polypeptide-associated complex (NAC) directly interact with the PolyQ repeat domain, thereby suppressing polyQ-mediated aggregation 44 , 62 – 64 . Furthermore, a recent study showed that another JDP, DNAJB1, together with Hsc70 and Apg2, binds to the PRD of mHttex1, and the trimeric chaperone system prevents and redisolves mHtt aggregates 65 . The S/T-rich region of DNAJB6 was suggested to form hydrogen bonds with the polyQ residues 62 , whereas the positively charged N terminus of βNAC is involved in interactions with the polyQ repeat domain 44 . These polyQ-binding domains in DNAJB6 and βNAC are low complexity linkers located between the JD and C-terminal substrate binding domain and N-terminal unstructured small domain (~ 40 aa), respectively 44 , 62 , 66 . In this study, we showed that the hydrophobic interactions between the α4 helix of hs PEX19 variants and the N17 domain of mHttex1 enable the suppression of mHttex1 aggregation. In contrast to DNAJB6 and βNAC that form electrostatic interactions or hydrogen bonds with mHttex1, the amphipathic N17 domain appears to dock into the hydrophobic farnesyl group-binding groove of hs PEX19-CTD 34 . The hydrophobic residues in the N17 domain are likely to be protected from aqueous cytosolic environments, thereby inhibiting the self-assembly of mHttex1. Together with these previous studies, our results further suggest that, depending on their mHttex1-binding domains, chaperones can employ different molecular mechanisms to prevent mHttex1 aggregation. Membrane protein chaperones recognize their cargo membrane proteins primarily based on the hydrophobicity and location of TMDs 67 , 68 . TMDs are typically between 15 and 30 amino acids long with widely variable hydrophobicity. Regardless of TMD location, PEX19 generally recognizes moderately hydrophobic TMDs in peroxisomal and mitochondrial membrane proteins 30 , 32 , 37 , 46 , 47 (Fig. 4 a). In contrast, SGTA (Sgt2 in yeast) and TRC40 (Get3 in yeast) preferentially bind to more hydrophobic TMDs located near the C-terminus in the ER TAs 32 , 48 , 68 – 70 . Consistent with the low hydrophobicity and N-terminal localization of the N17 domain of mHttex1 (Fig. 4 a), hs PEX19-WT exhibits a mild chaperone activity toward Httex1-51Q (Fig. 2 d), whereas SGTA was not able to suppress the aggregation of Httex1-51Q (Fig. 4 d). The hs PEX19 variants interact more efficiently with Httex1-51Q than hs PEX19-WT, thereby suppressing the formation of both SDS-insoluble larger aggregates and fibrils (Fig. 2 d, e). Despite the unaltered overall binding capacity of hs PEX19 variants to PEX26, the hydrophobic residue F255 at the α4 helix of hs PEX19 variants did not interact with PEX26 (Fig. 4 b-f). Since the α1 helix of hs PEX19 acts as the primary binding site of PMPs 32 , 34 , 50 , our results suggest that the α4 helix of hs PEX19 variants is a specific binding site for the N17 domain of Httex1 proteins. Alternatively, the α4 helix of hs PEX19 could be a unique structural feature to discriminate low hydrophobicity TMDs of membrane proteins. Nevertheless, further structural analysis on hs PEX19 variants would explain how hs PEX19 variants efficiently suppress mHttex1 aggregation. Accumulation of mHtt aggregates in the cytoplasm sequesters a variety of cytosolic proteins, thereby interfering with diverse cellular functions and endomembrane structures 20 , 21 , 23 , 71 , 72 . Several studies showed that cytoplasmic mHtt aggregates impair nucleocytoplasmic transport of proteins and mRNAs by sequestering nuclear-shutting factors to the aggregates 71 , 72 . Furthermore, cytoplasmic mHtt aggregates trap with the cytoskeletal transport system as well as other polyQ proteins in the cytosol, thus further disrupting axonal transport in a Drosophila HD model 73 . Despite the predominant nuclear localization of mHttex1 aggregates in Httex1-93Q expressing Drosophila , our results showed that overexpressing the hs PEX19-FV variant in the cytosol significantly reduces the nuclear aggregation of mHttex1 in both motor- and pan-neurons (Extended Data Fig. 7). Prior to the nuclear import of mHttex1, hs PEX19-FV could prevent the aggregation of mHttex1 in the cytosol (Extended Data Fig. 4 d, e). Together with previous studies 71 – 73 , our results suggest that maintaining a soluble form of mHttex1 assisted by molecular chaperones in the cytosol could also modulate the conformational quality of nuclear mHttex1. Mitigating any potential off-target effects caused by the artificial mutations on a target chaperone would be critical for further therapeutic applications. The identified hs PEX19 variants appear to be specific to HD, relative to proteins linked to other neurodegenerative diseases, potentially due to the highly conserved N17 sequences 74 and amphipathic helix property of Httex1. hs PEX19-WT and its variants were unable to suppress the aggregation of Ataxin3-78Q (Extended Data Fig. 5 a). In addition, hs PEX19-FV displayed only a very modest chaperone activity in TDP43 in vitro aggregation assays (Extended Data Fig. 6a). Substituting the E292 residue into the α4 helix of sc PEX19 variants with various hydrophobic amino acid residues led to different capacities for ameliorating mHttex1-induced toxicity in yeast (Fig. 1 c). These results further suggest that direct modulation of the amino acid sequences on the N17 domain-binding site of PEX19 variants could generate a higher substrate specificity for HD. Therefore, further tuning other amino acid sequences in the α4 helix of hs PEX19-FV would help to eliminate unidentified side effects caused by hs PEX19 variants for HD. The rescuing effects of hs PEX19-FV observed in mHttex1-expressing flies might not be entirely due to the increased chaperone action of hs PEX19-FV on the N17-mediated mHttex1 aggregates. We note that the PEX26 binding capacity of hs PEX19-FV was not drastically altered compared to hs PEX19-WT (Fig. 4 e, f), suggesting that the variant can act on peroxisomal membrane proteins as well as mHttex1. Indeed, given that a variety of cytosolic chaperones are sequestered to mHttex1 aggregates 20 , 21 , as also observed with hs PEX19-WT in Httex1-134Q expressing HEK293T cells (Extended Data Fig. 4 e), overexpression of hs PEX19-FV could assist PMP targeting to the peroxisome, thereby maintaining proper peroxisome biogenesis in mHttex1-expressing flies. Overall, our study demonstrates that engineering an ATP-independent membrane chaperone is a feasible approach to reducing N17-mediated mHttex1 aggregates in HD. Several tetratricopeptide repeat (TPR) domain-containing chaperones, i.e., ATP-independent chaperones involved in ER and mitochondrial membrane protein biogenesis, are known to have decreased expression levels in HD patients 7 . Furthermore, a recent study identified the TPR domain-containing chaperones, TTC1 and TOMM70A, as mitochondrial membrane protein biogenesis factors 75 . Thus, our approach could be readily applicable to design other ATP-independent membrane proteins that specifically reduce mHttex1 toxicity and maintain proper membrane protein biogenesis for other organelles. Furthermore, given that amphipathic helix-mediated aggregation was previously observed in α-Synuclein, islet amyloid polypeptide, and apolipoprotein C-II 76 – 79 , this approach may not be limited to HD but could also be used for other diseases such as Parkinson’s disease, diabetes, and cardiac amyloidosis. Therefore, ATP-independent membrane chaperones could serve as a design platform for therapeutic development that targets various diseases associated with misfolding and aggregation of amphipathic helix. Methods Plasmids To generate yeast integration plasmids for pRS306-Gal-FLAG-Httex1-25QΔP-GFP and pRS306-Gal-FLAG-Httex1-97QΔP-GFP, the insert genes for FLAG-Httex1-25QΔP-GFP and FLAG-Httex1-25QΔP-GFP were amplified from pYES2-FLAG-Httex1-25QΔP-GFP and pYES2-FLAG-Httex1-97Q-GFP (gift from F. Ulrich Hartl Lab) 19 , respectively. The vector backbone for pRS306-Gal gene was amplified from pRS306-Gal-NDC80-RFP-MPS1 (gift from Won-ki Huh Lab) 84 . Gibson assembly was performed to incorporate the insert genes into the pRS306-Gal vector. For pRS413-Gal- sc PEX19, the sc PEX19 gene was amplified from the isolated yeast genomic DNA and further incorporated into pRS413-Gal vector using Gibson assembly. For His 6 - sc PEX19 and His 6 - hs PEX19, the yeast PEX19 gene and the human PEX19 gene were cloned into pET-33b, respectively. Yeast strains Yeast strain used in this study is W303a (MATa, can1-100, his3-11,15, leu2-3,112, trp1-1, ura3-1, ade2-1; gift from Won-ki Huh Lab). To generate FLAG-Httex1-25QΔP-GFP and FLAG-Httex1-25QΔP-GFP integrated- W303a strains, the linearized pRS306-Gal-FLAG-Httex1-25QΔP-GFP and pRS306-Gal-FLAG-Httex1-97QΔP-GFP by NcoI were transformed into W303a strain and selected on SD media lacking Ura. Spotting assay Yeast cells were grown to mid-log phase in the selective media containing 2% raffinose and 0.1% glucose. Overnight cultured cells were diluted to OD 600 of 0.1. The diluted cells were spotted onto 3% galactose and 1% raffinose-containing plates in serial 5-fold dilutions. Equal spotting was confirmed by spotting the same diluted cells on plates containing 2% glucose. After 2–3 days of incubation at 30°C, the images were acquired using the iBright™ FL 1000 imaging system (Thermo Fisher Scientific). Yeast PEX19 library generation A random mutagenesis library was generated by error-prone PCR using a GeneMorph II mutagenesis kit (Agilent). The cloned wild-type sc PEX19 gene was used as a template, and the reactions were performed according to the manufacturer’s protocol. To generate diverse mutations, two different amounts of template (5 ng and 50 ng) were used in PCR reactions for 34 cycles, followed by two more sequential PCR reactions. The resulting PCR products in each reaction were re-amplified using Q5 High-Fidelity DNA Polymerase (New England Biolabs) and gel-purified using a QIAquick Gel Extraction Kit (Qiagen). The gel-purified PCR products were inserted into pRS413 vector using Gibson assembly. The Gibson assembly mixture was desalted and then transformed into DH5α competent cells using electroporation. The transformed colonies were pooled and purified using a Midi prep kit (MACHEREY-NAGEL). Yeast toxicity-based screening The sc PEX19 plasmid library was transformed into Httex1-97QΔP strain as described 85 , and the cells were spread onto 2% glucose-containing SD-His-Ura plate. Approximately 90,000 colonies were pooled and further cultured in SR-His-Ura media supplemented with 2% raffinose and 0.1% glucose overnight at 30°C. The culture cells were adjusted OD 600 to 0.004 and spread onto an SG-His-Ura plate (3% galactose and 1% raffinose). We used the sc PEX19-WT-transformed Httex1-97QΔP and Httex1-25QΔP cells as negative and positive controls, respectively. The selected colonies from the SG-His-Ura plate were further confirmed using the spotting assay. The sc PEX19 variants were amplified using colony PCR, and then mutation sites were identified with sequencing. The selective sc PEX19 variants were generated by Quick Change mutagenesis using pRS413-Gal- sc PEX19-WT as a template. Those plasmids were transformed into Httex1-97QΔP and Httex1-25QΔP cells and further confirmed their suppression activity using a spotting assay. Preparation of yeast cell extracts For total yeast cell extract, the cell pellets were resuspended in 100 µL of 0.3 M NaOH and incubated for 3 min at room temperature. After washing the cells with water, the cell pellets were resuspended in 100 µL of lysis buffer (20 mM Tris (pH 8.0), 150 mM NaCl, 2% SDS) supplemented with protease inhibitor cocktail (cOmplete mini, EDTA-free protease inhibitor cocktail, Roche) and then incubated at 95°C for 5 min. After centrifugation at 15,000 g for 5 min, the clarified lysate was subjected to Western blot analysis. To monitor SDS-insoluble 97Q aggregates in yeast, the cell extracts were prepared as described with a minor modification 86 . The cells were induced at OD 600 of 0.1 with 1% galactose and 3% raffinose for 4 h. The cell pellets were resuspended in 200 µL lysis buffer (25 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 5% glycerol) supplemented with protease inhibitor cocktail and benzonase, and then lysed by Distruptor Genie (Scientific industries) with glass beads. The extracts were clarified by centrifugation of 500 g for 5 min at 4°C. Protein expression and purification Expression and purification of GST-Httex1-51Q-WT and GST-Httex1-51Q-ΔN proteins were performed as described 18 . GST-Httex1-51Q-WT and GST-Httex1-51Q-ΔN were expressed BL21 Star™ (DE3) (Invitrogen) with 1 mM IPTG at 18℃ for 2.5 h. Cells were resuspended in PBS supplemented with 150 mM NaCl, 1 mM EDTA, and cOmplete™ EDTA-free protease inhibitor cocktail (Roche). After sonication, the lysate was centrifuged at 20,000g for 20 min at 4℃. The supernatant was incubated with glutathione agarose resin (Thermo Scientific) for 1 h at 4℃. The resin was washed with PBS containing 500 mM NaCl, 5 mM MgCl 2 , 2 mM ATP, and then the protein was eluted with 15 mM glutathione dissolved in PBS. After dialysis in the buffer (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5% glycerol), the purified GST-Httex1-51Q proteins were concentrated with Amicon® Ultra 30,000 MWCO centrifugal filters (Millipore) and 0.22 µm-filtered through prior to storing at -80℃. His 6 - hs PEX19 or His 6 - sc PEX19 proteins were expressed in BL21 Star™ (DE3) with 0.5 mM IPTG at 18℃ overnight. His 6 -SGTA, MBP-His 6 , and N17-MBP-His 6 proteins were expressed in BL21 StarTM (DE3) with 0.1 mM IPTG for 3 h at 37℃. Cells were resuspended in Buffer A (20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 5 mM imidazole, 2 mM 2-mercaptoethanol, 10% glycerol) supplemented with cOmplete™ EDTA-free protease inhibitor cocktail and lysed using sonication. The clarified lysate was incubated with Ni-NTA agarose resin (Qiagen), and the proteins were eluted with 300 mM imidazole dissolved in Buffer A. After dialysis in Buffer B (20 mM HEPES (pH 7.5), 150 mM NaCl, 2 mM 2-mercaptoethanol, 10% glycerol), the purified proteins were stored at -80℃. To site-specifically incorporate p -benzoyl-L-phenylalanine (Bpa) into His 6 - hs PEX19 or GST-Httex1-51Q proteins, the coding sequence for the residue F255 in the His 6 - hs PEX19 variants or the residue F11 in GST-Httex1-51Q was replaced with an amber codon (TAG) using npfu-special polymerase (Enzynomics) according to the manufacturer’s introduction. Expression plasmids for His 6 - hs PEX19-FV amb or His 6 - hs PEX19-FI amb , or GST-Httex1-51Q-F11 amb and tRNA CUA Opt synthetase 45 were co-transformed into BL21 Star™ (DE3) cells. The expression of tRNA synthetase was induced with 0.2% arabinose at OD 600 of 0.3. At OD 600 of 0.6, proteins were induced with 0.5 mM IPTG and 1 mM Bpa (Bachem) at 18℃ overnight. Bpa incorporation into the proteins was confirmed by SDS-PAGE analysis. His 6 - hs PEX19-FV Bpa , His 6 - hs PEX19-FI Bpa , and GST-Httex1-51Q-F11 Bpa were purified in the same way as their non-Bpa proteins. His 6 -Ataxin3-78Q (gift from Sheena Radford lab) was expressed in BL21 Star™ (DE3) with 0.5 mM IPTG at 30℃ for 3 h. His 6 -Ataxin3-78Q was purified using Ni-NTA, the same procedure used for His 6 -PEX19 proteins. The eluted proteins were loaded onto a superdex™ 200 increase 10/300 column (Cytiva), and the monomer fractions were collected and further concentrated with Amicon® Ultra 50,000 MWCO centrifugal filter (Millipore). The purified His 6 -Ataxin3-78Q protein was snap-frozen and used for the filter trap assay. TDP43-TEV-MBP-His 6 (Addgene plasmid #104480) was expressed BL21 Star™ (DE3) with 0.1 mM IPTG overnight at 18℃. The purification of TDP43-TEV-MBP-His 6 was carried out as described 52 with a minor modification. Briefly, the cells were resuspended in Buffer C (20 mM Tris-HCl (pH 8.0), 1 M NaCl, 5 mM imidazole, 2 mM 2-mercaptoethanol, 10% glycerol) and then sonicated. The bound TDP43-TEV-MBP-His 6 protein onto Ni-NTA resin was eluted with 300 mM imidazole dissolved in Buffer C. The eluted proteins were loaded onto a superdexTM 200 increase 10/300 column and further purified in Buffer D (20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 mM DTT). The purified TDP43-TEV-MBP-His 6 protein was concentrated using Amicon® Ultra 50,000 MWCO centrifugal filter and stored at -80℃. Expression and purification of 2×Strep-SUMO-PEX26 (237-305aa) were carried out as described in the previous study 83 . Briefly, the protein was induced with 0.1 mM IPTG in BL21 Star™ (DE3) (Invitrogen) at 37 ℃ for 1 h. Cells were lysed by incubating with 0.5% N,N-Dimethyl-1-Dodecanamine-N-Oxide (LDAO, Anatrace) and 1×CelLytic™ B Cell Lysis Reagent (Sigma) for 40 min at room temperature (25 ℃). The clarified lysate was then diluted 3-fold with Buffer A and loaded onto a Strep-Tactin Sepharose column (IBA Lifesciences). The proteins were eluted with 15 mM d -Desthiobiotin (Sigma) dissolved in the buffer (20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 2 mM 2-Mercaptoethanol, 0.05% LDAO,10% glycerol) and further dialyzed in the buffer (20 mM HEPES (pH 7.5), 200 mM NaCl, 10% glycerol, 0.05% LDAO). In vitro aggregation assay – Filter trap assay 3 µM of GST-TEV-Httex1-51Q-Stag proteins were mixed with 1.5 µM of hs PEX19 or sc PEX19 proteins in the 1X TEV reaction buffer (Invitrogen). The polyQ aggregation reaction was initiated by adding 0.05 Units/µL of AcTEV protease (Invitrogen) and further incubated at 30℃. For the in vitro aggregation of Ataxin-3-78Q, 30 µM of His 6 -Ataxin-3-78Q were incubated with the equimolar concentration of hs PEX19 protein in the reaction buffer (20 mM HEPES (pH 7.5), 25 mM NaCl, 2 mM DTT, 5% glycerol) at 37℃. Samples were quenched at the indicated time points by mixing an equal volume of the quench buffer (4% (w/v) SDS, 0.1 M DTT) and then boiling at 95 ℃ for 10 min. The quenched samples were filtered through a 0.22 µm cellulose acetate membrane (Hyundai Micro) and then washed with 0.1% SDS. The membrane was probed using an S-tag antibody (1:3,000 dilution, Invitrogen) and the secondary antibody IRDye800 (1: 15,000 dilution, LiCor). The membrane-trapped polyQ aggregates were detected using iBright™ FL1000 imaging system. In vitro aggregation assay – Turbidity assay 7.5 µM of TDP43-MBP-His 6 protein was mixed with 7.5 µM of hs PEX19-WT or hs PEX19-FV proteins in the reaction buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM DTT). After the addition of AcTEV protease, the optical density values at 395 nm were recorded using a BioTek Epoch 2 plate reader (Agilent). Negative-stain TEM analysis 3 µM of GST-TEV-Htt51Q-Stag proteins were mixed with 3 µM of hsPEX19 proteins in the 1X TEV reaction buffer supplemented with AcTEV protease and incubated at 30 ℃ for 15 h. A copper grid coated with a continuous carbon film (Electron Microscopy Sciences) was negatively glow-discharged at 15mA for 30 sec. 3 µL of protein samples were applied to a glow-discharged grid, incubated at room temperature for 3 min, and washed twice with distilled water and 0.75% uranyl formate once. The samples were negatively stained with 0.75% uranyl formate for 1 min with gentle shaking. The negatively stained specimens were examined under a FEI Tecnai™ G2 spirit microscope operated at 120 kV. Micrographs were collected using a FEI Eagle 4 K x 4 K CCD camera at a nominal magnification of 15,000X with an electron dose of ~ 30 e-/A2. Bpa crosslinking assay In vitro aggregation assay with hs PEX19-FV Bpa / hs PEX19-FI Bpa or Httex1-51Q-F11 Bpa was performed as described in the section of Filter trap assay. The reaction was stopped by freezing samples at the indicated time points, and frozen reaction aliquots were crosslinked on dry ice ∼4 cm away from a UVP B-100AP lamp (Analytik Jena) for 10 min. Crosslinked and uncrosslinked hs PEX19 or Httex1-51Q proteins were resolved on SDS-PAGE and probed with anti-PEX19 (1: 3,000 dilution, Novus Biologicals) and anti-S-tag (1: 3,000 dilution, Invitrogen) antibodies, respectively. HEK293 cell culture and plasmid transfection HEK293T cells were cultured in Dulbecco’s Modified Eagle Medium, GlutaMAX ™ (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco), 100 U/ml streptomycin, and 100 U/ml penicillin and incubated in a humidified chamber with 5% CO 2 at 37°C. For coexpression of Httex1-134Q-GFP and hs PEX19 proteins, 3x10 5 cells per well were seeded on a 6-well plate one day prior to the transfection. Transient transfections of plasmids (each 1.25 µg) were carried out with Polyethylenimine (Polysciences) according to the manufacturer's instruction. Filter trap assay using HEK293T cell lysates HEK293T cell lysates were prepared for filter trap assay as described previously 87 with minor modifications. Briefly, cells were harvested with PBS supplemented with 1% Triton X-100 and 1x protease inhibitor cocktail (Sigma-Aldrich) and further incubated on ice for 30 min. The cell lysates were bath-sonicated in ice water for 5 min. After supplementing with 1% SDS and 50 mM DTT, the cell lysates were heated at 95°C for 10 min and stored at -80°C for filter trap assay and Western blot analysis. Filter trap assays were carried out as described above in the section on in vitro aggregation assay. SDS-insoluble Httex1-134Q-GFP aggregates were probed with anti-GFP antibody (1: 3,000 dilution, Sigma) and quantified using iBright Analysis Software (Thermo Fisher Scientific Inc). To check protein expression levels, the cell lysates were loaded onto 10% Tris-glycine gels, and then Httex1-134Q-GFP, PEX19, and actin were probed in immunoblots with GFP (1: 3,000 dilution, Sigma), HA (1: 3,000 dilution, Genscript), and actin (1: 5,000 dilution, Invitrogen) antibodies, respectively. Live Cell imaging HEK293T cells were cultured and co-transfected with Httex1-134Q-GFP and hs PEX19-WT or its variants in a confocal 6-well plate (SPL Life Sciences). At 48 h post-transfection, the plate was inserted in an inverted Eclipse Ti-E microscope (Nikon) equipped with a stage-top incubator (37 ℃, 5% CO 2 ). Live cell images were acquired with a 20X 0.5 NA objective and an automated perfect focus system (PFS). Primary neuronal cell culture and transfection Striata were dissected from mouse pups aged postnatal day 1 (P1). Dissected tissues were digested in an enzyme solution containing 0.1% w/v Papain, 100 µg/mL DNase I, and 1 mM HEPES in Earle’s Balanced Salt Solution (EBSS) (Sigma) at 37 ℃ for 30 min. After incubation, the enzyme solution was carefully aspirated, and dissected tissues were rinsed with a Neurobasal A medium containing 20% FBS. The tissues were dissociated by mechanical trituration, and the isolated cells were resuspended in the neuro culture medium containing 2% v/v B-27 Supplement, 1 mM L-Glutamine, 1% Penicillin/Streptomycin in Neurobasal A medium (Gibco). 3x10 5 cells were plated on glass coverslips precoated with 0.1 mg/mL Poly-D-Lysine (Gibco) and 5 µg/mL Laminin (Gibco) in a 24-well plate. Striatal neurons were cultivated at 37 ℃ with 5% CO 2 in a humidified incubator and used for experiments at 7 days in vitro (DIV). The cells were cotransfected with 1 µg of each plasmid using Lipofectamine™ LTX Reagent with PLUS™ Reagent (Invitrogen). Immunofluorescence For HEK293T cells, transfection was carried out under the same conditions for live cell imaging. At 48 h post-transfection, cells were washed with PBS and fixed in 4% paraformaldehyde (PFA) for 10 min at room temperature. After washing with PBS twice, cells were permeabilized with 0.1% Triton X-100 for 15 min and then blocked with 2% BSA for 1 h at room temperature. Cells were incubated with HA Tag antibody (1:100 dilution, Invitrogen) for hs PEX19-WT and its variants proteins and PMP70 antibody (1:200 dilution, Invitrogen) in 0.1% BSA solution at 4°C for two overnights. After washing the cells with PBS, the cells were incubated with Alexa Fluor 647 secondary antibody (1:1000 dilution, Invitrogen) for hs PEX19 proteins and Alexa Fluor 568 secondary antibody (1:1000 dilution, Invitrogen) for PMP70 for 1 h at room temperature. To visualize nuclei, cells were additionally stained with 300 nM of DAPI (Invitrogen). Images were acquired with an inverted Eclipse Ti-E microscope (Nikon) with a 60×1.4 NA oil objective. At 48 h post-transfection, primary neuronal cells were washed with PBS and fixed 15 min in 4% PFA at room temperature. After washing with PBS twice, the cells were blocked with 10% Normal Donkey Serum (Jackson immunoresearch) in PBS containing 0.1% Triton X-100 for 1h at room temperature. Cells were incubated in the blocking solution anti-Map2 antibody (1:500 dilution, SYSY) overnight at 4°C. After washing with PBS, the cells were incubated with Cy3 secondary antibody (Jackson immunoresearch) for 1h at room temperature. Nuclei were stained with DAPI. TDP43-BiFC measurements HEK293 TDP43-BiFC cells were cultured in the same media supplemented with 100 µg/mL Geneticin (G418). All cells were maintained in a humidified chamber with 5% CO 2 at 37°C. HEK293 TDP43-BiFC cells were plated on a 96-well plate with an Opti-MEM medium (Invitrogen). After 12 h, the cells were transfected with 0.1 µg of hs PEX19-WT or hs PEX19-FV plasmid using Lipofectamine®2000 reagent (Invitrogen). At 13 h post-transfection, TDP43-BiFC cells were treated with Forskolin (30 µM) or Apicidine (1 µM). After 36 h, nuclei were counterstained with Hoechst 33342 (Thermo Scientific). Fluorescence images were automatically acquired using Operetta CLS (PerkinElmer) with a 20x water immersion objective (TDP43-BiFC; λ ex = 460–490 nm and λ em = 500–550 nm, Hoechst; λ ex = 355–385 nm and λ em = 430–500 nm). The fluorescence intensities of TDP43-BiFC were quantified using Harmony 4.9 software (PerkinElmer). Data for each replicate were collected from 20 fields of view per well in the 96-well plate. Drosophila melanogaster stocks The fly lines W 1118 (stock #5905), UAS-Httex1-20Q (stock #68412), UAS-Httex1-93Q (stock #68418), Elav-Gal4 (stock #8765), and D42-Gal4 (stock #8816) were obtained from the Bloomington Drosophila Stock Center (USA). All flies were maintained at 27°C. To generate transgenic fly lines, N-terminally HA-tagged hs PEX19-WT and hs PEX19-FV genes were subcloned into the pACU2- UAS vector (gift from Chun Han, Cornell University). The pACU2- UAS vector lacking the HA- hs PEX19 gene was used as a negative control. The UAS-pACU2 vector , UAS-hsPEX19-WT , and UAS-hsPEX19-FV transgenic fly lines were generated by BestGene, Inc. These hs PEX19 transgenic fly lines were crossed with Httex1 transgenic fly lines, and the genotypes of generated transgenic fly lines used in this study are listed in Supplementary Table 1. Climbing assay Ten to fifteen male flies were collected and transferred into an acrylic cylinder (3 cm diameter, 18 cm height) without the use of any CO 2 anesthesia but with cotton-sealed. Prior to the climbing assay, the collected flies were transferred into a new food vial within 24 h. The flies were acclimatized for 20 min in the cylinder. The climbing ability was measured by tapping the cylinder against a table, which was recorded for 1 min. A climbing index is the proportion of flies climbing > 5 cm from the bottom of the cylinder within 5 sec. Seven technical trials were conducted for each individual experiment, and the average of these trials was considered as one biological replicate. Lifespan assay A maximum of 15 male flies were collected within 24 h after pupal eclosion (APE) in a food vial and transferred to a new vial every two days. The number of dead flies was counted daily until the flies died. Lifespan data were plotted as Kaplan-Meier survival curves and statistical analyses were performed using the log-rank (Mantel-Cox) test. Immunohistochemistry Male fly heads were dissected to extract brains in Schneider's insect medium (Sigma-Aldrich). The brains were fixed with 3.7% formaldehyde for 20 min at room temperature. After washing with the washing buffer (0.3% Triton X-100 in PBS) for 10 min (a total of six washes), the brain samples were incubated in the blocking buffer (5% Normal Donkey Serum in PBS containing 0.3% Triton X-100) for 1 h at room temperature with gentle shaking. To stain Httex1-93Q and HA- hs PEX19 proteins, the samples were incubated with Huntingtin (mEM48) mouse (1:200 dilution, Sigma-Aldrich) and HA rabbit (1: 200 dilution, Cell Signaling) primary antibodies at 4°C overnight, respectively. After washing with the washing buffer, the samples were further incubated with rabbit Alexa Fluor 555 (1: 200 dilution, Thermo Fisher Scientific) and mouse Alexa Fluor488 (1: 200 dilution, Invitrogen) secondary antibodies for 2 h at room temperature. The stained samples were mounted onto slides using Antifade Mounting Medium with DAPI (VectorLabs). All stained brain samples were taken at 400X magnification using 40X water immersion objective, acquired by Zeiss LSM700 confocal microscopy. Confocal microscopy images were set to a threshold to eliminate non-puncta fluorescence signals using Zeiss ZEN software. The same threshold settings were applied to all the images of the experiment. The total number of puncta in the region of interest was calculated using Fiji software. Quantification and statistical analysis Statistical analysis was conducted using GraphPad Prism Software. As indicated in Figure legends, we used Log-rank (Mantel-Cox) test, one-way ANOVA with Tukey post-hoc test, and two-way ANOVA with Tukey post-hoc test. All data were shown as mean ± SD or SEM, and p values were represented with asterisks: * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001. Declarations Acknowledgments We thank S.-h. Park and K. Shen for valuable discussions on yeast culture and TEM sample preparation. We thank the Research Solution Center at the Institute for Basic Science for the confocal microscopy facility. We thank W.-k. Huh, F.U. Hartl, J. Frydman, and S.E. Radford for sharing the W303a yeast strain or plasmids. This work was supported by grants from the Institute for Basic Science (IBS-R030-Y1 to H.C. and IBS-R030-C1 to H.M.K.) and the National Research Foundation (NRF) grant funded by the Korea government (MSIT) (RS-2023-00261784 to Y.K.K. and 2022R1C1C100714612 to S.L.). Author contributions H.C. conceived the study. H.C., S.B.L., and H.M.K. designed the study. J.O., C.C., E.S.K., K.W.M., M.K., H.K., H.C.J., S.H.A., N.L., H.S.B, and H.C. carried out all experiments. J.O., C.C., E.S.K., K.W.M., M.K., H.K., H.C.J., N.L., S.L., and H.C. performed data analysis. H.C., S.B.L., H.M.K., S.L., and Y.K.K. supervised the study. H.C., J.O., E.S.K., K.W.M., M.K., H.K., S.L. drafted the manuscript. H.C., S.B.L., H.M.K. edited the manuscript. All authors read and approved the final version of the manuscript. Competing interests The authors declare no competing interests. References Hartl, F. U., Bracher, A. & Hayer-Hartl, M. Molecular chaperones in protein folding and proteostasis. Nature 475 , 324–332 (2011). Kim, Y. E., Hipp, M. S., Bracher, A., Hayer-Hartl, M. & Hartl, F. U. Molecular chaperone functions in protein folding and proteostasis. Annu Rev Biochem 82 , 323–355 (2013). Ellis, R. J. & Minton, A. P. Protein aggregation in crowded environments. Biol Chem 387 , 485–497 (2006). Yu, I. et al. Biomolecular interactions modulate macromolecular structure and dynamics in atomistic model of a bacterial cytoplasm. Elife 5 , e19274 (2016). Balchin, D., Hayer-Hartl, M. & Hartl, F. U. In vivo aspects of protein folding and quality control. Science 353 , aac4354 (2016). Nollen, E. A. A. et al. Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation. Proc Natl Acad Sci U S A 101 , 6403–6408 (2004). Brehme, M. et al. A chaperome subnetwork safeguards proteostasis in aging and neurodegenerative disease. Cell Rep 9 , 1135–1150 (2014). Shemesh, N. et al. The landscape of molecular chaperones across human tissues reveals a layered architecture of core and variable chaperones. Nat Commun 12 , 2180 (2021). Walther, D. M. et al. Widespread Proteome Remodeling and Aggregation in Aging C. elegans. Cell 161 , 919–932 (2015). Iadanza, M. G., Jackson, M. P., Hewitt, E. W., Ranson, N. A. & Radford, S. E. A new era for understanding amyloid structures and disease. Nat Rev Mol Cell Biol 19 , 755–773 (2018). Hipp, M. S., Kasturi, P. & Hartl, F. U. The proteostasis network and its decline in ageing. Nat Rev Mol Cell Biol 20 , 421–435 (2019). A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. The Huntington’s Disease Collaborative Research Group. Cell 72 , 971–983 (1993). DiFiglia, M. et al. Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science 277 , 1990–1993 (1997). Scherzinger, E. et al. Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo. Cell 90 , 549–558 (1997). Gusella, J. F. & MacDonald, M. E. Molecular genetics: Unmasking polyglutamine triggers in neurodegenerative disease. Nat Rev Neurosci 1 , 109–115 (2000). Tam, S. et al. The chaperonin TRiC blocks a huntingtin sequence element that promotes the conformational switch to aggregation. Nat Struct Mol Biol 16 , 1279–1285 (2009). Crick, S. L., Ruff, K. M., Garai, K., Frieden, C. & Pappu, R. V. Unmasking the roles of N- and C-terminal flanking sequences from exon 1 of huntingtin as modulators of polyglutamine aggregation. Proc Natl Acad Sci U S A 110 , 20075–20080 (2013). Shen, K. et al. Control of the structural landscape and neuronal proteotoxicity of mutant Huntingtin by domains flanking the polyQ tract. Elife 5 , e18065 (2016). Gruber, A. et al. Molecular and structural architecture of polyQ aggregates in yeast. Proc Natl Acad Sci U S A 115 , E3446–E3453 (2018). Kim, Y. E. et al. Soluble Oligomers of PolyQ-Expanded Huntingtin Target a Multiplicity of Key Cellular Factors. Mol Cell 63 , 951–964 (2016). Riguet, N. et al. Nuclear and cytoplasmic huntingtin inclusions exhibit distinct biochemical composition, interactome and ultrastructural properties. Nat Commun 12 , 6579 (2021). Gidalevitz, T., Ben-Zvi, A., Ho, K. H., Brignull, H. R. & Morimoto, R. I. Progressive Disruption of Cellular Protein Folding in Models of Polyglutamine Diseases. Science 311 , 1471–1474 (2006). Bäuerlein, F. J. B. et al. In Situ Architecture and Cellular Interactions of PolyQ Inclusions. Cell 171 , 179-187.e10 (2017). Jackrel, M. E. et al. Potentiated Hsp104 variants antagonize diverse proteotoxic misfolding events. Cell 156 , 170–182 (2014). Wang, J. D., Herman, C., Tipton, K. A., Gross, C. A. & Weissman, J. S. Directed evolution of substrate-optimized GroEL/S chaperonins. Cell 111 , 1027–1039 (2002). Quan, S. et al. Genetic selection designed to stabilize proteins uncovers a chaperone called Spy. Nat Struct Mol Biol 18 , 262–269 (2011). Mack, K. L. et al. Tuning Hsp104 specificity to selectively detoxify α-synuclein. Mol Cell 83 , 3314-3332.e9 (2023). Tariq, A. et al. Mining Disaggregase Sequence Space to Safely Counter TDP-43, FUS, and α-Synuclein Proteotoxicity. Cell Rep 28 , 2080-2095.e6 (2019). Sathyanarayanan, U. et al. ATP hydrolysis by yeast Hsp104 determines protein aggregate dissolution and size in vivo. Nat Commun 11 , 5226 (2020). Jones, J. M., Morrell, J. C. & Gould, S. J. PEX19 is a predominantly cytosolic chaperone and import receptor for class 1 peroxisomal membrane proteins. J Cell Biol 164 , 57–67 (2004). Yagita, Y., Hiromasa, T. & Fujiki, Y. Tail-anchored PEX26 targets peroxisomes via a PEX19-dependent and TRC40-independent class I pathway. J Cell Biol 200 , 651–666 (2013). Chen, Y. et al. Hydrophobic handoff for direct delivery of peroxisome tail-anchored proteins. Nat Commun 5 , 5790 (2014). Ripaud, L. et al. Overexpression of Q-rich prion-like proteins suppresses polyQ cytotoxicity and alters the polyQ interactome. Proc Natl Acad Sci U S A 111 , 18219–18224 (2014). Emmanouilidis, L. et al. Allosteric modulation of peroxisomal membrane protein recognition by farnesylation of the peroxisomal import receptor PEX19. Nat Commun 8 , 14635 (2017). Sato, Y. et al. Structural basis for docking of peroxisomal membrane protein carrier Pex19p onto its receptor Pex3p. EMBO J 29 , 4083–4093 (2010). Schmidt, F. et al. Insights into peroxisome function from the structure of PEX3 in complex with a soluble fragment of PEX19. J Biol Chem 285 , 25410–25417 (2010). Liu, Y., Yagita, Y. & Fujiki, Y. Assembly of Peroxisomal Membrane Proteins via the Direct Pex19p-Pex3p Pathway. Traffic 17 , 433–455 (2016). Rucktäschel, R. et al. Farnesylation of pex19p is required for its structural integrity and function in peroxisome biogenesis. J Biol Chem 284 , 20885–20896 (2009). Tam, S., Geller, R., Spiess, C. & Frydman, J. The chaperonin TRiC controls polyglutamine aggregation and toxicity through subunit-specific interactions. Nat Cell Biol 8 , 1155–1162 (2006). Rozales, K. et al. Differential roles for DNAJ isoforms in HTT-polyQ and FUS aggregation modulation revealed by chaperone screens. Nat Commun 13 , 516 (2022). Sivanandam, V. N. et al. The aggregation-enhancing huntingtin N-terminus is helical in amyloid fibrils. J Am Chem Soc 133 , 4558–4566 (2011). Elena-Real, C. A. et al. The structure of pathogenic huntingtin exon 1 defines the bases of its aggregation propensity. Nat Struct Mol Biol 30 , 309–320 (2023). Scarff, C. A. et al. Examination of Ataxin-3 (atx-3) Aggregation by Structural Mass Spectrometry Techniques: A Rationale for Expedited Aggregation upon Polyglutamine (polyQ) Expansion. Mol Cell Proteomics 14 , 1241–1253 (2015). Shen, K. et al. Dual Role of Ribosome-Binding Domain of NAC as a Potent Suppressor of Protein Aggregation and Aging-Related Proteinopathies. Mol Cell 74 , 729-741.e7 (2019). Young, T. S., Ahmad, I., Yin, J. A. & Schultz, P. G. An enhanced system for unnatural amino acid mutagenesis in E. coli. J. Mol. Biol. 395 , 361–374 (2010). Costello, J. L. et al. Predicting the targeting of tail-anchored proteins to subcellular compartments in mammalian cells. J Cell Sci 130 , 1675–1687 (2017). Fransen, M., Wylin, T., Brees, C., Mannaerts, G. P. & Van Veldhoven, P. P. Human pex19p binds peroxisomal integral membrane proteins at regions distinct from their sorting sequences. Mol Cell Biol 21 , 4413–4424 (2001). Shao, S., Rodrigo-Brenni, M. C., Kivlen, M. H. & Hegde, R. S. Mechanistic basis for a molecular triage reaction. Science 355 , 298–302 (2017). Guna, A., Volkmar, N., Christianson, J. C. & Hegde, R. S. The ER membrane protein complex is a transmembrane domain insertase. Science 359 , 470–473 (2018). Schueller, N. et al. The peroxisomal receptor Pex19p forms a helical mPTS recognition domain. EMBO J 29 , 2491–2500 (2010). Kulak, N. A., Pichler, G., Paron, I., Nagaraj, N. & Mann, M. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat Methods 11 , 319–324 (2014). Hallegger, M. et al. TDP-43 condensation properties specify its RNA-binding and regulatory repertoire. Cell 184 , 4680-4696.e22 (2021). Neumann, M. et al. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314 , 130–133 (2006). Brady, O. A., Meng, P., Zheng, Y., Mao, Y. & Hu, F. Regulation of TDP-43 aggregation by phosphorylation and p62/SQSTM1. J Neurochem 116 , 248–259 (2011). Neumann, M. et al. Phosphorylation of S409/410 of TDP-43 is a consistent feature in all sporadic and familial forms of TDP-43 proteinopathies. Acta Neuropathol 117 , 137–149 (2009). Cohen, T. J. et al. An acetylation switch controls TDP-43 function and aggregation propensity. Nat Commun 6 , 5845 (2015). Lokireddy, S., Kukushkin, N. V. & Goldberg, A. L. cAMP-induced phosphorylation of 26S proteasomes on Rpn6/PSMD11 enhances their activity and the degradation of misfolded proteins. Proc Natl Acad Sci U S A 112 , E7176-7185 (2015). Han, J. W. et al. Apicidin, a histone deacetylase inhibitor, inhibits proliferation of tumor cells via induction of p21WAF1/Cip1 and gelsolin. Cancer Res 60 , 6068–6074 (2000). Li, H., Li, S. H., Yu, Z. X., Shelbourne, P. & Li, X. J. Huntingtin aggregate-associated axonal degeneration is an early pathological event in Huntington’s disease mice. J Neurosci 21 , 8473–8481 (2001). Li, H., Li, S. H., Johnston, H., Shelbourne, P. F. & Li, X. J. Amino-terminal fragments of mutant huntingtin show selective accumulation in striatal neurons and synaptic toxicity. Nat Genet 25 , 385–389 (2000). Monsellier, E., Redeker, V., Ruiz-Arlandis, G., Bousset, L. & Melki, R. Molecular interaction between the chaperone Hsc70 and the N-terminal flank of huntingtin exon 1 modulates aggregation. J Biol Chem 290 , 2560–2576 (2015). Kakkar, V. et al. The S/T-Rich Motif in the DNAJB6 Chaperone Delays Polyglutamine Aggregation and the Onset of Disease in a Mouse Model. Mol Cell 62 , 272–283 (2016). Gillis, J. et al. The DNAJB6 and DNAJB8 protein chaperones prevent intracellular aggregation of polyglutamine peptides. J Biol Chem 288 , 17225–17237 (2013). Månsson, C. et al. DNAJB6 is a peptide-binding chaperone which can suppress amyloid fibrillation of polyglutamine peptides at substoichiometric molar ratios. Cell Stress Chaperones 19 , 227–239 (2014). Ayala Mariscal, S. M. et al. Identification of a HTT-specific binding motif in DNAJB1 essential for suppression and disaggregation of HTT. Nat Commun 13 , 4692 (2022). Karamanos, T. K., Tugarinov, V. & Clore, G. M. Unraveling the structure and dynamics of the human DNAJB6b chaperone by NMR reveals insights into Hsp40-mediated proteostasis. Proc Natl Acad Sci U S A 116 , 21529–21538 (2019). Hegde, R. S. & Keenan, R. J. The mechanisms of integral membrane protein biogenesis. Nat Rev Mol Cell Biol 23 , 107–124 (2022). Chio, U. S., Cho, H. & Shan, S. Mechanisms of Tail-Anchored Membrane Protein Targeting and Insertion. Annu Rev Cell Dev Biol 33 , 417–438 (2017). Rao, M. et al. Multiple selection filters ensure accurate tail-anchored membrane protein targeting. Elife 5 , (2016). Cho, H. et al. Dynamic stability of Sgt2 enables selective and privileged client handover in a chaperone triad. Nat Commun 15 , 134 (2024). Woerner, A. C. et al. Cytoplasmic protein aggregates interfere with nucleocytoplasmic transport of protein and RNA. Science 351 , 173–176 (2016). Gasset-Rosa, F. et al. Polyglutamine-Expanded Huntingtin Exacerbates Age-Related Disruption of Nuclear Integrity and Nucleocytoplasmic Transport. Neuron 94 , 48-57.e4 (2017). Lee, W.-C. M., Yoshihara, M. & Littleton, J. T. Cytoplasmic aggregates trap polyglutamine-containing proteins and block axonal transport in a Drosophila model of Huntington’s disease. Proc Natl Acad Sci U S A 101 , 3224–3229 (2004). Tartari, M. et al. Phylogenetic comparison of huntingtin homologues reveals the appearance of a primitive polyQ in sea urchin. Mol Biol Evol 25 , 330–338 (2008). Muthukumar, G. et al. Triaging of α-helical proteins to the mitochondrial outer membrane by distinct chaperone machinery based on substrate topology. Mol Cell S1097-2765(24)00095–9 (2024) doi:10.1016/j.molcel.2024.01.028. Zhu, M. & Fink, A. L. Lipid binding inhibits alpha-synuclein fibril formation. J Biol Chem 278 , 16873–16877 (2003). Burré, J., Sharma, M. & Südhof, T. C. Definition of a molecular pathway mediating α-synuclein neurotoxicity. J Neurosci 35 , 5221–5232 (2015). MacRaild, C. A., Howlett, G. J. & Gooley, P. R. The structure and interactions of human apolipoprotein C-II in dodecyl phosphocholine. Biochemistry 43 , 8084–8093 (2004). Apostolidou, M., Jayasinghe, S. A. & Langen, R. Structure of α-Helical Membrane-bound Human Islet Amyloid Polypeptide and Its Implications for Membrane-mediated Misfolding. J Biol Chem 283 , 17205–17210 (2008). Robert, X. & Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res 42 , W320-324 (2014). Madeira, F. et al. Search and sequence analysis tools services from EMBL-EBI in 2022. Nucleic Acids Res 50 , W276–W279 (2022). Kyte, J. & Doolittle, R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol 157 , 105–132 (1982). Oh, J., Kim, D. K., Ahn, S. H., Kim, H. M. & Cho, H. A dual role of the conserved PEX19 helix in safeguarding peroxisomal membrane proteins. iScience 27 , (2024). Lim, G. & Huh, W.-K. Rad52 phosphorylation by Ipl1 and Mps1 contributes to Mps1 kinetochore localization and spindle assembly checkpoint regulation. Proc Natl Acad Sci U S A 114 , E9261–E9270 (2017). Gietz, R. D. & Schiestl, R. H. Large-scale high-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2 , 38–41 (2007). Klaips, C. L., Gropp, M. H. M., Hipp, M. S. & Hartl, F. U. Sis1 potentiates the stress response to protein aggregation and elevated temperature. Nat Commun 11 , 6271 (2020). Tashiro, E. et al. Prefoldin protects neuronal cells from polyglutamine toxicity by preventing aggregation formation. J Biol Chem 288 , 19958–19972 (2013). Additional Declarations There is NO Competing Interest. Supplementary Files HttSupplementaryInfov1.docx ExtendedDataFig.docx Cite Share Download PDF Status: Published Journal Publication published 17 Jan, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4292547","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":297105851,"identity":"ec5819fb-93e9-4ca2-8e6f-e4bd24bf8e8a","order_by":0,"name":"Hyunju 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variants that suppress cellular toxicity of mHttex1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Yeast toxicity-based screening to identify mHttex1 suppressors. \u003cem\u003esc\u003c/em\u003ePEX19 plasmid library generated by random mutagenesis was transformed into Httex1-97QΔP-GFP-integrated yeast cells. Both \u003cem\u003esc\u003c/em\u003ePEX19 variants and Httex1-97QΔP-GFP are under the control of the \u003cem\u003eGAL1\u003c/em\u003e promoter. The sequences of identified \u003cem\u003esc\u003c/em\u003ePEX19 mutants, m1 and m2, are shown, and the common mutation sites were highlighted in red.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ec\u003c/strong\u003e, Growth test of Httex1-25QΔP-GFP- and Httex1-97QΔP-GFP-integrated yeast cells expressing \u003cem\u003esc\u003c/em\u003ePEX19-WT and its \u003cem\u003esc\u003c/em\u003ePEX19 variants. Five-fold serial dilutions of cells were spotted on galactose plates to coexpress Httex1-25QΔP or Httex1-97QΔP and the indicated \u003cem\u003esc\u003c/em\u003ePEX19 proteins (Right) or on glucose plates as loading controls (Left).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e, Confocal microscopy images of Httex1-25QΔP-GFP and Httex1-97QΔP-GFP cells upon coexpression of \u003cem\u003esc\u003c/em\u003ePEX19-WT, \u003cem\u003esc\u003c/em\u003ePEX19-FV, and \u003cem\u003esc\u003c/em\u003ePEX19-FI. Scale bar: 10 µm\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e, \u003cstrong\u003ef\u003c/strong\u003e, (\u003cstrong\u003ee\u003c/strong\u003e) Representative image of Western blot monitoring SDS-insouble and SDS-soluble Httex1-97QΔP-GFP proteins and (\u003cstrong\u003ef\u003c/strong\u003e) quantification of SDS-insoluble protein in \u003cstrong\u003ee\u003c/strong\u003e. Yeast cells were lysed using glass beads, and then the total cell lysates were analyzed using Western blot. N-terminally Flag-tagged Httex1-97QΔP-GFP was probed using Flag antibody. PGK1 serves as a loading control. Data in \u003cstrong\u003ef\u003c/strong\u003e are shown as mean ± SD, with three biological replicates (n=3).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4292547/v1/0da6de858e2e5d30ab277f42.png"},{"id":56642905,"identity":"64c98f84-ceda-4ab9-ba63-5bd6bc26dd47","added_by":"auto","created_at":"2024-05-17 06:26:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":182059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ehs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ePEX19 variants suppress mHttex1 aggregation \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and in mammalian cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Multiple sequence alignment of the α4 helix of the PEX19 sequence with various species. The alignment was performed using the Clustal Omega and displayed with ESPript 3\u003csup\u003e80,81\u003c/sup\u003e. Conserved sequences of \u003cem\u003esc\u003c/em\u003ePEX19-L288 and \u003cem\u003esc\u003c/em\u003ePEX19-E292 are highlighted as green boxes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e, NMR structure of \u003cem\u003ehs\u003c/em\u003ePEX19-CTD (161-299 aa) (PDB 5LNF)\u003csup\u003e34\u003c/sup\u003e. Two conserved residues, M255 and Q259, are located at the α4 helix of \u003cem\u003ehs\u003c/em\u003ePEX19 and are shown in magenta. The M255 residue of \u003cem\u003ehs\u003c/em\u003ePEX19 is known to bind its C-terminally modified farnesyl group (cyan)\u003csup\u003e34\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e, \u003cem\u003eIn vitro\u003c/em\u003e aggregation assay of Httex1-51Q in the absence and presence of \u003cem\u003esc\u003c/em\u003ePEX19 (\u003cstrong\u003ec\u003c/strong\u003e) and \u003cem\u003ehs\u003c/em\u003ePEX19 (\u003cstrong\u003ed\u003c/strong\u003e) proteins. 3 μM of GST-TEV-Httex1-51Q-Stag and 1.5 μM of PEX19 proteins were incubated at 30℃, and after the addition of TEV protease, samples were quenched at the indicated time points. Filter trap assay was performed with the quenched samples as outlined in Extended Data Fig. 3a.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e, Negatively stained transmission electron micrograph (TEM) images of Httex1-51Q in the absence and presence of \u003cem\u003ehs\u003c/em\u003ePEX19 proteins. Equimolar concentrations (3 µM) of GST-TEV-Httex1-51Q and \u003cem\u003ehs\u003c/em\u003ePEX19 proteins were incubated for 15 h at 30℃, and the samples were processed for TEM analysis. Scale bar: 500 nm\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e, Confocal microscopy images of HEK293T cells coexpressing Httex1-19Q-GFP or Httex1-134Q-GFP and \u003cem\u003ehs\u003c/em\u003ePEX19. Empty vector control (denoted as vector control) was used as a negative control. Scale bar: 50 µm\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg\u003c/strong\u003e and \u003cstrong\u003eh\u003c/strong\u003e, (\u003cstrong\u003eg\u003c/strong\u003e) Representative image of the filter trap assays monitoring the SDS-insoluble Httex1-134Q-GFP aggregates in HEK293T cells upon coexpression of \u003cem\u003ehs\u003c/em\u003ePEX19 proteins. (\u003cstrong\u003eh\u003c/strong\u003e) Quantification of the images in \u003cstrong\u003eg\u003c/strong\u003e and their replicates. Data in \u003cstrong\u003eg\u003c/strong\u003e are shown as mean ± SD, with three biological replicates (n=3).\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4292547/v1/8fb3adb9d20dcec4f6965212.png"},{"id":56642906,"identity":"bcb5703e-d09c-4171-a7ea-e455f49cff68","added_by":"auto","created_at":"2024-05-17 06:26:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":124445,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe α4 helix of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ehs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ePEX19 variants directly interacts with the N17 domain of mHttex1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, N-terminal GST-tagged Httex1-51Q-WT or Httex1-51Q-ΔN were used to monitor Htt51Q aggregation or interaction between \u003cem\u003ehs\u003c/em\u003ePEX19 and Httex1-51Q proteins \u003cem\u003ein vitro\u003c/em\u003e. The helical wheel illustrates the distribution of hydrophobic amino acids in the amphipathic helix of the N17 domain of Httex1-51Q. The sequences of hydrophobic amino acids in the N17 domain are also highlighted in red.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e, \u003cem\u003eIn vitro\u003c/em\u003e aggregation assay of Httex1-51Q-ΔN in the absence and presence of \u003cem\u003ehs\u003c/em\u003ePEX19 variants. The assay was carried out as described in \u003cstrong\u003eFig. 2d\u003c/strong\u003e and \u003cstrong\u003eExtended Data Fig. 3a\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e, Schematic representation of Bpa crosslinking assay. GST-TEV-Httex1-51Q and \u003cem\u003ehs\u003c/em\u003ePEX19\u003csup\u003eBpa \u003c/sup\u003eor GST-TEV-Httex1-51\u003csup\u003eBpa\u003c/sup\u003e and \u003cem\u003ehs\u003c/em\u003ePEX19\u003csup\u003e \u003c/sup\u003e\u0026nbsp;were incubated in the presence of TEV protease at 30 °C. After 3 h, the samples were frozen and analyzed by UV crosslinking at -20 °C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e, Bpa crosslinking assay to monitor the direct association of \u003cem\u003ehs\u003c/em\u003ePEX19\u003csup\u003eBpa\u003c/sup\u003e with Httex1-51Q-WT or Httex1-51Q-ΔN. 3 μM of \u003cem\u003ehs\u003c/em\u003ePE+X19-FV\u003csup\u003eBpa\u003c/sup\u003e or \u003cem\u003ehs\u003c/em\u003ePEX19-FI\u003csup\u003eBpa\u003c/sup\u003e was incubated with equal molar concentration of Httex1-51Q-WT or Httex1-51Q-ΔN for 3 h at 30°C. Crosslinked samples were analyzed using Western blots probed with PEX19 (\u003cstrong\u003ed\u003c/strong\u003e) and S-tag (51Q) (\u003cstrong\u003ee\u003c/strong\u003e) antibodies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e, Bpa crosslinking assay to monitor the intermolecular interaction of Httex1-51Q-F11\u003csup\u003eBpa\u003c/sup\u003e with \u003cem\u003ehs\u003c/em\u003ePEX19-WT and its \u003cem\u003ehs\u003c/em\u003ePEX19 variants. 3 μM of Httex1-51Q-F11\u003csup\u003eBpa\u003c/sup\u003e was incubated with 1.5 μM of \u003cem\u003ehs\u003c/em\u003ePEX19 proteins for 3 h at 30°C. Crosslinked samples were subjected to Western blot analysis against PEX19 (\u003cstrong\u003ef\u003c/strong\u003e) and S-tag (51Q) (\u003cstrong\u003eg\u003c/strong\u003e) antibodies.\u0026nbsp;\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4292547/v1/e8fe3407ccf3512a919fbe07.png"},{"id":56643341,"identity":"fed87773-c116-4496-9285-49bd7746b834","added_by":"auto","created_at":"2024-05-17 06:34:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":145723,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe α4 helix of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ehs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ePEX19 variants is a specific binding site for the N17 domain of Httex1-51Q.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Sequences of the N17 domain of Httex1-51Q and TMDs of peroxisomal and mitochondrial membrane proteins and their Grand Average of Hydropathy (GRAVY) scores\u003csup\u003e82\u003c/sup\u003e. All listed membrane proteins are known to interact with \u003cem\u003ehs\u003c/em\u003ePEX19 during their targeting to peroxisome or mitochondria\u003csup\u003e30,37,46,47\u003c/sup\u003e. PEX26, PEX11B, and ACBD5 are peroxisomal tail-anchored membrane proteins (TAs) that contain TMDs near the C-terminus, whereas Fis1 is a dually localized TA in mitochondria and peroxisomes\u003csup\u003e46,47\u003c/sup\u003e. PEX13 and PMP34 are multi-spanning peroxisomal membrane proteins (PMPs)\u003csup\u003e30,47\u003c/sup\u003e. ATAD1 is an N-terminal signal-anchored membrane protein localized in mitochondria and peroxisome\u003csup\u003e37\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e, Schematic representation of Httex1-51Q, N17-MBP, MBP-WT, and PEX26. The isolated N17 sequence was N-terminally fused to MBP (maltose binding protein). The recombinant PEX26 protein contains the N-terminal 2×Strep-tagged SUMO domain and the PEX26 targeting sequences (237-305 aa) encompassing the TMD and C-terminal charged tail of PEX26\u003csup\u003e83\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e, Bpa crosslinking assay of \u003cem\u003ehs\u003c/em\u003ePEX19\u003csup\u003eBpa\u003c/sup\u003e with MBP-WT or N17-MBP. 3 µM of \u003cem\u003ehs\u003c/em\u003ePEX19-FV\u003csup\u003eBpa\u003c/sup\u003e or \u003cem\u003ehs\u003c/em\u003ePEX19-FI\u003csup\u003eBpa\u003c/sup\u003e was incubated with 3 µM of MBP-WT or N17-MBP at room temperature for 5 min. The Bpa crosslinking assay was carried out as described in Methods, and the crosslinked product was detected using the PEX19 antibody.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e, \u003cem\u003eIn vitro \u003c/em\u003eaggregation assay to monitor the chaperone activity of \u003cem\u003ehs\u003c/em\u003ePEX19-FV and SGTA toward Httex1-51Q-WT. 3 µM of Httex1-51Q-WT was incubated 1.5 µM of \u003cem\u003ehs\u003c/em\u003ePEX19-FV or SGTA at 30°C. The quenched samples were subjected to the filter trap assay.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e, \u003cstrong\u003ef\u003c/strong\u003e, Representative images of His\u003csub\u003e6\u003c/sub\u003e-PEX19 pulldown assay with PEX26 in \u003cstrong\u003ee\u003c/strong\u003e and their quantification in \u003cstrong\u003ef\u003c/strong\u003e. 150 nM of PEX26 was incubated with 600 nM of \u003cem\u003ehs\u003c/em\u003ePEX19 proteins at room temperature. After 5 min, His\u003csub\u003e6\u003c/sub\u003e-PEX19 pulldown assays were carried out using Talon resin and subjected to western blot analysis. I, FT, and E denote total input, flowthrough, and elution, respectively. The amounts of PEX26 bound to \u003cem\u003ehs\u003c/em\u003ePEX19 were analyzed by western blot against Strep and His antibodies. Data in \u003cstrong\u003ef\u003c/strong\u003e are shown as mean ± SEM, with n=2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg\u003c/strong\u003e-\u003cstrong\u003ei\u003c/strong\u003e, Bpa crosslinking assay of \u003cem\u003ehs\u003c/em\u003ePEX19\u003csup\u003eBpa\u003c/sup\u003e with either Httex1-51Q or PEX26. The Bpa crosslinking assays with Httex1-51Q were carried out under the same conditions as \u003cstrong\u003eFig. 3f\u003c/strong\u003e and \u003cstrong\u003e3g\u003c/strong\u003e. Prior to the Bpa crosslinking assay, 0.75\u0026nbsp; µM of PEX26 was incubated with 3 µM of \u003cem\u003ehs\u003c/em\u003ePEX19\u003csup\u003eBpa\u003c/sup\u003e at room temperature for 5 min. “*” represents the SDS-resistant PEX26 dimers in (\u003cstrong\u003ei\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4292547/v1/6f296adb03c5b9abf8710621.png"},{"id":56642909,"identity":"90280cc7-a14d-4c62-a510-ccbcf3bd8cbe","added_by":"auto","created_at":"2024-05-17 06:26:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":303420,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ehs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ePEX19-FV mitigates mHttex1-induced neurodegenerative phenotypes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Confocal microscopy images of mouse striatal neurons coexpressing Httex1-19Q-GFP and Httex1-134Q-GFP with vector control, \u003cem\u003ehs\u003c/em\u003ePEX19-WT, or \u003cem\u003ehs\u003c/em\u003ePEX19-FV. Httex1-19Q-GFP or Httex1-134Q-GFP and Map2 (dendrite marker) were stained using GFP and Map2 antibodies, respectively. Scale bar: 20 µm. Inset scale bar: 5 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb,\u003c/strong\u003e Climbing ability of 12-day-old adult flies (\u003cem\u003eW\u003c/em\u003e\u003csup\u003e\u003cem\u003e1118\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eHttex1-20Q\u003c/em\u003e, and \u003cem\u003eHttex1-93Q\u003c/em\u003e) expressing vector control, \u003cem\u003ehs\u003c/em\u003ePEX19-WT, and \u003cem\u003ehs\u003c/em\u003ePEX19-FV in motor neurons. The data in \u003cstrong\u003eb\u003c/strong\u003e are shown as violin plots with mean and quartiles (n=102~127 adult flies). Statistical significance was evaluated using the two-way ANOVA with Tukey post-hoc test. **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, ns=not significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e, Climbing ability of 10-day-old adult flies (\u003cem\u003eW\u003c/em\u003e\u003csup\u003e\u003cem\u003e1118\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eHttex1-20Q\u003c/em\u003e, and \u003cem\u003eHttex1-93Q\u003c/em\u003e) expressing vector control, \u003cem\u003ehs\u003c/em\u003ePEX19-WT, and \u003cem\u003ehs\u003c/em\u003ePEX19-FV in pan-neurons. The data in \u003cstrong\u003ec\u003c/strong\u003e are shown as violin plots with mean and quartiles (n=103~130 adult flies). Statistical significance was evaluated using two-way ANOVA with Tukey post-hoc test. **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, ns=not significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed-f\u003c/strong\u003e, Lifespan analysis of \u003cem\u003eW\u003c/em\u003e\u003csup\u003e\u003cem\u003e1118\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eHttex1-20Q\u003c/em\u003e, and \u003cem\u003eHttex1-93Q Drosophila\u003c/em\u003e expressing vector control, \u003cem\u003ehs\u003c/em\u003ePEX19-WT, and \u003cem\u003ehs\u003c/em\u003ePEX19-FV in pan-neurons. Lifespan data are plotted as Kaplan-Meier survival curves, and \u003cem\u003ep\u003c/em\u003e-values were determined using the Log-rank (Mantel-Cox) test. All data in (D), (E), and (F) are shown as mean values, with n=112~164 adult flies. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, ns=not significant.\u003c/p\u003e\n\u003cp\u003eGenotypes of \u003cem\u003eDrosophila\u003c/em\u003e used in \u003cstrong\u003eb\u003c/strong\u003e-\u003cstrong\u003ef\u003c/strong\u003e are listed in Supplementary Table 1.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4292547/v1/2f600f28cc61b95283911955.png"},{"id":74127724,"identity":"d02701a4-df29-4a07-aa47-91d0998d3369","added_by":"auto","created_at":"2025-01-18 08:05:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2633004,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4292547/v1/1b2383a8-afda-480b-b0ec-8893343c59be.pdf"},{"id":56643340,"identity":"410e3769-ca5e-47f4-84e4-5092f98e98a0","added_by":"auto","created_at":"2024-05-17 06:34:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20813,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"HttSupplementaryInfov1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4292547/v1/1409db29e234c505cf652c72.docx"},{"id":56642908,"identity":"f5761780-830e-42c1-84d0-0594507d71e6","added_by":"auto","created_at":"2024-05-17 06:26:54","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":9682447,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedDataFig.docx","url":"https://assets-eu.researchsquare.com/files/rs-4292547/v1/136041c98ae39739f6867e9d.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Engineering a membrane protein chaperone to ameliorate the proteotoxicity of mutant huntingtin","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMaintaining proper protein homeostasis is essential for healthy cells. However, the cell is under continuous risk from newly synthesized proteins that might expose hydrophobic surfaces in the crowded cellular environment, leading to protein misfolding and aggregation\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. To overcome these problems, cells invest in a sophisticated integrative chaperone network that supports accurate \u003cem\u003ede novo\u003c/em\u003e protein folding, facilitates refolding of misfolded proteins, and prevents protein aggregation\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. However, environmental stresses, genetic mutations, and aging can reduce the overall capacity of molecular chaperones, resulting in the accumulation of toxic aggregates and misfolded proteins in cells\u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Such aggregates eventually lead to various diseases, including neurodegenerative diseases and type 2 diabetes\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHuntington\u0026rsquo;s disease (HD) is the most common dominantly inherited neurodegenerative disorder and is caused by the abnormal expansion of CAG (polyQ) repeats in exon 1 of the huntingtin gene (Httex1)\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The length of polyQ repeats in the mutant Httex1 (mHttex1 with \u0026gt;\u0026thinsp;36 repeats) positively correlates with an increasing propensity to form aggregates and correlates inversely with the age of disease onset\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Aggregation of the polyQ repeat domain is also mediated by its flanking domains, the N-terminal conserved N17 domain and the C-terminal proline-rich domain (PRD). The N17 domain stimulates mHttex1 aggregation, whereas the PRD inhibits it\u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Accumulation of mHttex1 aggregates in the nucleus and cytoplasm impairs the proteostasis network and disrupts cellular endomembranes, thus leading to dysregulation of diverse cellular processes including transcription, mitochondrial respiration, ER homeostasis, vesicular trafficking, and axonal transport\u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOne suggested approach to correcting protein misfolding and removing pathological aggregates involves engineering a molecular chaperone to increase chaperone capacity in affected cells\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Indeed, the yeast AAA\u0026thinsp;+\u0026thinsp;protein disaggregase, Hsp104, has been engineered to rescue the proteotoxicity of TDP43, FUS, and α-synuclein for amyotrophic lateral sclerosis (ALS) and Parkinson\u0026rsquo;s disease\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. However, most chaperones, including Hsp104, require subunit assembly, oligomerization, co-chaperones, or cofactors, such as ATP and metal ions, for their optimal activities\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Thus, engineered chaperones that rely on cellular ATP concentrations and the expression levels of their subunits and co-chaperones\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e may complicate therapeutic applications.\u003c/p\u003e \u003cp\u003ePEX19, an ATP-independent cytosolic chaperone, mediates the targeting of peroxisomal membrane proteins (PMPs) during peroxisome biogenesis\u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Importantly, PEX19 does not require any co-chaperone or cofactors for its chaperone activity. Therefore, we hypothesized that PEX19 could be readily engineered to provide a robust approach for mitigating mHttex1 proteotoxicity. Here, using yeast toxicity-based screening\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e with a random mutant library, we isolated yeast PEX19 (\u003cem\u003esc\u003c/em\u003ePEX19) variants that suppress the proteotoxicity of mHttex1 aggregates. Using this information, we engineered the equivalent human PEX19 (\u003cem\u003ehs\u003c/em\u003ePEX19) variants and found that they also potently suppress toxic mHttex1 aggregates. Biochemical assays revealed that the isolated \u003cem\u003ehs\u003c/em\u003ePEX19 variants directly bind the hydrophobic side of the amphipathic helix at the N17 domain of mHttex1, thereby preventing mHttex1 aggregation. Overexpression of the \u003cem\u003ehs\u003c/em\u003ePEX19 variant further rescued mHttex1-induced neurite degeneration in mouse striatal neurons and improved both the climbing ability and lifespan of flies expressing mHttex1-93Q. Altogether, our study suggests that fine-tuning the sequences of ATP-independent membrane protein chaperones could be a feasible approach to designing therapeutic chaperones for HD and potentially other diseases linked to protein aggregation.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEngineered\u003c/b\u003e \u003cb\u003esc\u003c/b\u003e\u003cb\u003ePEX19 variants suppress the toxicity of mHttex1 in yeast\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo isolate an \u003cem\u003esc\u003c/em\u003ePEX19 mutant gene that suppresses the cellular toxicity of mHttex1 protein, we used the yeast toxicity-based screening method\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Deletion of PRD in Httex1-97Q enhances its polyQ-induced toxicity in yeast\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. This mutant is more optimal for screening since it results in a larger difference in cell viability compared to expressing non-toxic Httex1-25Q. To this end, we generated yeast strains carrying chromosomally integrated Httex1 genes (Httex1-25QΔP and Httex1-97QΔP), which encode an N-terminal FLAG tag, the first 17 amino acids of Httex1 (N17 domain), 25 or 97 repeats of glutamine, and a C-terminal GFP gene under the control of the galactose-inducible promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Expression of the wild-type \u003cem\u003esc\u003c/em\u003ePEX19 did not alter the cellular toxicity of Httex1-97QΔP when compared with the empty vector control (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). We randomly mutated the entire \u003cem\u003esc\u003c/em\u003ePEX19 gene and screened the \u003cem\u003esc\u003c/em\u003ePEX19 plasmid library against Httex1-97QΔP toxicity. Among approximately 90,000 transformants, 21 colonies were able to grow on galactose plates. After assessing the cell viability of those colonies, we found that two \u003cem\u003esc\u003c/em\u003ePEX19 variants, m1 and m2, effectively suppressed the cellular toxicity of Httex1-97QΔP in yeast (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe isolated \u003cem\u003esc\u003c/em\u003ePEX19 variants share two common mutation sites, L288F and E292V (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Therefore, we hypothesized that the mutation of these two sites accounts for the ability of \u003cem\u003esc\u003c/em\u003ePEX19 variants to rescue Httex1-97QΔP-induced toxicity in yeast. To test this hypothesis, we generated a double mutant \u003cem\u003esc\u003c/em\u003ePEX19-L288F/E292V. The results of the spotting assay showed that \u003cem\u003esc\u003c/em\u003ePEX19-L288F/E292V is sufficient to suppress the cellular toxicity of Httex1-97QΔP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). In contrast, coexpression of the single mutants of \u003cem\u003esc\u003c/em\u003ePEX19-L288F or \u003cem\u003esc\u003c/em\u003ePEX19-E292V with Httex1-97QΔP did not restore cell viability (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), suggesting that \u003cem\u003esc\u003c/em\u003ePEX19-L288F/E292V is a minimally mutated suppressor of polyQ-induced toxicity in yeast. In addition, we substituted E292 with other hydrophobic amino acids on the \u003cem\u003esc\u003c/em\u003ePEX19 variant. We found that only \u003cem\u003esc\u003c/em\u003ePEX19-L288F/E292I suppressed Httex1-97QΔP-induced toxicity to the same degree as \u003cem\u003esc\u003c/em\u003ePEX19-L288F/E292V (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), possibly due to the structural similarity between the valine and isoleucine side chains. Therefore, we identified two \u003cem\u003esc\u003c/em\u003ePEX19 variants, \u003cem\u003esc\u003c/em\u003ePEX19-L288F/E292V (\u003cem\u003esc\u003c/em\u003ePEX19-FV) and \u003cem\u003esc\u003c/em\u003ePEX19-L288F/E292I (\u003cem\u003esc\u003c/em\u003ePEX19-FI), that potently suppress polyQ toxicity in yeast.\u003c/p\u003e \u003cp\u003eConsistent with the results obtained with the spotting assay, microscopy and Western blot analyses showed that \u003cem\u003esc\u003c/em\u003ePEX19-FV and \u003cem\u003esc\u003c/em\u003ePEX19-FI significantly reduced the aggregation of Httex1-97QΔP proteins compared to \u003cem\u003esc\u003c/em\u003ePEX19-WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-f). Over 50% of Httex1-97QΔP was found in SDS-insoluble aggregates in \u003cem\u003esc\u003c/em\u003ePEX19-WT expressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, f). In contrast, overexpression of \u003cem\u003esc\u003c/em\u003ePEX19-FV and \u003cem\u003esc\u003c/em\u003ePEX19-FI drastically reduced the relative amount of SDS-insoluble 97Q aggregate and simultaneously increased SDS-soluble 97Q protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, f, and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-d). This enhancement of Httex1-97QΔP solubility by \u003cem\u003esc\u003c/em\u003ePEX19-FV and \u003cem\u003esc\u003c/em\u003ePEX19-FI is not due to different expression levels of PEX19 in the cells (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, e).\u003c/p\u003e \u003cp\u003eDuring protein targeting to peroxisome membranes, farnesylation of the C-terminal cysteine residue in the PEX19-CaaX motif increases the binding affinity of PMPs, effectively preventing PMP aggregation in the cytosol\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In addition, the PMP-bound PEX19 is recruited to the peroxisomal membrane by PEX3, which interacts with an N-terminal αa helix in PEX19\u003csup\u003e35\u0026ndash;37\u003c/sup\u003e. Noticeably, the levels of farnesylated PEX19 were significantly reduced in \u003cem\u003esc\u003c/em\u003ePEX19-FV and \u003cem\u003esc\u003c/em\u003ePEX19-FI overexpressing cells (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, f). Therefore, we tested whether two major aspects of PEX19-mediated PMP targeting, farnesylation and interaction with PEX3\u003csup\u003e34\u0026ndash;36,38\u003c/sup\u003e, are crucial for ameliorating polyQ-induced cellular toxicity in yeast. To this end, we introduced two further mutations, a farnesylation-defective mutation \u003cem\u003esc\u003c/em\u003ePEX19-C339S and a PEX3 binding-defective mutation \u003cem\u003esc\u003c/em\u003ePEX19-ΔN, into \u003cem\u003esc\u003c/em\u003ePEX19-WT and the toxicity-reducing variants (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The results of a spotting assay showed that coexpression of \u003cem\u003esc\u003c/em\u003ePEX19-FV/C339S, \u003cem\u003esc\u003c/em\u003ePEX19-FV/ΔN, \u003cem\u003esc\u003c/em\u003ePEX19-FI/C339S, and \u003cem\u003esc\u003c/em\u003ePEX19-FI/ΔN with Httex1-97QΔP did not alter cell growth compared to \u003cem\u003esc\u003c/em\u003ePEX19-FV and \u003cem\u003esc\u003c/em\u003ePEX19-FI (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c). Therefore, both farnesylation and recruitment of PEX19 to the peroxisomal membrane by binding to PEX3 are dispensable for \u003cem\u003esc\u003c/em\u003ePEX19-FV and \u003cem\u003esc\u003c/em\u003ePEX19-FI to suppress the cellular toxicity of Httex1-97QΔP in yeast.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ehs\u003c/b\u003e \u003cb\u003ePEX19 variants suppress mHttex1 aggregation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBoth mutated residues (L288F/E292V and L288F/E292I) are located in the α4 helix of PEX19 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, green highlighted box). Sequence alignment analysis showed that these residues are highly conserved from Human (M255/Q259) to Arabidopsis (M202/Q206) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In addition, the M255 residue of \u003cem\u003ehs\u003c/em\u003ePEX19 directly interacts with the farnesyl group in its C-terminal end\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), suggesting that this residue could be important for substrate recognition. Due to their highly homologous sequences, we hypothesized that introducing identical mutations (M255F/Q259V or M255F/Q259I) into \u003cem\u003ehs\u003c/em\u003ePEX19 could also enhance suppression of mHttex1 aggregation.\u003c/p\u003e \u003cp\u003eTo test whether both purified \u003cem\u003esc\u003c/em\u003ePEX19 and \u003cem\u003ehs\u003c/em\u003ePEX19 variants directly prevent Httex1-51Q aggregation \u003cem\u003ein vitro\u003c/em\u003e, we used the well-established filter trap assay that detects heat-stable, SDS-insoluble aggregates\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In this assay, the N-terminal Httex1-51Q can be exposed by cleaving off a GST-tag using TEV protease, thus initiating polyQ aggregation (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In the absence of a chaperone, Httex1-51Q readily formed SDS-insoluble aggregates at 3 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d). In contrast, \u003cem\u003esc\u003c/em\u003ePEX19-FV and \u003cem\u003esc\u003c/em\u003ePEX19-FI effectively suppressed aggregation of the purified Httex1-51Q protein, while \u003cem\u003esc\u003c/em\u003ePEX19-WT was insufficient to prevent Httex1-51Q aggregation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Similar to \u003cem\u003esc\u003c/em\u003ePEX19 variants, \u003cem\u003ehs\u003c/em\u003ePEX19 variants effectively prevented aggregation of Httex1-51Q protein \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). This enhanced chaperone activity of \u003cem\u003ehs\u003c/em\u003ePEX19 variants was not due to different TEV cleavage efficiency caused by their mutations (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). In addition, our negatively stained transmission electron micrograph (TEM) analysis showed that \u003cem\u003ehs\u003c/em\u003ePEX19-WT was not sufficient to prevent Httex1-51Q fibril formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). In contrast, \u003cem\u003ehs\u003c/em\u003ePEX19-FV completely suppressed the formation of Httex1-51Q fibrils at 15 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Consistent with the results of the filter trap assay (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), \u003cem\u003ehs\u003c/em\u003ePEX19-FI also prevented fibril formation by Httex1-51Q (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, lower), although in some cases, TEM analysis of \u003cem\u003ehs\u003c/em\u003ePEX19-FI revealed both larger Httex1-51Q aggregates and small fibril fragments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, upper). Indeed, \u003cem\u003ehs\u003c/em\u003ePEX19 variants were unable to redissolve preformed Httex1-51Q aggregates when added at 3 h, suggesting that they do not have a disaggregase activity (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Therefore, we conclude that \u003cem\u003ehs\u003c/em\u003ePEX19 variants function as a holdase that prevents the initial aggregation process of Httex1-51Q.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo test whether \u003cem\u003ehs\u003c/em\u003ePEX19 variants are also effective in reducing mHttex1 aggregation in a mammalian HD model, we coexpressed \u003cem\u003ehs\u003c/em\u003ePEX19 variants with Httex1-19Q-GFP or Httex1-134Q-GFP in HEK293T cells\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Overexpression of \u003cem\u003ehs\u003c/em\u003ePEX19-FV and \u003cem\u003ehs\u003c/em\u003ePEX19-FI at ~\u0026thinsp;3-fold over endogenous PEX19 levels strongly prevented the aggregation of Httex1-134Q, as demonstrated by both fluorescence microscopy analysis and the filter trap assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef-h and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, c). In contrast, overexpression of \u003cem\u003ehs\u003c/em\u003ePEX19-WT reduced the Httex1-134Q aggregates by ~\u0026thinsp;50% on average, suggesting that \u003cem\u003ehs\u003c/em\u003ePEX19-WT itself exhibits a mild chaperone activity toward polyQ proteins as supported by the \u003cem\u003ein vitro\u003c/em\u003e aggregation assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg, h). The difference in rescuing effects observed in \u003cem\u003ehs\u003c/em\u003ePEX19 variants relative to their wild-type protein was not due to different expression levels of exogenous PEX19 or Httex1-134Q (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-c). Critically, overexpression of \u003cem\u003ehs\u003c/em\u003ePEX19 variants did not perturb the peroxisomal localization of the peroxisomal membrane protein PMP70, suggesting that this approach is unlikely to interfere with peroxisome biogenesis (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, e). Therefore, these data demonstrate that the substitution of two conserved residues on the α4 helix of \u003cem\u003ehs\u003c/em\u003ePEX19 significantly increases its chaperone activity toward mHttex1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePEX19 variants bind the N17 domain of mHttex1\u003c/h2\u003e \u003cp\u003eThe N17 domain of Httex1 has an amphipathic helical property, which contributes to the initiation and acceleration of mHttex1 aggregation\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Furthermore, a recent study suggested that structural coupling between the N17 and polyQ repeat domains stabilizes the helical content of Httex1 and accelerates aggregation\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Deletion of the N17 domain of Httex1-51Q (Httex1-51Q-ΔN) delays the kinetics of Httex1-51Q aggregation\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Given that \u003cem\u003ehs\u003c/em\u003ePEX19 variants generate a more hydrophobic environment at their C-terminal domain (CTD) than \u003cem\u003ehs\u003c/em\u003ePEX19-WT, we hypothesized that they bind to the hydrophobic amino acids in mHttex1, possibly at the N17 domain of mHttex1. Thus, we tested whether \u003cem\u003ehs\u003c/em\u003ePEX19 variants also suppress Httex1-51Q-ΔN aggregation \u003cem\u003ein vitro\u003c/em\u003e. In contrast to Httex1-51Q-WT, \u003cem\u003ehs\u003c/em\u003ePEX19 variants were unable to suppress the aggregation of Httex1-51Q-ΔN (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed vs 3b). Furthermore, \u003cem\u003ehs\u003c/em\u003ePEX19 variants did not suppress the aggregation of another polyQ repeat protein, Ataxin3 (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Since Ataxin3-78Q has only the polyQ-repeat domain in common with Httex1\u003csup\u003e43,44\u003c/sup\u003e, it is plausible that \u003cem\u003ehs\u003c/em\u003ePEX19 variants do not target this polyQ-repeat domain (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Taken together, these results suggest that the N17 domain could be the primary recognition site of \u003cem\u003ehs\u003c/em\u003ePEX19 variants within the Httex1-51Q protein.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo check whether the mutated hydrophobic residues at the \u003cem\u003ehs\u003c/em\u003ePEX19 variants directly interact with Httex1-51Q, we used the Bpa crosslinking assay that uses a photocrosslinker, p-benzoyl-l-phenylalanine (Bpa) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). We site-specifically incorporated Bpa into F255 at the \u003cem\u003ehs\u003c/em\u003ePEX19 variants using amber suppression\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Suppression of Httex1-51Q aggregation by \u003cem\u003ehs\u003c/em\u003ePEX19-FV\u003csup\u003eBpa\u003c/sup\u003e and \u003cem\u003ehs\u003c/em\u003ePEX19-FI\u003csup\u003eBpa\u003c/sup\u003e was also observed at 3 and 6 h, albeit to a lesser extent than the unincorporated \u003cem\u003ehs\u003c/em\u003ePEX19 variants (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb vs Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). At 3 h incubation, \u003cem\u003ehs\u003c/em\u003ePEX19-FVxHttex1-51Q or \u003cem\u003ehs\u003c/em\u003ePEX19-FIxHttex1-51Q crosslink at ~\u0026thinsp;70 kDa was readily detectable, whereas there was no observed crosslinked band in the presence of Httex1-51Q-ΔN (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, e). Therefore, these results indicate that the F255 residue in the \u003cem\u003ehs\u003c/em\u003ePEX19 variants specifically binds to the N17 domain of Httex1-51Q.\u003c/p\u003e \u003cp\u003eWe further tested whether the hydrophobic amino acid residues in the N-terminal amphipathic helix of mHttex1 also bind the \u003cem\u003ehs\u003c/em\u003ePEX19 variants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). To minimize structural perturbation, we incorporated Bpa at the F11 residue on Httex1-51Q among seven hydrophobic amino acid residues (Fig, 3a). Bpa incorporation on Httex1-51Q did not alter the aggregation kinetics (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec vs Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Similar to Httex1-51Q-WT, both \u003cem\u003ehs\u003c/em\u003ePEX19-FV and \u003cem\u003ehs\u003c/em\u003ePEX19-FI suppressed the aggregation of Httex1-51Q-F11\u003csup\u003eBpa\u003c/sup\u003e more efficiently than \u003cem\u003ehs\u003c/em\u003ePEX19-WT (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). In the presence of \u003cem\u003ehs\u003c/em\u003ePEX19 variants, two distinct \u003cem\u003ehs\u003c/em\u003ePEX19-Httex1-51Q crosslink bands at ~\u0026thinsp;70 and ~\u0026thinsp;80 kDa were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, g), suggesting that Httex1-51Q -F11\u003csup\u003eBpa\u003c/sup\u003e binds \u003cem\u003ehs\u003c/em\u003ePEX19 variants, possibly with two different conformations. In contrast, \u003cem\u003ehs\u003c/em\u003ePEX19-WT resulted in a distinct crosslinked band at ~\u0026thinsp;80 kDa and a weak diffuse band at ~\u0026thinsp;70 kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, g). Consistent with its mild chaperone activity, \u003cem\u003ehs\u003c/em\u003ePEX19-WT also binds to Httex1-51Q-F11\u003csup\u003eBpa\u003c/sup\u003e, but likely with one dominant conformation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, g). These observed differences in the aggregation and Bpa crosslinking assays are not due to different TEV cleavage efficiency (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Therefore, our data demonstrate that the F11 hydrophobic residue on Httex1-51Q directly interacts with \u003cem\u003ehs\u003c/em\u003ePEX19 and consistent with the results in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, its variants increase this interaction.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe α4 helix of\u003c/b\u003e \u003cb\u003ehs\u003c/b\u003e\u003cb\u003ePEX19 variants serves as a specific binding site for the N17 domain of mHttex1\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePEX19 binds to the moderately hydrophobic transmembrane domains (TMDs) of peroxisomal and mitochondrial membrane proteins\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In addition, PEX19 interacts with TMDs located in diverse topologies of membrane proteins, multi-spanning PMPs, tail-anchored membrane proteins (TAs), and N-terminal signal-anchored membrane proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Since \u003cem\u003ehs\u003c/em\u003ePEX19 binds to these moderately hydrophobic TMDs, we hypothesized that \u003cem\u003ehs\u003c/em\u003ePEX19 variants might also interact with the isolated N17 domain of Httex1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). To test this, we fused the N17 domain of Httex1 to the N-terminus of the Maltose binding protein (MBP) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The \u003cem\u003ehs\u003c/em\u003ePEX19-FV\u003csup\u003eBpa\u003c/sup\u003e and \u003cem\u003ehs\u003c/em\u003ePEX19-FI\u003csup\u003eBpa\u003c/sup\u003e proteins readily crosslinked to the N17-MBP protein, whereas no crosslinked band appeared in the presence of wild-type MBP protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). These results suggest that the N17 domain of Httex1 is a minimum recognition motif for \u003cem\u003ehs\u003c/em\u003ePEX19 variants that allows suppression of the mHttex1 aggregation. In contrast to \u003cem\u003ehs\u003c/em\u003ePEX19 variants, SGTA, a cytosolic co-chaperone that binds highly hydrophobic TMDs of ER TAs\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, was unable to suppress the aggregation of Httex1-51Q (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Collectively, our results suggest that mutations on the α4 helix of \u003cem\u003ehs\u003c/em\u003ePEX19 enable binding to the relatively low hydrophobic N17 domain of mHttex1.\u003c/p\u003e \u003cp\u003eSeveral studies suggested that the α1 helix of PEX19-CTD serves as a binding site of PMPs\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Given that the Httex1-51Q binds to the α4 helix of \u003cem\u003ehs\u003c/em\u003ePEX19 variants, we checked whether the \u003cem\u003ehs\u003c/em\u003ePEX19 variants also interact with a \u003cem\u003ebona fide\u003c/em\u003e PEX19 substrate, the peroxisomal TA, PEX26\u003csup\u003e31\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, f). At an approximately 3-fold excess concentration of the endogenous \u003cem\u003ehs\u003c/em\u003ePEX19\u003csup\u003e51\u003c/sup\u003e, the amounts of PEX26 loaded onto \u003cem\u003ehs\u003c/em\u003ePEX19 variants were comparable to \u003cem\u003ehs\u003c/em\u003ePEX19-WT, indicating that these mutations on the α4 helix of \u003cem\u003ehs\u003c/em\u003ePEX19 do not largely alter the overall binding capacity of PEX26 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, f). In contrast to Httex1-51Q, both \u003cem\u003ehs\u003c/em\u003ePEX19-FV\u003csup\u003eBpa\u003c/sup\u003e and \u003cem\u003ehs\u003c/em\u003ePEX19-FI\u003csup\u003eBpa\u003c/sup\u003e did not crosslink to PEX26 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg-i). These results suggest that F255 and V259/I259 mutations on \u003cem\u003ehs\u003c/em\u003ePEX19 could create a specific binding site for the N17 domain of Httex1-51Q, eventually resulting in robust suppression activity of Httex1-51Q aggregation.\u003c/p\u003e \u003cp\u003eWe tested whether \u003cem\u003ehs\u003c/em\u003ePEX19-FV also prevents aggregation of a non-polyQ protein, TDP43, which is associated with another neurodegenerative disease, ALS. To this end, we performed an established \u003cem\u003ein vitro\u003c/em\u003e aggregation assay using the purified TDP43-TEV-MBP-His\u003csub\u003e6\u003c/sub\u003e protein\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. The addition of TEV protease enables initiation of TDP43 aggregation (Extended Data Fig.\u0026nbsp;6a, black). In contrast to Httex1-51Q aggregation in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, incubation with \u003cem\u003ehs\u003c/em\u003ePEX19-WT or \u003cem\u003ehs\u003c/em\u003ePEX19-FV exhibited only a minor delay in TDP43 aggregation kinetics (Extended Data Fig.\u0026nbsp;6a, blue and red). To further monitor TDP43 aggregation in cells, we generated a stable HEK293 cell line (TDP43-BiFC) that expresses both TDP43-VN and TDP43-VC. Given that phosphorylation and acetylation on TDP43 promote its aggregation\u003csup\u003e\u003cspan additionalcitationids=\"CR54 CR55\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, we used Forskolin as a phosphorylation activator and Apicidin as an acetylation-inducing agent for TDP43\u003csup\u003e57,58\u003c/sup\u003e. Treatment with either Forskolin or Apicidin significantly increased the fluorescence intensities of TDP43-BiFC in the cytosol (Extended Data Fig.\u0026nbsp;6b, c). Overexpression of \u003cem\u003ehs\u003c/em\u003ePEX19-WT or \u003cem\u003ehs\u003c/em\u003ePEX19-FV showed at most a minor rescue of Forskolin or Apicidin-induced cytosolic TDP43 aggregation in HEK293 cells (Extended Data Fig.\u0026nbsp;6d-g). Together with Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, we conclude that \u003cem\u003ehs\u003c/em\u003ePEX19-FV selectively suppresses the aggregation of mHttex1 \u003cem\u003ein vitro\u003c/em\u003e and in mammalian cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003ehs\u003c/b\u003e \u003cb\u003ePEX19-FV rescues HD-associated phenotypes\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo test whether \u003cem\u003ehs\u003c/em\u003ePEX19-FV protects striatal neurons from mHttex1 proteotoxicity, we coexpressed Httex1-134Q-GFP with \u003cem\u003ehs\u003c/em\u003ePEX19-WT or \u003cem\u003ehs\u003c/em\u003ePEX19-FV at 7 days \u003cem\u003ein vitro\u003c/em\u003e (DIV) in primary striatal neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). In contrast to Httex1-19Q-GFP- and vector control- coexpressing striatal neurons, at 48 h post-transfection, we observed largely fragmented neurites in the striatal neurons when coexpressed with Httex1-134Q-GFP and vector control, suggesting that mHttex1 induces neuritic degeneration\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Striatal neurons coexpressing Httex1-134Q-GFP and \u003cem\u003ehs\u003c/em\u003ePEX19-FV exhibited unfragmented healthy neurites, while partially fragmented neurites were observed in the Httex1-134Q-GFP-and \u003cem\u003ehs\u003c/em\u003ePEX19-WT-coexpressing neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). These results suggest that \u003cem\u003ehs\u003c/em\u003ePEX19-FV effectively protects neuritic degeneration in mHttex1-expressing mouse striatal neurons.\u003c/p\u003e \u003cp\u003eWe next tested whether the \u003cem\u003ehs\u003c/em\u003ePEX19-FV variant could rescue HD-associated phenotypes in \u003cem\u003eDrosophila\u003c/em\u003e HD models. To this end, we generated transgenic fly lines expressing pACU2 empty vector (vector control), \u003cem\u003ehs\u003c/em\u003ePEX19-WT, or \u003cem\u003ehs\u003c/em\u003ePEX19-FV and coexpressed Httex1-20Q or Httex1-93Q under the control of \u003cem\u003eElav-GAL4\u003c/em\u003e (pan-neurons) or \u003cem\u003eD42-GAL4\u003c/em\u003e (motor neurons) drivers (Supplementary Table\u0026nbsp;1). As a negative control, we used the \u003cem\u003eW\u003c/em\u003e\u003csup\u003e\u003cem\u003e1118\u003c/em\u003e\u003c/sup\u003e fly line which does not carry a Httex1 transgene. Compared to \u003cem\u003eW\u003c/em\u003e\u003csup\u003e\u003cem\u003e1118\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/vector control\u003c/em\u003e and \u003cem\u003eHttex1-20Q/vector control\u003c/em\u003e flies, motor- or pan-neuronal Httex1-93Q overexpression in \u003cem\u003eHttex1-93Q/vector control\u003c/em\u003e flies led to a significant defect in their locomotion capacities (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, c). In contrast to \u003cem\u003ehs\u003c/em\u003ePEX19-WT, \u003cem\u003ehs\u003c/em\u003ePEX19-FV expression partially restored the impaired climbing ability of flies overexpressing Httex1-93Q in motor- and pan-neurons. Consistent with these results, the numbers of Httex1-93Q-positive puncta in \u003cem\u003eHttex1-93Q/hsPEX19-FV\u003c/em\u003e flies were significantly reduced in both motor- and pan-neurons compared to \u003cem\u003eHttex1-93Q/vector control\u003c/em\u003e flies (Extended Data Fig.\u0026nbsp;7a-f). Furthermore, despite the exclusive cytosolic localization of \u003cem\u003ehs\u003c/em\u003ePEX19-FV, \u003cem\u003eHttex1-93Q/hsPEX19-FV\u003c/em\u003e flies displayed nuclear-localized soluble Httex1-93Q in both motor- and pan-neurons (Extended Data Fig.\u0026nbsp;7g, h). Overexpression of \u003cem\u003ehs\u003c/em\u003ePEX19-FV significantly increased the lifespan of flies expressing Httex1-93Q, whereas it did not affect the \u003cem\u003eW\u003c/em\u003e\u003csup\u003e\u003cem\u003e1118\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eHttex1-20Q\u003c/em\u003e flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed-f). Taken together, \u003cem\u003ehs\u003c/em\u003ePEX19-FV provides effective neuroprotection in both mouse striatal neurons and Httex1-93Q-expressing flies.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, we used the yeast toxicity-based screening method to identify two yeast PEX19 variants, \u003cem\u003esc\u003c/em\u003ePEX19-FV (L288F/E292V) and \u003cem\u003esc\u003c/em\u003ePEX19-FI (L288F/E292I), that rescue the toxicity of mHttex1 in yeast. Since the sites of these mutations in the α4 helix of PEX19 are highly conserved, we further generated the human variants \u003cem\u003ehs\u003c/em\u003ePEX19-FV (M255F/Q259V) and \u003cem\u003ehs\u003c/em\u003ePEX19-FI (M255F/Q259I). We confirmed that \u003cem\u003ehs\u003c/em\u003ePEX19 variants effectively suppress mHttex1 aggregation \u003cem\u003ein vivo\u003c/em\u003e and in mammalian cells. The mutated phenylalanine residue in the α4 helix of \u003cem\u003ehs\u003c/em\u003ePEX19 variants directly interacts with the N17 domain of mHttex1, thereby preventing aggregation of mHttex1. Finally, our results demonstrate that \u003cem\u003ehs\u003c/em\u003ePEX19-FV rescues mHttex1-induced neuritic degeneration in primary striatal neurons and HD-associated behavioral deficits and lifespan in the \u003cem\u003eDrosophila\u003c/em\u003e HD model.\u003c/p\u003e \u003cp\u003eSeveral chaperones have been identified as mHttex1 aggregation suppressors, which target different domains of mHttex1. Previous studies showed that the TRiC chaperonin and Hsc70 chaperone bind to the N17 domain of mHttex1, thereby preventing mHttex1 aggregation\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. In addition, two J-domain proteins (JDPs), DNAJB6 and DNAJB8, and the β subunit of the nascent polypeptide-associated complex (NAC) directly interact with the PolyQ repeat domain, thereby suppressing polyQ-mediated aggregation\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan additionalcitationids=\"CR63\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Furthermore, a recent study showed that another JDP, DNAJB1, together with Hsc70 and Apg2, binds to the PRD of mHttex1, and the trimeric chaperone system prevents and redisolves mHtt aggregates\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. The S/T-rich region of DNAJB6 was suggested to form hydrogen bonds with the polyQ residues\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e, whereas the positively charged N terminus of βNAC is involved in interactions with the polyQ repeat domain\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. These polyQ-binding domains in DNAJB6 and βNAC are low complexity linkers located between the JD and C-terminal substrate binding domain and N-terminal unstructured small domain (~\u0026thinsp;40 aa), respectively\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. In this study, we showed that the hydrophobic interactions between the α4 helix of \u003cem\u003ehs\u003c/em\u003ePEX19 variants and the N17 domain of mHttex1 enable the suppression of mHttex1 aggregation. In contrast to DNAJB6 and βNAC that form electrostatic interactions or hydrogen bonds with mHttex1, the amphipathic N17 domain appears to dock into the hydrophobic farnesyl group-binding groove of \u003cem\u003ehs\u003c/em\u003ePEX19-CTD\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The hydrophobic residues in the N17 domain are likely to be protected from aqueous cytosolic environments, thereby inhibiting the self-assembly of mHttex1. Together with these previous studies, our results further suggest that, depending on their mHttex1-binding domains, chaperones can employ different molecular mechanisms to prevent mHttex1 aggregation.\u003c/p\u003e \u003cp\u003eMembrane protein chaperones recognize their cargo membrane proteins primarily based on the hydrophobicity and location of TMDs\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. TMDs are typically between 15 and 30 amino acids long with widely variable hydrophobicity. Regardless of TMD location, PEX19 generally recognizes moderately hydrophobic TMDs in peroxisomal and mitochondrial membrane proteins\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In contrast, SGTA (Sgt2 in yeast) and TRC40 (Get3 in yeast) preferentially bind to more hydrophobic TMDs located near the C-terminus in the ER TAs\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan additionalcitationids=\"CR69\" citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Consistent with the low hydrophobicity and N-terminal localization of the N17 domain of mHttex1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), \u003cem\u003ehs\u003c/em\u003ePEX19-WT exhibits a mild chaperone activity toward Httex1-51Q (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), whereas SGTA was not able to suppress the aggregation of Httex1-51Q (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The \u003cem\u003ehs\u003c/em\u003ePEX19 variants interact more efficiently with Httex1-51Q than \u003cem\u003ehs\u003c/em\u003ePEX19-WT, thereby suppressing the formation of both SDS-insoluble larger aggregates and fibrils (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, e). Despite the unaltered overall binding capacity of \u003cem\u003ehs\u003c/em\u003ePEX19 variants to PEX26, the hydrophobic residue F255 at the α4 helix of \u003cem\u003ehs\u003c/em\u003ePEX19 variants did not interact with PEX26 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-f). Since the α1 helix of \u003cem\u003ehs\u003c/em\u003ePEX19 acts as the primary binding site of PMPs\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, our results suggest that the α4 helix of \u003cem\u003ehs\u003c/em\u003ePEX19 variants is a specific binding site for the N17 domain of Httex1 proteins. Alternatively, the α4 helix of \u003cem\u003ehs\u003c/em\u003ePEX19 could be a unique structural feature to discriminate low hydrophobicity TMDs of membrane proteins. Nevertheless, further structural analysis on \u003cem\u003ehs\u003c/em\u003ePEX19 variants would explain how \u003cem\u003ehs\u003c/em\u003ePEX19 variants efficiently suppress mHttex1 aggregation.\u003c/p\u003e \u003cp\u003eAccumulation of mHtt aggregates in the cytoplasm sequesters a variety of cytosolic proteins, thereby interfering with diverse cellular functions and endomembrane structures\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e,\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. Several studies showed that cytoplasmic mHtt aggregates impair nucleocytoplasmic transport of proteins and mRNAs by sequestering nuclear-shutting factors to the aggregates\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e,\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. Furthermore, cytoplasmic mHtt aggregates trap with the cytoskeletal transport system as well as other polyQ proteins in the cytosol, thus further disrupting axonal transport in a \u003cem\u003eDrosophila\u003c/em\u003e HD model\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. Despite the predominant nuclear localization of mHttex1 aggregates in Httex1-93Q expressing \u003cem\u003eDrosophila\u003c/em\u003e, our results showed that overexpressing the \u003cem\u003ehs\u003c/em\u003ePEX19-FV variant in the cytosol significantly reduces the nuclear aggregation of mHttex1 in both motor- and pan-neurons (Extended Data Fig.\u0026nbsp;7). Prior to the nuclear import of mHttex1, \u003cem\u003ehs\u003c/em\u003ePEX19-FV could prevent the aggregation of mHttex1 in the cytosol (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, e). Together with previous studies\u003csup\u003e\u003cspan additionalcitationids=\"CR72\" citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e, our results suggest that maintaining a soluble form of mHttex1 assisted by molecular chaperones in the cytosol could also modulate the conformational quality of nuclear mHttex1.\u003c/p\u003e \u003cp\u003eMitigating any potential off-target effects caused by the artificial mutations on a target chaperone would be critical for further therapeutic applications. The identified \u003cem\u003ehs\u003c/em\u003ePEX19 variants appear to be specific to HD, relative to proteins linked to other neurodegenerative diseases, potentially due to the highly conserved N17 sequences\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e and amphipathic helix property of Httex1. \u003cem\u003ehs\u003c/em\u003ePEX19-WT and its variants were unable to suppress the aggregation of Ataxin3-78Q (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). In addition, \u003cem\u003ehs\u003c/em\u003ePEX19-FV displayed only a very modest chaperone activity in TDP43 \u003cem\u003ein vitro\u003c/em\u003e aggregation assays (Extended Data Fig.\u0026nbsp;6a). Substituting the E292 residue into the α4 helix of \u003cem\u003esc\u003c/em\u003ePEX19 variants with various hydrophobic amino acid residues led to different capacities for ameliorating mHttex1-induced toxicity in yeast (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). These results further suggest that direct modulation of the amino acid sequences on the N17 domain-binding site of PEX19 variants could generate a higher substrate specificity for HD. Therefore, further tuning other amino acid sequences in the α4 helix of \u003cem\u003ehs\u003c/em\u003ePEX19-FV would help to eliminate unidentified side effects caused by \u003cem\u003ehs\u003c/em\u003ePEX19 variants for HD.\u003c/p\u003e \u003cp\u003eThe rescuing effects of \u003cem\u003ehs\u003c/em\u003ePEX19-FV observed in mHttex1-expressing flies might not be entirely due to the increased chaperone action of \u003cem\u003ehs\u003c/em\u003ePEX19-FV on the N17-mediated mHttex1 aggregates. We note that the PEX26 binding capacity of \u003cem\u003ehs\u003c/em\u003ePEX19-FV was not drastically altered compared to \u003cem\u003ehs\u003c/em\u003ePEX19-WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, f), suggesting that the variant can act on peroxisomal membrane proteins as well as mHttex1. Indeed, given that a variety of cytosolic chaperones are sequestered to mHttex1 aggregates\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, as also observed with \u003cem\u003ehs\u003c/em\u003ePEX19-WT in Httex1-134Q expressing HEK293T cells (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee), overexpression of \u003cem\u003ehs\u003c/em\u003ePEX19-FV could assist PMP targeting to the peroxisome, thereby maintaining proper peroxisome biogenesis in mHttex1-expressing flies.\u003c/p\u003e \u003cp\u003eOverall, our study demonstrates that engineering an ATP-independent membrane chaperone is a feasible approach to reducing N17-mediated mHttex1 aggregates in HD. Several tetratricopeptide repeat (TPR) domain-containing chaperones, i.e., ATP-independent chaperones involved in ER and mitochondrial membrane protein biogenesis, are known to have decreased expression levels in HD patients\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Furthermore, a recent study identified the TPR domain-containing chaperones, TTC1 and TOMM70A, as mitochondrial membrane protein biogenesis factors\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. Thus, our approach could be readily applicable to design other ATP-independent membrane proteins that specifically reduce mHttex1 toxicity and maintain proper membrane protein biogenesis for other organelles. Furthermore, given that amphipathic helix-mediated aggregation was previously observed in α-Synuclein, islet amyloid polypeptide, and apolipoprotein C-II\u003csup\u003e\u003cspan additionalcitationids=\"CR77 CR78\" citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e, this approach may not be limited to HD but could also be used for other diseases such as Parkinson\u0026rsquo;s disease, diabetes, and cardiac amyloidosis. Therefore, ATP-independent membrane chaperones could serve as a design platform for therapeutic development that targets various diseases associated with misfolding and aggregation of amphipathic helix.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePlasmids\u003c/h2\u003e \u003cp\u003eTo generate yeast integration plasmids for pRS306-Gal-FLAG-Httex1-25QΔP-GFP and pRS306-Gal-FLAG-Httex1-97QΔP-GFP, the insert genes for FLAG-Httex1-25QΔP-GFP and FLAG-Httex1-25QΔP-GFP were amplified from pYES2-FLAG-Httex1-25QΔP-GFP and pYES2-FLAG-Httex1-97Q-GFP (gift from F. Ulrich Hartl Lab)\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, respectively. The vector backbone for pRS306-Gal gene was amplified from pRS306-Gal-NDC80-RFP-MPS1 (gift from Won-ki Huh Lab)\u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. Gibson assembly was performed to incorporate the insert genes into the pRS306-Gal vector. For pRS413-Gal-\u003cem\u003esc\u003c/em\u003ePEX19, the \u003cem\u003esc\u003c/em\u003ePEX19 gene was amplified from the isolated yeast genomic DNA and further incorporated into pRS413-Gal vector using Gibson assembly. For His\u003csub\u003e6\u003c/sub\u003e-\u003cem\u003esc\u003c/em\u003ePEX19 and His\u003csub\u003e6\u003c/sub\u003e-\u003cem\u003ehs\u003c/em\u003ePEX19, the yeast PEX19 gene and the human PEX19 gene were cloned into pET-33b, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eYeast strains\u003c/h2\u003e \u003cp\u003eYeast strain used in this study is \u003cem\u003eW303a\u003c/em\u003e (MATa, can1-100, his3-11,15, leu2-3,112, trp1-1, ura3-1, ade2-1; gift from Won-ki Huh Lab). To generate FLAG-Httex1-25QΔP-GFP and FLAG-Httex1-25QΔP-GFP integrated-\u003cem\u003eW303a\u003c/em\u003e strains, the linearized pRS306-Gal-FLAG-Httex1-25QΔP-GFP and pRS306-Gal-FLAG-Httex1-97QΔP-GFP by NcoI were transformed into \u003cem\u003eW303a\u003c/em\u003e strain and selected on SD media lacking Ura.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSpotting assay\u003c/h2\u003e \u003cp\u003eYeast cells were grown to mid-log phase in the selective media containing 2% raffinose and 0.1% glucose. Overnight cultured cells were diluted to OD\u003csub\u003e600\u003c/sub\u003e of 0.1. The diluted cells were spotted onto 3% galactose and 1% raffinose-containing plates in serial 5-fold dilutions. Equal spotting was confirmed by spotting the same diluted cells on plates containing 2% glucose. After 2\u0026ndash;3 days of incubation at 30\u0026deg;C, the images were acquired using the iBright\u0026trade; FL 1000 imaging system (Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eYeast PEX19 library generation\u003c/h2\u003e \u003cp\u003eA random mutagenesis library was generated by error-prone PCR using a GeneMorph II mutagenesis kit (Agilent). The cloned wild-type \u003cem\u003esc\u003c/em\u003ePEX19 gene was used as a template, and the reactions were performed according to the manufacturer\u0026rsquo;s protocol. To generate diverse mutations, two different amounts of template (5 ng and 50 ng) were used in PCR reactions for 34 cycles, followed by two more sequential PCR reactions. The resulting PCR products in each reaction were re-amplified using Q5 High-Fidelity DNA Polymerase (New England Biolabs) and gel-purified using a QIAquick Gel Extraction Kit (Qiagen). The gel-purified PCR products were inserted into pRS413 vector using Gibson assembly. The Gibson assembly mixture was desalted and then transformed into DH5α competent cells using electroporation. The transformed colonies were pooled and purified using a Midi prep kit (MACHEREY-NAGEL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eYeast toxicity-based screening\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003esc\u003c/em\u003ePEX19 plasmid library was transformed into Httex1-97QΔP strain as described\u003csup\u003e\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e, and the cells were spread onto 2% glucose-containing SD-His-Ura plate. Approximately 90,000 colonies were pooled and further cultured in SR-His-Ura media supplemented with 2% raffinose and 0.1% glucose overnight at 30\u0026deg;C. The culture cells were adjusted OD\u003csub\u003e600\u003c/sub\u003e to 0.004 and spread onto an SG-His-Ura plate (3% galactose and 1% raffinose). We used the \u003cem\u003esc\u003c/em\u003ePEX19-WT-transformed Httex1-97QΔP and Httex1-25QΔP cells as negative and positive controls, respectively. The selected colonies from the SG-His-Ura plate were further confirmed using the spotting assay. The \u003cem\u003esc\u003c/em\u003ePEX19 variants were amplified using colony PCR, and then mutation sites were identified with sequencing. The selective \u003cem\u003esc\u003c/em\u003ePEX19 variants were generated by Quick Change mutagenesis using pRS413-Gal-\u003cem\u003esc\u003c/em\u003ePEX19-WT as a template. Those plasmids were transformed into Httex1-97QΔP and Httex1-25QΔP cells and further confirmed their suppression activity using a spotting assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of yeast cell extracts\u003c/h2\u003e \u003cp\u003eFor total yeast cell extract, the cell pellets were resuspended in 100 \u0026micro;L of 0.3 M NaOH and incubated for 3 min at room temperature. After washing the cells with water, the cell pellets were resuspended in 100 \u0026micro;L of lysis buffer (20 mM Tris (pH 8.0), 150 mM NaCl, 2% SDS) supplemented with protease inhibitor cocktail (cOmplete mini, EDTA-free protease inhibitor cocktail, Roche) and then incubated at 95\u0026deg;C for 5 min. After centrifugation at 15,000 g for 5 min, the clarified lysate was subjected to Western blot analysis.\u003c/p\u003e \u003cp\u003eTo monitor SDS-insoluble 97Q aggregates in yeast, the cell extracts were prepared as described with a minor modification\u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u003c/sup\u003e. The cells were induced at OD\u003csub\u003e600\u003c/sub\u003e of 0.1 with 1% galactose and 3% raffinose for 4 h. The cell pellets were resuspended in 200 \u0026micro;L lysis buffer (25 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 5% glycerol) supplemented with protease inhibitor cocktail and benzonase, and then lysed by Distruptor Genie (Scientific industries) with glass beads. The extracts were clarified by centrifugation of 500 g for 5 min at 4\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eProtein expression and purification\u003c/h2\u003e \u003cp\u003eExpression and purification of GST-Httex1-51Q-WT and GST-Httex1-51Q-ΔN proteins were performed as described\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. GST-Httex1-51Q-WT and GST-Httex1-51Q-ΔN were expressed BL21 Star\u0026trade; (DE3) (Invitrogen) with 1 mM IPTG at 18℃ for 2.5 h. Cells were resuspended in PBS supplemented with 150 mM NaCl, 1 mM EDTA, and cOmplete\u0026trade; EDTA-free protease inhibitor cocktail (Roche). After sonication, the lysate was centrifuged at 20,000g for 20 min at 4℃. The supernatant was incubated with glutathione agarose resin (Thermo Scientific) for 1 h at 4℃. The resin was washed with PBS containing 500 mM NaCl, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 2 mM ATP, and then the protein was eluted with 15 mM glutathione dissolved in PBS. After dialysis in the buffer (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5% glycerol), the purified GST-Httex1-51Q proteins were concentrated with Amicon\u0026reg; Ultra 30,000 MWCO centrifugal filters (Millipore) and 0.22 \u0026micro;m-filtered through prior to storing at -80℃.\u003c/p\u003e \u003cp\u003eHis\u003csub\u003e6\u003c/sub\u003e-\u003cem\u003ehs\u003c/em\u003ePEX19 or His\u003csub\u003e6\u003c/sub\u003e-\u003cem\u003esc\u003c/em\u003ePEX19 proteins were expressed in BL21 Star\u0026trade; (DE3) with 0.5 mM IPTG at 18℃ overnight. His\u003csub\u003e6\u003c/sub\u003e-SGTA, MBP-His\u003csub\u003e6\u003c/sub\u003e, and N17-MBP-His\u003csub\u003e6\u003c/sub\u003e proteins were expressed in BL21 StarTM (DE3) with 0.1 mM IPTG for 3 h at 37℃. Cells were resuspended in Buffer A (20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 5 mM imidazole, 2 mM 2-mercaptoethanol, 10% glycerol) supplemented with cOmplete\u0026trade; EDTA-free protease inhibitor cocktail and lysed using sonication. The clarified lysate was incubated with Ni-NTA agarose resin (Qiagen), and the proteins were eluted with 300 mM imidazole dissolved in Buffer A. After dialysis in Buffer B (20 mM HEPES (pH 7.5), 150 mM NaCl, 2 mM 2-mercaptoethanol, 10% glycerol), the purified proteins were stored at -80℃.\u003c/p\u003e \u003cp\u003eTo site-specifically incorporate \u003cem\u003ep\u003c/em\u003e-benzoyl-L-phenylalanine (Bpa) into His\u003csub\u003e6\u003c/sub\u003e-\u003cem\u003ehs\u003c/em\u003ePEX19 or GST-Httex1-51Q proteins, the coding sequence for the residue F255 in the His\u003csub\u003e6\u003c/sub\u003e-\u003cem\u003ehs\u003c/em\u003ePEX19 variants or the residue F11 in GST-Httex1-51Q was replaced with an amber codon (TAG) using npfu-special polymerase (Enzynomics) according to the manufacturer\u0026rsquo;s introduction. Expression plasmids for His\u003csub\u003e6\u003c/sub\u003e-\u003cem\u003ehs\u003c/em\u003ePEX19-FV\u003csup\u003eamb\u003c/sup\u003e or His\u003csub\u003e6\u003c/sub\u003e-\u003cem\u003ehs\u003c/em\u003ePEX19-FI\u003csup\u003eamb\u003c/sup\u003e, or GST-Httex1-51Q-F11\u003csup\u003eamb\u003c/sup\u003e and tRNA\u003csub\u003eCUA\u003c/sub\u003e\u003csup\u003eOpt\u003c/sup\u003e synthetase\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e were co-transformed into BL21 Star\u0026trade; (DE3) cells. The expression of tRNA synthetase was induced with 0.2% arabinose at OD\u003csub\u003e600\u003c/sub\u003e of 0.3. At OD\u003csub\u003e600\u003c/sub\u003e of 0.6, proteins were induced with 0.5 mM IPTG and 1 mM Bpa (Bachem) at 18℃ overnight. Bpa incorporation into the proteins was confirmed by SDS-PAGE analysis. His\u003csub\u003e6\u003c/sub\u003e-\u003cem\u003ehs\u003c/em\u003ePEX19-FV\u003csup\u003eBpa\u003c/sup\u003e, His\u003csub\u003e6\u003c/sub\u003e-\u003cem\u003ehs\u003c/em\u003ePEX19-FI\u003csup\u003eBpa\u003c/sup\u003e, and GST-Httex1-51Q-F11\u003csup\u003eBpa\u003c/sup\u003e were purified in the same way as their non-Bpa proteins.\u003c/p\u003e \u003cp\u003eHis\u003csub\u003e6\u003c/sub\u003e-Ataxin3-78Q (gift from Sheena Radford lab) was expressed in BL21 Star\u0026trade; (DE3) with 0.5 mM IPTG at 30℃ for 3 h. His\u003csub\u003e6\u003c/sub\u003e-Ataxin3-78Q was purified using Ni-NTA, the same procedure used for His\u003csub\u003e6\u003c/sub\u003e-PEX19 proteins. The eluted proteins were loaded onto a superdex\u0026trade; 200 increase 10/300 column (Cytiva), and the monomer fractions were collected and further concentrated with Amicon\u0026reg; Ultra 50,000 MWCO centrifugal filter (Millipore). The purified His\u003csub\u003e6\u003c/sub\u003e-Ataxin3-78Q protein was snap-frozen and used for the filter trap assay.\u003c/p\u003e \u003cp\u003eTDP43-TEV-MBP-His\u003csub\u003e6\u003c/sub\u003e (Addgene plasmid #104480) was expressed BL21 Star\u0026trade; (DE3) with 0.1 mM IPTG overnight at 18℃. The purification of TDP43-TEV-MBP-His\u003csub\u003e6\u003c/sub\u003e was carried out as described\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e with a minor modification. Briefly, the cells were resuspended in Buffer C (20 mM Tris-HCl (pH 8.0), 1 M NaCl, 5 mM imidazole, 2 mM 2-mercaptoethanol, 10% glycerol) and then sonicated. The bound TDP43-TEV-MBP-His\u003csub\u003e6\u003c/sub\u003e protein onto Ni-NTA resin was eluted with 300 mM imidazole dissolved in Buffer C. The eluted proteins were loaded onto a superdexTM 200 increase 10/300 column and further purified in Buffer D (20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 mM DTT). The purified TDP43-TEV-MBP-His\u003csub\u003e6\u003c/sub\u003e protein was concentrated using Amicon\u0026reg; Ultra 50,000 MWCO centrifugal filter and stored at -80℃.\u003c/p\u003e \u003cp\u003eExpression and purification of 2\u0026times;Strep-SUMO-PEX26 (237-305aa) were carried out as described in the previous study\u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e. Briefly, the protein was induced with 0.1 mM IPTG in BL21 Star\u0026trade; (DE3) (Invitrogen) at 37 ℃ for 1 h. Cells were lysed by incubating with 0.5% N,N-Dimethyl-1-Dodecanamine-N-Oxide (LDAO, Anatrace) and 1\u0026times;CelLytic\u0026trade; B Cell Lysis Reagent (Sigma) for 40 min at room temperature (25 ℃). The clarified lysate was then diluted 3-fold with Buffer A and loaded onto a Strep-Tactin Sepharose column (IBA Lifesciences). The proteins were eluted with 15 mM \u003cem\u003ed\u003c/em\u003e-Desthiobiotin (Sigma) dissolved in the buffer (20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 2 mM 2-Mercaptoethanol, 0.05% LDAO,10% glycerol) and further dialyzed in the buffer (20 mM HEPES (pH 7.5), 200 mM NaCl, 10% glycerol, 0.05% LDAO).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro aggregation assay \u0026ndash; Filter trap assay\u003c/h2\u003e \u003cp\u003e3 \u0026micro;M of GST-TEV-Httex1-51Q-Stag proteins were mixed with 1.5 \u0026micro;M of \u003cem\u003ehs\u003c/em\u003ePEX19 or \u003cem\u003esc\u003c/em\u003ePEX19 proteins in the 1X TEV reaction buffer (Invitrogen). The polyQ aggregation reaction was initiated by adding 0.05 Units/\u0026micro;L of AcTEV protease (Invitrogen) and further incubated at 30℃. For the \u003cem\u003ein vitro\u003c/em\u003e aggregation of Ataxin-3-78Q, 30 \u0026micro;M of His\u003csub\u003e6\u003c/sub\u003e-Ataxin-3-78Q were incubated with the equimolar concentration of \u003cem\u003ehs\u003c/em\u003ePEX19 protein in the reaction buffer (20 mM HEPES (pH 7.5), 25 mM NaCl, 2 mM DTT, 5% glycerol) at 37℃. Samples were quenched at the indicated time points by mixing an equal volume of the quench buffer (4% (w/v) SDS, 0.1 M DTT) and then boiling at 95 ℃ for 10 min. The quenched samples were filtered through a 0.22 \u0026micro;m cellulose acetate membrane (Hyundai Micro) and then washed with 0.1% SDS. The membrane was probed using an S-tag antibody (1:3,000 dilution, Invitrogen) and the secondary antibody IRDye800 (1: 15,000 dilution, LiCor). The membrane-trapped polyQ aggregates were detected using iBright\u0026trade; FL1000 imaging system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro aggregation assay \u0026ndash; Turbidity assay\u003c/h2\u003e \u003cp\u003e7.5 \u0026micro;M of TDP43-MBP-His\u003csub\u003e6\u003c/sub\u003e protein was mixed with 7.5 \u0026micro;M of \u003cem\u003ehs\u003c/em\u003ePEX19-WT or \u003cem\u003ehs\u003c/em\u003ePEX19-FV proteins in the reaction buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM DTT). After the addition of AcTEV protease, the optical density values at 395 nm were recorded using a BioTek Epoch 2 plate reader (Agilent).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eNegative-stain TEM analysis\u003c/h2\u003e \u003cp\u003e3 \u0026micro;M of GST-TEV-Htt51Q-Stag proteins were mixed with 3 \u0026micro;M of hsPEX19 proteins in the 1X TEV reaction buffer supplemented with AcTEV protease and incubated at 30 ℃ for 15 h. A copper grid coated with a continuous carbon film (Electron Microscopy Sciences) was negatively glow-discharged at 15mA for 30 sec. 3 \u0026micro;L of protein samples were applied to a glow-discharged grid, incubated at room temperature for 3 min, and washed twice with distilled water and 0.75% uranyl formate once. The samples were negatively stained with 0.75% uranyl formate for 1 min with gentle shaking. The negatively stained specimens were examined under a FEI Tecnai\u0026trade; G2 spirit microscope operated at 120 kV. Micrographs were collected using a FEI Eagle 4 K x 4 K CCD camera at a nominal magnification of 15,000X with an electron dose of ~\u0026thinsp;30 e-/A2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBpa crosslinking assay\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e aggregation assay with \u003cem\u003ehs\u003c/em\u003ePEX19-FV\u003csup\u003eBpa\u003c/sup\u003e/\u003cem\u003ehs\u003c/em\u003ePEX19-FI\u003csup\u003eBpa\u003c/sup\u003e or Httex1-51Q-F11\u003csup\u003eBpa\u003c/sup\u003e was performed as described in the section of Filter trap assay. The reaction was stopped by freezing samples at the indicated time points, and frozen reaction aliquots were crosslinked on dry ice \u0026sim;4 cm away from a UVP B-100AP lamp (Analytik Jena) for 10 min. Crosslinked and uncrosslinked \u003cem\u003ehs\u003c/em\u003ePEX19 or Httex1-51Q proteins were resolved on SDS-PAGE and probed with anti-PEX19 (1: 3,000 dilution, Novus Biologicals) and anti-S-tag (1: 3,000 dilution, Invitrogen) antibodies, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHEK293 cell culture and plasmid transfection\u003c/h2\u003e \u003cp\u003eHEK293T cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium, GlutaMAX\u003csup\u003e\u0026trade;\u003c/sup\u003e (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco), 100 U/ml streptomycin, and 100 U/ml penicillin and incubated in a humidified chamber with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. For coexpression of Httex1-134Q-GFP and \u003cem\u003ehs\u003c/em\u003ePEX19 proteins, 3x10\u003csup\u003e5\u003c/sup\u003e cells per well were seeded on a 6-well plate one day prior to the transfection. Transient transfections of plasmids (each 1.25 \u0026micro;g) were carried out with Polyethylenimine (Polysciences) according to the manufacturer's instruction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eFilter trap assay using HEK293T cell lysates\u003c/h2\u003e \u003cp\u003eHEK293T cell lysates were prepared for filter trap assay as described previously\u003csup\u003e\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e with minor modifications. Briefly, cells were harvested with PBS supplemented with 1% Triton X-100 and 1x protease inhibitor cocktail (Sigma-Aldrich) and further incubated on ice for 30 min. The cell lysates were bath-sonicated in ice water for 5 min. After supplementing with 1% SDS and 50 mM DTT, the cell lysates were heated at 95\u0026deg;C for 10 min and stored at -80\u0026deg;C for filter trap assay and Western blot analysis. Filter trap assays were carried out as described above in the section on \u003cem\u003ein vitro\u003c/em\u003e aggregation assay. SDS-insoluble Httex1-134Q-GFP aggregates were probed with anti-GFP antibody (1: 3,000 dilution, Sigma) and quantified using iBright Analysis Software (Thermo Fisher Scientific Inc). To check protein expression levels, the cell lysates were loaded onto 10% Tris-glycine gels, and then Httex1-134Q-GFP, PEX19, and actin were probed in immunoblots with GFP (1: 3,000 dilution, Sigma), HA (1: 3,000 dilution, Genscript), and actin (1: 5,000 dilution, Invitrogen) antibodies, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eLive Cell imaging\u003c/h2\u003e \u003cp\u003eHEK293T cells were cultured and co-transfected with Httex1-134Q-GFP and \u003cem\u003ehs\u003c/em\u003ePEX19-WT or its variants in a confocal 6-well plate (SPL Life Sciences). At 48 h post-transfection, the plate was inserted in an inverted Eclipse Ti-E microscope (Nikon) equipped with a stage-top incubator (37 ℃, 5% CO\u003csub\u003e2\u003c/sub\u003e). Live cell images were acquired with a 20X 0.5 NA objective and an automated perfect focus system (PFS).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003ePrimary neuronal cell culture and transfection\u003c/h2\u003e \u003cp\u003eStriata were dissected from mouse pups aged postnatal day 1 (P1). Dissected tissues were digested in an enzyme solution containing 0.1% w/v Papain, 100 \u0026micro;g/mL DNase I, and 1 mM HEPES in Earle\u0026rsquo;s Balanced Salt Solution (EBSS) (Sigma) at 37 ℃ for 30 min. After incubation, the enzyme solution was carefully aspirated, and dissected tissues were rinsed with a Neurobasal A medium containing 20% FBS. The tissues were dissociated by mechanical trituration, and the isolated cells were resuspended in the neuro culture medium containing 2% v/v B-27 Supplement, 1 mM L-Glutamine, 1% Penicillin/Streptomycin in Neurobasal A medium (Gibco). 3x10\u003csup\u003e5\u003c/sup\u003e cells were plated on glass coverslips precoated with 0.1 mg/mL Poly-D-Lysine (Gibco) and 5 \u0026micro;g/mL Laminin (Gibco) in a 24-well plate. Striatal neurons were cultivated at 37 ℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e in a humidified incubator and used for experiments at 7 days in vitro (DIV). The cells were cotransfected with 1 \u0026micro;g of each plasmid using Lipofectamine\u0026trade; LTX Reagent with PLUS\u0026trade; Reagent (Invitrogen).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eFor HEK293T cells, transfection was carried out under the same conditions for live cell imaging. At 48 h post-transfection, cells were washed with PBS and fixed in 4% paraformaldehyde (PFA) for 10 min at room temperature. After washing with PBS twice, cells were permeabilized with 0.1% Triton X-100 for 15 min and then blocked with 2% BSA for 1 h at room temperature. Cells were incubated with HA Tag antibody (1:100 dilution, Invitrogen) for \u003cem\u003ehs\u003c/em\u003ePEX19-WT and its variants proteins and PMP70 antibody (1:200 dilution, Invitrogen) in 0.1% BSA solution at 4\u0026deg;C for two overnights. After washing the cells with PBS, the cells were incubated with Alexa Fluor 647 secondary antibody (1:1000 dilution, Invitrogen) for \u003cem\u003ehs\u003c/em\u003ePEX19 proteins and Alexa Fluor 568 secondary antibody (1:1000 dilution, Invitrogen) for PMP70 for 1 h at room temperature. To visualize nuclei, cells were additionally stained with 300 nM of DAPI (Invitrogen). Images were acquired with an inverted Eclipse Ti-E microscope (Nikon) with a 60\u0026times;1.4 NA oil objective.\u003c/p\u003e \u003cp\u003eAt 48 h post-transfection, primary neuronal cells were washed with PBS and fixed 15 min in 4% PFA at room temperature. After washing with PBS twice, the cells were blocked with 10% Normal Donkey Serum (Jackson immunoresearch) in PBS containing 0.1% Triton X-100 for 1h at room temperature. Cells were incubated in the blocking solution anti-Map2 antibody (1:500 dilution, SYSY) overnight at 4\u0026deg;C. After washing with PBS, the cells were incubated with Cy3 secondary antibody (Jackson immunoresearch) for 1h at room temperature. Nuclei were stained with DAPI.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eTDP43-BiFC measurements\u003c/h2\u003e \u003cp\u003eHEK293 TDP43-BiFC cells were cultured in the same media supplemented with 100 \u0026micro;g/mL Geneticin (G418). All cells were maintained in a humidified chamber with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. HEK293 TDP43-BiFC cells were plated on a 96-well plate with an Opti-MEM medium (Invitrogen). After 12 h, the cells were transfected with 0.1 \u0026micro;g of \u003cem\u003ehs\u003c/em\u003ePEX19-WT or \u003cem\u003ehs\u003c/em\u003ePEX19-FV plasmid using Lipofectamine\u0026reg;2000 reagent (Invitrogen). At 13 h post-transfection, TDP43-BiFC cells were treated with Forskolin (30 \u0026micro;M) or Apicidine (1 \u0026micro;M). After 36 h, nuclei were counterstained with Hoechst 33342 (Thermo Scientific). Fluorescence images were automatically acquired using Operetta CLS (PerkinElmer) with a 20x water immersion objective (TDP43-BiFC; λ\u003csub\u003eex\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;460\u0026ndash;490 nm and λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;500\u0026ndash;550 nm, Hoechst; λ\u003csub\u003eex\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;355\u0026ndash;385 nm and λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;430\u0026ndash;500 nm). The fluorescence intensities of TDP43-BiFC were quantified using Harmony 4.9 software (PerkinElmer). Data for each replicate were collected from 20 fields of view per well in the 96-well plate.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eDrosophila melanogaster stocks\u003c/h2\u003e \u003cp\u003eThe fly lines \u003cem\u003eW\u003c/em\u003e\u003csup\u003e\u003cem\u003e1118\u003c/em\u003e\u003c/sup\u003e (stock #5905), \u003cem\u003eUAS-Httex1-20Q\u003c/em\u003e (stock #68412), \u003cem\u003eUAS-Httex1-93Q\u003c/em\u003e (stock #68418), \u003cem\u003eElav-Gal4\u003c/em\u003e (stock #8765), and \u003cem\u003eD42-Gal4\u003c/em\u003e (stock #8816) were obtained from the Bloomington \u003cem\u003eDrosophila\u003c/em\u003e Stock Center (USA). All flies were maintained at 27\u0026deg;C.\u003c/p\u003e \u003cp\u003eTo generate transgenic fly lines, N-terminally HA-tagged \u003cem\u003ehs\u003c/em\u003ePEX19-WT and \u003cem\u003ehs\u003c/em\u003ePEX19-FV genes were subcloned into the pACU2-\u003cem\u003eUAS\u003c/em\u003e vector (gift from Chun Han, Cornell University). The pACU2-\u003cem\u003eUAS\u003c/em\u003e vector lacking the HA-\u003cem\u003ehs\u003c/em\u003ePEX19 gene was used as a negative control. The \u003cem\u003eUAS-pACU2 vector\u003c/em\u003e, \u003cem\u003eUAS-hsPEX19-WT\u003c/em\u003e, and \u003cem\u003eUAS-hsPEX19-FV\u003c/em\u003e transgenic fly lines were generated by BestGene, Inc. These \u003cem\u003ehs\u003c/em\u003ePEX19 transgenic fly lines were crossed with Httex1 transgenic fly lines, and the genotypes of generated transgenic fly lines used in this study are listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eClimbing assay\u003c/h2\u003e \u003cp\u003eTen to fifteen male flies were collected and transferred into an acrylic cylinder (3 cm diameter, 18 cm height) without the use of any CO\u003csub\u003e2\u003c/sub\u003e anesthesia but with cotton-sealed. Prior to the climbing assay, the collected flies were transferred into a new food vial within 24 h. The flies were acclimatized for 20 min in the cylinder. The climbing ability was measured by tapping the cylinder against a table, which was recorded for 1 min. A climbing index is the proportion of flies climbing\u0026thinsp;\u0026gt;\u0026thinsp;5 cm from the bottom of the cylinder within 5 sec. Seven technical trials were conducted for each individual experiment, and the average of these trials was considered as one biological replicate.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eLifespan assay\u003c/h2\u003e \u003cp\u003eA maximum of 15 male flies were collected within 24 h after pupal eclosion (APE) in a food vial and transferred to a new vial every two days. The number of dead flies was counted daily until the flies died. Lifespan data were plotted as Kaplan-Meier survival curves and statistical analyses were performed using the log-rank (Mantel-Cox) test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eMale fly heads were dissected to extract brains in Schneider's insect medium (Sigma-Aldrich). The brains were fixed with 3.7% formaldehyde for 20 min at room temperature. After washing with the washing buffer (0.3% Triton X-100 in PBS) for 10 min (a total of six washes), the brain samples were incubated in the blocking buffer (5% Normal Donkey Serum in PBS containing 0.3% Triton X-100) for 1 h at room temperature with gentle shaking. To stain Httex1-93Q and HA-\u003cem\u003ehs\u003c/em\u003ePEX19 proteins, the samples were incubated with Huntingtin (mEM48) mouse (1:200 dilution, Sigma-Aldrich) and HA rabbit (1: 200 dilution, Cell Signaling) primary antibodies at 4\u0026deg;C overnight, respectively. After washing with the washing buffer, the samples were further incubated with rabbit Alexa Fluor 555 (1: 200 dilution, Thermo Fisher Scientific) and mouse Alexa Fluor488 (1: 200 dilution, Invitrogen) secondary antibodies for 2 h at room temperature. The stained samples were mounted onto slides using Antifade Mounting Medium with DAPI (VectorLabs). All stained brain samples were taken at 400X magnification using 40X water immersion objective, acquired by Zeiss LSM700 confocal microscopy. Confocal microscopy images were set to a threshold to eliminate non-puncta fluorescence signals using Zeiss ZEN software. The same threshold settings were applied to all the images of the experiment. The total number of puncta in the region of interest was calculated using Fiji software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eQuantification and statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was conducted using GraphPad Prism Software. As indicated in Figure legends, we used Log-rank (Mantel-Cox) test, one-way ANOVA with Tukey post-hoc test, and two-way ANOVA with Tukey post-hoc test. All data were shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD or SEM, and \u003cem\u003ep\u003c/em\u003e values were represented with asterisks: *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; and ****\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank S.-h. Park and K. Shen for valuable discussions on yeast culture and TEM sample preparation. We thank the Research Solution Center at the Institute for Basic Science for the confocal microscopy facility. We thank W.-k. Huh, F.U. Hartl, J. Frydman, and S.E. Radford for sharing the \u003cem\u003eW303a\u003c/em\u003e yeast strain or plasmids. This work was supported by grants from the Institute for Basic Science (IBS-R030-Y1 to H.C. and\u0026nbsp;IBS-R030-C1 to H.M.K.) and the National Research Foundation (NRF) grant funded by the Korea government (MSIT) (RS-2023-00261784 to Y.K.K. and 2022R1C1C100714612 to S.L.).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.C. conceived the study. H.C., S.B.L., and H.M.K. designed the study. J.O., C.C., E.S.K., K.W.M., M.K., H.K., H.C.J., S.H.A., N.L., H.S.B, and H.C. carried out all experiments. J.O., C.C., E.S.K., K.W.M., M.K., H.K., H.C.J., N.L., S.L., and H.C. performed data analysis. H.C., S.B.L., H.M.K., S.L., and Y.K.K. supervised the study. H.C., J.O., E.S.K., K.W.M., M.K., H.K., S.L. drafted the manuscript. H.C., S.B.L., H.M.K. edited the manuscript. All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eHartl, F. U., Bracher, A. \u0026amp; Hayer-Hartl, M. Molecular chaperones in protein folding and proteostasis. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e475\u003c/strong\u003e, 324\u0026ndash;332 (2011).\u003c/li\u003e\n \u003cli\u003eKim, Y. E., Hipp, M. S., Bracher, A., Hayer-Hartl, M. \u0026amp; Hartl, F. U. Molecular chaperone functions in protein folding and proteostasis. \u003cem\u003eAnnu Rev Biochem\u003c/em\u003e \u003cstrong\u003e82\u003c/strong\u003e, 323\u0026ndash;355 (2013).\u003c/li\u003e\n \u003cli\u003eEllis, R. J. \u0026amp; Minton, A. P. Protein aggregation in crowded environments. \u003cem\u003eBiol Chem\u003c/em\u003e \u003cstrong\u003e387\u003c/strong\u003e, 485\u0026ndash;497 (2006).\u003c/li\u003e\n \u003cli\u003eYu, I. \u003cem\u003eet al.\u003c/em\u003e Biomolecular interactions modulate macromolecular structure and dynamics in atomistic model of a bacterial cytoplasm. \u003cem\u003eElife\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, e19274 (2016).\u003c/li\u003e\n \u003cli\u003eBalchin, D., Hayer-Hartl, M. \u0026amp; Hartl, F. U. In vivo aspects of protein folding and quality control. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e353\u003c/strong\u003e, aac4354 (2016).\u003c/li\u003e\n \u003cli\u003eNollen, E. A. A. \u003cem\u003eet al.\u003c/em\u003e Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e101\u003c/strong\u003e, 6403\u0026ndash;6408 (2004).\u003c/li\u003e\n \u003cli\u003eBrehme, M. \u003cem\u003eet al.\u003c/em\u003e A chaperome subnetwork safeguards proteostasis in aging and neurodegenerative disease. \u003cem\u003eCell Rep\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 1135\u0026ndash;1150 (2014).\u003c/li\u003e\n \u003cli\u003eShemesh, N. \u003cem\u003eet al.\u003c/em\u003e The landscape of molecular chaperones across human tissues reveals a layered architecture of core and variable chaperones. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 2180 (2021).\u003c/li\u003e\n \u003cli\u003eWalther, D. M. \u003cem\u003eet al.\u003c/em\u003e Widespread Proteome Remodeling and Aggregation in Aging C. elegans. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e161\u003c/strong\u003e, 919\u0026ndash;932 (2015).\u003c/li\u003e\n \u003cli\u003eIadanza, M. G., Jackson, M. P., Hewitt, E. W., Ranson, N. A. \u0026amp; Radford, S. E. A new era for understanding amyloid structures and disease. \u003cem\u003eNat Rev Mol Cell Biol\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 755\u0026ndash;773 (2018).\u003c/li\u003e\n \u003cli\u003eHipp, M. S., Kasturi, P. \u0026amp; Hartl, F. U. The proteostasis network and its decline in ageing. \u003cem\u003eNat Rev Mol Cell Biol\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 421\u0026ndash;435 (2019).\u003c/li\u003e\n \u003cli\u003eA novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington\u0026rsquo;s disease chromosomes. The Huntington\u0026rsquo;s Disease Collaborative Research Group. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e72\u003c/strong\u003e, 971\u0026ndash;983 (1993).\u003c/li\u003e\n \u003cli\u003eDiFiglia, M. \u003cem\u003eet al.\u003c/em\u003e Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e277\u003c/strong\u003e, 1990\u0026ndash;1993 (1997).\u003c/li\u003e\n \u003cli\u003eScherzinger, E. \u003cem\u003eet al.\u003c/em\u003e Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e90\u003c/strong\u003e, 549\u0026ndash;558 (1997).\u003c/li\u003e\n \u003cli\u003eGusella, J. F. \u0026amp; MacDonald, M. E. Molecular genetics: Unmasking polyglutamine triggers in neurodegenerative disease. \u003cem\u003eNat Rev Neurosci\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 109\u0026ndash;115 (2000).\u003c/li\u003e\n \u003cli\u003eTam, S. \u003cem\u003eet al.\u003c/em\u003e The chaperonin TRiC blocks a huntingtin sequence element that promotes the conformational switch to aggregation. \u003cem\u003eNat Struct Mol Biol\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 1279\u0026ndash;1285 (2009).\u003c/li\u003e\n \u003cli\u003eCrick, S. L., Ruff, K. M., Garai, K., Frieden, C. \u0026amp; Pappu, R. V. Unmasking the roles of N- and C-terminal flanking sequences from exon 1 of huntingtin as modulators of polyglutamine aggregation. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e110\u003c/strong\u003e, 20075\u0026ndash;20080 (2013).\u003c/li\u003e\n \u003cli\u003eShen, K. \u003cem\u003eet al.\u003c/em\u003e Control of the structural landscape and neuronal proteotoxicity of mutant Huntingtin by domains flanking the polyQ tract. \u003cem\u003eElife\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, e18065 (2016).\u003c/li\u003e\n \u003cli\u003eGruber, A. \u003cem\u003eet al.\u003c/em\u003e Molecular and structural architecture of polyQ aggregates in yeast. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e115\u003c/strong\u003e, E3446\u0026ndash;E3453 (2018).\u003c/li\u003e\n \u003cli\u003eKim, Y. E. \u003cem\u003eet al.\u003c/em\u003e Soluble Oligomers of PolyQ-Expanded Huntingtin Target a Multiplicity of Key Cellular Factors. \u003cem\u003eMol Cell\u003c/em\u003e \u003cstrong\u003e63\u003c/strong\u003e, 951\u0026ndash;964 (2016).\u003c/li\u003e\n \u003cli\u003eRiguet, N. \u003cem\u003eet al.\u003c/em\u003e Nuclear and cytoplasmic huntingtin inclusions exhibit distinct biochemical composition, interactome and ultrastructural properties. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 6579 (2021).\u003c/li\u003e\n \u003cli\u003eGidalevitz, T., Ben-Zvi, A., Ho, K. H., Brignull, H. R. \u0026amp; Morimoto, R. I. Progressive Disruption of Cellular Protein Folding in Models of Polyglutamine Diseases. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e311\u003c/strong\u003e, 1471\u0026ndash;1474 (2006).\u003c/li\u003e\n \u003cli\u003eB\u0026auml;uerlein, F. J. B. \u003cem\u003eet al.\u003c/em\u003e In Situ Architecture and Cellular Interactions of PolyQ Inclusions. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e171\u003c/strong\u003e, 179-187.e10 (2017).\u003c/li\u003e\n \u003cli\u003eJackrel, M. E. \u003cem\u003eet al.\u003c/em\u003e Potentiated Hsp104 variants antagonize diverse proteotoxic misfolding events. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e156\u003c/strong\u003e, 170\u0026ndash;182 (2014).\u003c/li\u003e\n \u003cli\u003eWang, J. D., Herman, C., Tipton, K. A., Gross, C. A. \u0026amp; Weissman, J. S. Directed evolution of substrate-optimized GroEL/S chaperonins. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e111\u003c/strong\u003e, 1027\u0026ndash;1039 (2002).\u003c/li\u003e\n \u003cli\u003eQuan, S. \u003cem\u003eet al.\u003c/em\u003e Genetic selection designed to stabilize proteins uncovers a chaperone called Spy. \u003cem\u003eNat Struct Mol Biol\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 262\u0026ndash;269 (2011).\u003c/li\u003e\n \u003cli\u003eMack, K. L. \u003cem\u003eet al.\u003c/em\u003e Tuning Hsp104 specificity to selectively detoxify \u0026alpha;-synuclein. \u003cem\u003eMol Cell\u003c/em\u003e \u003cstrong\u003e83\u003c/strong\u003e, 3314-3332.e9 (2023).\u003c/li\u003e\n \u003cli\u003eTariq, A. \u003cem\u003eet al.\u003c/em\u003e Mining Disaggregase Sequence Space to Safely Counter TDP-43, FUS, and \u0026alpha;-Synuclein Proteotoxicity. \u003cem\u003eCell Rep\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 2080-2095.e6 (2019).\u003c/li\u003e\n \u003cli\u003eSathyanarayanan, U. \u003cem\u003eet al.\u003c/em\u003e ATP hydrolysis by yeast Hsp104 determines protein aggregate dissolution and size in vivo. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 5226 (2020).\u003c/li\u003e\n \u003cli\u003eJones, J. M., Morrell, J. C. \u0026amp; Gould, S. J. PEX19 is a predominantly cytosolic chaperone and import receptor for class 1 peroxisomal membrane proteins. \u003cem\u003eJ Cell Biol\u003c/em\u003e \u003cstrong\u003e164\u003c/strong\u003e, 57\u0026ndash;67 (2004).\u003c/li\u003e\n \u003cli\u003eYagita, Y., Hiromasa, T. \u0026amp; Fujiki, Y. Tail-anchored PEX26 targets peroxisomes via a PEX19-dependent and TRC40-independent class I pathway. \u003cem\u003eJ Cell Biol\u003c/em\u003e \u003cstrong\u003e200\u003c/strong\u003e, 651\u0026ndash;666 (2013).\u003c/li\u003e\n \u003cli\u003eChen, Y. \u003cem\u003eet al.\u003c/em\u003e Hydrophobic handoff for direct delivery of peroxisome tail-anchored proteins. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 5790 (2014).\u003c/li\u003e\n \u003cli\u003eRipaud, L. \u003cem\u003eet al.\u003c/em\u003e Overexpression of Q-rich prion-like proteins suppresses polyQ cytotoxicity and alters the polyQ interactome. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e111\u003c/strong\u003e, 18219\u0026ndash;18224 (2014).\u003c/li\u003e\n \u003cli\u003eEmmanouilidis, L. \u003cem\u003eet al.\u003c/em\u003e Allosteric modulation of peroxisomal membrane protein recognition by farnesylation of the peroxisomal import receptor PEX19. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 14635 (2017).\u003c/li\u003e\n \u003cli\u003eSato, Y. \u003cem\u003eet al.\u003c/em\u003e Structural basis for docking of peroxisomal membrane protein carrier Pex19p onto its receptor Pex3p. \u003cem\u003eEMBO J\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 4083\u0026ndash;4093 (2010).\u003c/li\u003e\n \u003cli\u003eSchmidt, F. \u003cem\u003eet al.\u003c/em\u003e Insights into peroxisome function from the structure of PEX3 in complex with a soluble fragment of PEX19. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e285\u003c/strong\u003e, 25410\u0026ndash;25417 (2010).\u003c/li\u003e\n \u003cli\u003eLiu, Y., Yagita, Y. \u0026amp; Fujiki, Y. Assembly of Peroxisomal Membrane Proteins via the Direct Pex19p-Pex3p Pathway. \u003cem\u003eTraffic\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 433\u0026ndash;455 (2016).\u003c/li\u003e\n \u003cli\u003eRuckt\u0026auml;schel, R. \u003cem\u003eet al.\u003c/em\u003e Farnesylation of pex19p is required for its structural integrity and function in peroxisome biogenesis. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e284\u003c/strong\u003e, 20885\u0026ndash;20896 (2009).\u003c/li\u003e\n \u003cli\u003eTam, S., Geller, R., Spiess, C. \u0026amp; Frydman, J. The chaperonin TRiC controls polyglutamine aggregation and toxicity through subunit-specific interactions. \u003cem\u003eNat Cell Biol\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 1155\u0026ndash;1162 (2006).\u003c/li\u003e\n \u003cli\u003eRozales, K. \u003cem\u003eet al.\u003c/em\u003e Differential roles for DNAJ isoforms in HTT-polyQ and FUS aggregation modulation revealed by chaperone screens. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 516 (2022).\u003c/li\u003e\n \u003cli\u003eSivanandam, V. N. \u003cem\u003eet al.\u003c/em\u003e The aggregation-enhancing huntingtin N-terminus is helical in amyloid fibrils. \u003cem\u003eJ Am Chem Soc\u003c/em\u003e \u003cstrong\u003e133\u003c/strong\u003e, 4558\u0026ndash;4566 (2011).\u003c/li\u003e\n \u003cli\u003eElena-Real, C. A. \u003cem\u003eet al.\u003c/em\u003e The structure of pathogenic huntingtin exon 1 defines the bases of its aggregation propensity. \u003cem\u003eNat Struct Mol Biol\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 309\u0026ndash;320 (2023).\u003c/li\u003e\n \u003cli\u003eScarff, C. A. \u003cem\u003eet al.\u003c/em\u003e Examination of Ataxin-3 (atx-3) Aggregation by Structural Mass Spectrometry Techniques: A Rationale for Expedited Aggregation upon Polyglutamine (polyQ) Expansion. \u003cem\u003eMol Cell Proteomics\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1241\u0026ndash;1253 (2015).\u003c/li\u003e\n \u003cli\u003eShen, K. \u003cem\u003eet al.\u003c/em\u003e Dual Role of Ribosome-Binding Domain of NAC as a Potent Suppressor of Protein Aggregation and Aging-Related Proteinopathies. \u003cem\u003eMol Cell\u003c/em\u003e \u003cstrong\u003e74\u003c/strong\u003e, 729-741.e7 (2019).\u003c/li\u003e\n \u003cli\u003eYoung, T. S., Ahmad, I., Yin, J. A. \u0026amp; Schultz, P. G. An enhanced system for unnatural amino acid mutagenesis in E. coli. \u003cem\u003eJ. Mol. Biol.\u003c/em\u003e \u003cstrong\u003e395\u003c/strong\u003e, 361\u0026ndash;374 (2010).\u003c/li\u003e\n \u003cli\u003eCostello, J. L. \u003cem\u003eet al.\u003c/em\u003e Predicting the targeting of tail-anchored proteins to subcellular compartments in mammalian cells. \u003cem\u003eJ Cell Sci\u003c/em\u003e \u003cstrong\u003e130\u003c/strong\u003e, 1675\u0026ndash;1687 (2017).\u003c/li\u003e\n \u003cli\u003eFransen, M., Wylin, T., Brees, C., Mannaerts, G. P. \u0026amp; Van Veldhoven, P. P. Human pex19p binds peroxisomal integral membrane proteins at regions distinct from their sorting sequences. \u003cem\u003eMol Cell Biol\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 4413\u0026ndash;4424 (2001).\u003c/li\u003e\n \u003cli\u003eShao, S., Rodrigo-Brenni, M. C., Kivlen, M. H. \u0026amp; Hegde, R. S. Mechanistic basis for a molecular triage reaction. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e355\u003c/strong\u003e, 298\u0026ndash;302 (2017).\u003c/li\u003e\n \u003cli\u003eGuna, A., Volkmar, N., Christianson, J. C. \u0026amp; Hegde, R. S. The ER membrane protein complex is a transmembrane domain insertase. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e359\u003c/strong\u003e, 470\u0026ndash;473 (2018).\u003c/li\u003e\n \u003cli\u003eSchueller, N. \u003cem\u003eet al.\u003c/em\u003e The peroxisomal receptor Pex19p forms a helical mPTS recognition domain. \u003cem\u003eEMBO J\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 2491\u0026ndash;2500 (2010).\u003c/li\u003e\n \u003cli\u003eKulak, N. A., Pichler, G., Paron, I., Nagaraj, N. \u0026amp; Mann, M. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. \u003cem\u003eNat Methods\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 319\u0026ndash;324 (2014).\u003c/li\u003e\n \u003cli\u003eHallegger, M. \u003cem\u003eet al.\u003c/em\u003e TDP-43 condensation properties specify its RNA-binding and regulatory repertoire. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e184\u003c/strong\u003e, 4680-4696.e22 (2021).\u003c/li\u003e\n \u003cli\u003eNeumann, M. \u003cem\u003eet al.\u003c/em\u003e Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e314\u003c/strong\u003e, 130\u0026ndash;133 (2006).\u003c/li\u003e\n \u003cli\u003eBrady, O. A., Meng, P., Zheng, Y., Mao, Y. \u0026amp; Hu, F. Regulation of TDP-43 aggregation by phosphorylation and p62/SQSTM1. \u003cem\u003eJ Neurochem\u003c/em\u003e \u003cstrong\u003e116\u003c/strong\u003e, 248\u0026ndash;259 (2011).\u003c/li\u003e\n \u003cli\u003eNeumann, M. \u003cem\u003eet al.\u003c/em\u003e Phosphorylation of S409/410 of TDP-43 is a consistent feature in all sporadic and familial forms of TDP-43 proteinopathies. \u003cem\u003eActa Neuropathol\u003c/em\u003e \u003cstrong\u003e117\u003c/strong\u003e, 137\u0026ndash;149 (2009).\u003c/li\u003e\n \u003cli\u003eCohen, T. J. \u003cem\u003eet al.\u003c/em\u003e An acetylation switch controls TDP-43 function and aggregation propensity. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 5845 (2015).\u003c/li\u003e\n \u003cli\u003eLokireddy, S., Kukushkin, N. V. \u0026amp; Goldberg, A. L. cAMP-induced phosphorylation of 26S proteasomes on Rpn6/PSMD11 enhances their activity and the degradation of misfolded proteins. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e112\u003c/strong\u003e, E7176-7185 (2015).\u003c/li\u003e\n \u003cli\u003eHan, J. W. \u003cem\u003eet al.\u003c/em\u003e Apicidin, a histone deacetylase inhibitor, inhibits proliferation of tumor cells via induction of p21WAF1/Cip1 and gelsolin. \u003cem\u003eCancer Res\u003c/em\u003e \u003cstrong\u003e60\u003c/strong\u003e, 6068\u0026ndash;6074 (2000).\u003c/li\u003e\n \u003cli\u003eLi, H., Li, S. H., Yu, Z. X., Shelbourne, P. \u0026amp; Li, X. J. Huntingtin aggregate-associated axonal degeneration is an early pathological event in Huntington\u0026rsquo;s disease mice. \u003cem\u003eJ Neurosci\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 8473\u0026ndash;8481 (2001).\u003c/li\u003e\n \u003cli\u003eLi, H., Li, S. H., Johnston, H., Shelbourne, P. F. \u0026amp; Li, X. J. Amino-terminal fragments of mutant huntingtin show selective accumulation in striatal neurons and synaptic toxicity. \u003cem\u003eNat Genet\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 385\u0026ndash;389 (2000).\u003c/li\u003e\n \u003cli\u003eMonsellier, E., Redeker, V., Ruiz-Arlandis, G., Bousset, L. \u0026amp; Melki, R. Molecular interaction between the chaperone Hsc70 and the N-terminal flank of huntingtin exon 1 modulates aggregation. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e290\u003c/strong\u003e, 2560\u0026ndash;2576 (2015).\u003c/li\u003e\n \u003cli\u003eKakkar, V. \u003cem\u003eet al.\u003c/em\u003e The S/T-Rich Motif in the DNAJB6 Chaperone Delays Polyglutamine Aggregation and the Onset of Disease in a Mouse Model. \u003cem\u003eMol Cell\u003c/em\u003e \u003cstrong\u003e62\u003c/strong\u003e, 272\u0026ndash;283 (2016).\u003c/li\u003e\n \u003cli\u003eGillis, J. \u003cem\u003eet al.\u003c/em\u003e The DNAJB6 and DNAJB8 protein chaperones prevent intracellular aggregation of polyglutamine peptides. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e288\u003c/strong\u003e, 17225\u0026ndash;17237 (2013).\u003c/li\u003e\n \u003cli\u003eM\u0026aring;nsson, C. \u003cem\u003eet al.\u003c/em\u003e DNAJB6 is a peptide-binding chaperone which can suppress amyloid fibrillation of polyglutamine peptides at substoichiometric molar ratios. \u003cem\u003eCell Stress Chaperones\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 227\u0026ndash;239 (2014).\u003c/li\u003e\n \u003cli\u003eAyala Mariscal, S. M. \u003cem\u003eet al.\u003c/em\u003e Identification of a HTT-specific binding motif in DNAJB1 essential for suppression and disaggregation of HTT. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 4692 (2022).\u003c/li\u003e\n \u003cli\u003eKaramanos, T. K., Tugarinov, V. \u0026amp; Clore, G. M. Unraveling the structure and dynamics of the human DNAJB6b chaperone by NMR reveals insights into Hsp40-mediated proteostasis. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e116\u003c/strong\u003e, 21529\u0026ndash;21538 (2019).\u003c/li\u003e\n \u003cli\u003eHegde, R. S. \u0026amp; Keenan, R. J. The mechanisms of integral membrane protein biogenesis. \u003cem\u003eNat Rev Mol Cell Biol\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 107\u0026ndash;124 (2022).\u003c/li\u003e\n \u003cli\u003eChio, U. S., Cho, H. \u0026amp; Shan, S. Mechanisms of Tail-Anchored Membrane Protein Targeting and Insertion. \u003cem\u003eAnnu Rev Cell Dev Biol\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 417\u0026ndash;438 (2017).\u003c/li\u003e\n \u003cli\u003eRao, M. \u003cem\u003eet al.\u003c/em\u003e Multiple selection filters ensure accurate tail-anchored membrane protein targeting. \u003cem\u003eElife\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, (2016).\u003c/li\u003e\n \u003cli\u003eCho, H. \u003cem\u003eet al.\u003c/em\u003e Dynamic stability of Sgt2 enables selective and privileged client handover in a chaperone triad. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 134 (2024).\u003c/li\u003e\n \u003cli\u003eWoerner, A. C. \u003cem\u003eet al.\u003c/em\u003e Cytoplasmic protein aggregates interfere with nucleocytoplasmic transport of protein and RNA. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e351\u003c/strong\u003e, 173\u0026ndash;176 (2016).\u003c/li\u003e\n \u003cli\u003eGasset-Rosa, F. \u003cem\u003eet al.\u003c/em\u003e Polyglutamine-Expanded Huntingtin Exacerbates Age-Related Disruption of Nuclear Integrity and Nucleocytoplasmic Transport. \u003cem\u003eNeuron\u003c/em\u003e \u003cstrong\u003e94\u003c/strong\u003e, 48-57.e4 (2017).\u003c/li\u003e\n \u003cli\u003eLee, W.-C. M., Yoshihara, M. \u0026amp; Littleton, J. T. Cytoplasmic aggregates trap polyglutamine-containing proteins and block axonal transport in a Drosophila model of Huntington\u0026rsquo;s disease. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e101\u003c/strong\u003e, 3224\u0026ndash;3229 (2004).\u003c/li\u003e\n \u003cli\u003eTartari, M. \u003cem\u003eet al.\u003c/em\u003e Phylogenetic comparison of huntingtin homologues reveals the appearance of a primitive polyQ in sea urchin. \u003cem\u003eMol Biol Evol\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 330\u0026ndash;338 (2008).\u003c/li\u003e\n \u003cli\u003eMuthukumar, G. \u003cem\u003eet al.\u003c/em\u003e Triaging of \u0026alpha;-helical proteins to the mitochondrial outer membrane by distinct chaperone machinery based on substrate topology. \u003cem\u003eMol Cell\u003c/em\u003e S1097-2765(24)00095\u0026ndash;9 (2024) doi:10.1016/j.molcel.2024.01.028.\u003c/li\u003e\n \u003cli\u003eZhu, M. \u0026amp; Fink, A. L. Lipid binding inhibits alpha-synuclein fibril formation. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e278\u003c/strong\u003e, 16873\u0026ndash;16877 (2003).\u003c/li\u003e\n \u003cli\u003eBurr\u0026eacute;, J., Sharma, M. \u0026amp; S\u0026uuml;dhof, T. C. Definition of a molecular pathway mediating \u0026alpha;-synuclein neurotoxicity. \u003cem\u003eJ Neurosci\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 5221\u0026ndash;5232 (2015).\u003c/li\u003e\n \u003cli\u003eMacRaild, C. A., Howlett, G. J. \u0026amp; Gooley, P. R. The structure and interactions of human apolipoprotein C-II in dodecyl phosphocholine. \u003cem\u003eBiochemistry\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, 8084\u0026ndash;8093 (2004).\u003c/li\u003e\n \u003cli\u003eApostolidou, M., Jayasinghe, S. A. \u0026amp; Langen, R. Structure of \u0026alpha;-Helical Membrane-bound Human Islet Amyloid Polypeptide and Its Implications for Membrane-mediated Misfolding. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e283\u003c/strong\u003e, 17205\u0026ndash;17210 (2008).\u003c/li\u003e\n \u003cli\u003eRobert, X. \u0026amp; Gouet, P. Deciphering key features in protein structures with the new ENDscript server. \u003cem\u003eNucleic Acids Res\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, W320-324 (2014).\u003c/li\u003e\n \u003cli\u003eMadeira, F. \u003cem\u003eet al.\u003c/em\u003e Search and sequence analysis tools services from EMBL-EBI in 2022. \u003cem\u003eNucleic Acids Res\u003c/em\u003e \u003cstrong\u003e50\u003c/strong\u003e, W276\u0026ndash;W279 (2022).\u003c/li\u003e\n \u003cli\u003eKyte, J. \u0026amp; Doolittle, R. F. A simple method for displaying the hydropathic character of a protein. \u003cem\u003eJ Mol Biol\u003c/em\u003e \u003cstrong\u003e157\u003c/strong\u003e, 105\u0026ndash;132 (1982).\u003c/li\u003e\n \u003cli\u003eOh, J., Kim, D. K., Ahn, S. H., Kim, H. M. \u0026amp; Cho, H. A dual role of the conserved PEX19 helix in safeguarding peroxisomal membrane proteins. \u003cem\u003eiScience\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, (2024).\u003c/li\u003e\n \u003cli\u003eLim, G. \u0026amp; Huh, W.-K. Rad52 phosphorylation by Ipl1 and Mps1 contributes to Mps1 kinetochore localization and spindle assembly checkpoint regulation. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e114\u003c/strong\u003e, E9261\u0026ndash;E9270 (2017).\u003c/li\u003e\n \u003cli\u003eGietz, R. D. \u0026amp; Schiestl, R. H. Large-scale high-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. \u003cem\u003eNat Protoc\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 38\u0026ndash;41 (2007).\u003c/li\u003e\n \u003cli\u003eKlaips, C. L., Gropp, M. H. M., Hipp, M. S. \u0026amp; Hartl, F. U. Sis1 potentiates the stress response to protein aggregation and elevated temperature. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 6271 (2020).\u003c/li\u003e\n \u003cli\u003eTashiro, E. \u003cem\u003eet al.\u003c/em\u003e Prefoldin protects neuronal cells from polyglutamine toxicity by preventing aggregation formation. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e288\u003c/strong\u003e, 19958\u0026ndash;19972 (2013).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4292547/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4292547/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eToxic protein aggregates are associated with various neurodegenerative diseases, including Huntington\u0026rsquo;s disease (HD). Since no current treatment delays the progression of HD, we developed a mechanistic approach to preventing mutant huntingtin (mHttex1) aggregation. Here, we engineered the ATP-independent cytosolic chaperone PEX19, which targets peroxisomal membrane proteins to peroxisomes, to remove mHttex1 aggregates. Using yeast toxicity-based screening with a random mutant library, we identified two yeast PEX19 (\u003cem\u003esc\u003c/em\u003ePEX19) variants and engineered equivalent mutations into human PEX19 (\u003cem\u003ehs\u003c/em\u003ePEX19). These variants prevented mHttex1 aggregation \u003cem\u003ein vitro\u003c/em\u003e and in cellular HD models. The mutated hydrophobic residue in the α4 helix of \u003cem\u003ehs\u003c/em\u003ePEX19 variants binds to the N17 domain of mHttex1, thereby inhibiting the initial aggregation process. Overexpression of the \u003cem\u003ehs\u003c/em\u003ePEX19-FV variant rescues HD-associated phenotypes in primary striatal neurons and in \u003cem\u003eDrosophila\u003c/em\u003e. Overall, our data reveal that engineering ATP-independent membrane protein chaperones is a promising therapeutic approach for rational targeting of mHttex1 aggregation in HD.\u003c/p\u003e","manuscriptTitle":"Engineering a membrane protein chaperone to ameliorate the proteotoxicity of mutant huntingtin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-17 06:26:49","doi":"10.21203/rs.3.rs-4292547/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9eefcbc9-377a-4f89-94eb-743a2de4e98d","owner":[],"postedDate":"May 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":31338967,"name":"Biological sciences/Biochemistry/Protein folding/Protein aggregation"},{"id":31338968,"name":"Biological sciences/Biochemistry/Protein folding/Chaperones"},{"id":31338969,"name":"Biological sciences/Biological techniques/Molecular engineering/Protein design"}],"tags":[],"updatedAt":"2025-01-18T08:05:09+00:00","versionOfRecord":{"articleIdentity":"rs-4292547","link":"https://doi.org/10.1038/s41467-025-56030-6","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-01-17 05:00:00","publishedOnDateReadable":"January 17th, 2025"},"versionCreatedAt":"2024-05-17 06:26:49","video":"","vorDoi":"10.1038/s41467-025-56030-6","vorDoiUrl":"https://doi.org/10.1038/s41467-025-56030-6","workflowStages":[]},"version":"v1","identity":"rs-4292547","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4292547","identity":"rs-4292547","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}