NS1 binding protein regulates stress granule dynamics and clearance by inhibiting p62 ubiquitination

NS1 binding protein regulates stress granule dynamics and clearance by inhibiting p62 ubiquitination | 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 NS1 binding protein regulates stress granule dynamics and clearance by inhibiting p62 ubiquitination Jin-A Lee, Pureum Jeon, Hyunji Ham, Haneul Choi, Semin Park, Jae-Woo Jang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4380078/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Dec, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract NS1 binding protein (NS1-BP), a non-structural NS1-binding protein of influenza A virus, regulates viral or host RNA processing/export, cancer progression, or neurite/dendritic spine regulation. However, its precise roles in stress-induced responses without viral infection are largely unknown. Therefore, this study aims to investigate the novel roles of NS1-BP, which interact with GABARAP subfamily proteins, including LC3-interacting region-containing proteins, in regulating stress granules (SGs) during oxidative stress. NS1-BP interacts with core SG components and localizes to GABARAP-containing SGs during oxidative stress. Moreover, it associates with p62, acting as an adaptor for selective autophagy via its Kelch-motif and ubiquitin-associated domain in p62 in a stress-dependent manner. NS1-BP knockout (KO) HeLa cells demonstrated altered SG dynamics, mirroring observation in p62 KO or GABARAP triple KO cells, indicating impaired autophagic SG degradation. NS1-BP KO cells, compared to wild-type (WT) cells, showed increased p62 ubiquitination, leading to autophagic p62 degradation, while NS1-BP overexpression reduces p62 ubiquitination. In NS1-BP KO cells, overexpression of p62 WT, not p62 K420R or K435R, restored SGs size and number. Additionally, amyotrophic lateral sclerosis (ALS)-induced pluripotent stem cell-derived motor neurons showed reduced NS1-BP levels, resulting in SG morphology dysregulation. Our findings reveal the novel role of NS1-BP in negatively regulating p62 ubiquitination, influencing SG dynamics and clearance during oxidative stress. This highlights its relevance to ALS pathogenesis associated with SGs. Biological sciences/Cell biology Biological sciences/Molecular biology Stress granules ubiquitin p62/SQSTM1 GABARAP autophagy amyotrophic lateral sclerosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Autophagy is cellular recycling, breaking down damaged proteins and organelles for energy and cellular repair 1 . It aids adaptation to stress conditions, including nutrient shortage, oxidative stress, and infection 2, 3 . Regulated by different signals, autophagy maintains cell health, preventing toxic build-up. Conversely, stress granules (SGs) are dynamic structures that form in cell cytoplasm in response to stress, including heat shock, viral infection, or oxidative stress 4 . Comprising RNA and RNA-binding proteins, they store mRNA temporarily, shielding it from degradation. When stress subsides, SGs aid mRNA translation and regulate gene expression and stress response. SGs are rapidly assembled and disassembled by various chaperons, valosin-containing protein (VCP), or RNA helicase 5, 6 . Some specific or prolonged stress conditions also facilitate SG degradation. Over the past two decades, approximately 100 proteins have been identified in the SG interactome 7, 8, 9 . Some with low complexity regions enable multiple interactions with various binding partners 10 . SGs serve as signalling platforms, aiding the coordination of cellular processes during stress, including apoptosis, cell growth, and metabolic control 11 . Dysregulated SG dynamics can impair RNA metabolism and protein homeostasis 12 , potentially inducing neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) 13, 14 . Autophagy and SGs interact closely. Autophagy, activated during stress, clears damaged or aggregated proteins, including those in SGs 2 , regulating their assembly and disassembly 6, 15, 16 . This interplay enables cells to adapt to stress and maintain cellular homeostasis. Recent evidence associates SGs with autophagy, suggesting a molecular connection 7, 17, 18 . Autophagy components, including VCP, p62/SQSTM1 (from hereon p62), mammalian ATG8s (mATG8s), and Unc-51-like kinase 1/2 (ULK1/2), indicating their role in SG regulation or clearance. For example, VCP regulates selective autophagy involved in SGs degradation, and mutations in VCP impair autophagy-related functions 6, 17 . Additionally, ULK1/2, associated with autophagy, regulate SG disassembly through phosphorylation and activation of VCP/p97 18 . Moreover, SG homeostasis is influenced by tripartite motif-containing protein 21 (TRIM21)-mediated ubiquitination of Ras-GTPase-activating protein (GAP)-binding protein 1 (G3BP1) and autophagy-dependent SG elimination 19 . Among autophagy regulators of SGs, p62 stands out as a multifunctional protein. It aids in degrading ubiquitinated proteins via autophagy and participates in various signalling pathways such as nutrient sensing, oxidative stress, infection, immunity, and inflammation 20 . Acting as an autophagic receptor/adaptor, p62 interacts with ubiquitinated cargo via its ubiquitin association (UBA) domain and recruits them to the growing autophagosome membrane through its LC3-interacting (LIR) motif 21, 22, 23 . Recent research indicates that p62-droplets also facilitate autophagosome formation and counter oxidative stress 24 . Additionally, the C9ORF72-p62 complex aids in the autophagic clearance of SG via arginine methylation 15 . Proteomic analyses have revealed a network of proteins associated with SGs, including mATG8 proteins, such as LC3B and GABARAP, key constituents of these granules 7 . These proteins interact with LIR motif-containing proteins, including p62, acting as adaptors/receptors, and recruit selective cargos or autophagic machinery proteins to autophagosomes during selective autophagy 25, 26 . However, the specific mechanisms by which p62 and mATG8 proteins selectively recognize SGs and regulate autophagic SG clearance remain unclear. Identifying the components in SGs that interact with p62 and mATG8 proteins and how they recruit autophagic machinery for autophagosome formation under various stress conditions is essential. Recent findings indicate that various types of selective autophagy employ receptors/adaptors with LIR motifs to recognize and tether specific substrates to phagophores. Recently, LC3 subfamily- or GABARAP subfamily-selective LIR motifs have been identified, along with several proteins that bind to either the LC3 subfamily or GABARAP subfamily, aiming to characterize their function in selective autophagy 27, 28 . Therefore, this study aims to investigate NS1-BP, a protein characterized as containing an LIR motif that specifically interacts with GABARAP subfamily proteins; however, not with LC3 subfamily proteins. Co-immunoprecipitation (co-IP) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis revealed NS1-BP interaction with SG components, including Ataxin2, Poly(A)-binding protein (PABP), T-cell-restricted intracellular antigen-1 (TIA-1), and G3BP1. NS1-BP was observed to localize to GABARAP-containing SGs during oxidative stress. Furthermore, it strongly associates with p62, acting as an adaptor/receptor for selective autophagy in an oxidative stress-dependent manner. NS1-BP KO cells demonstrated increased SG size and decreased SG numbers, akin to observation in p62 KO cells or GABARAP triple KO (TKO) cells, suggesting impaired autophagic clearance. Additionally, NS1-BP KO cells showed increased p62 ubiquitination, leading to p62 autophagic degradation, while NS1-BP overexpression reduced p62 ubiquitination. Additionally, wild-type (WT) p62 expression, not p62 mutants (K420R or K435R), rescued p62 ubiquitination and the cellular phenotype in NS1-BP KO cells. NS1-BP KO cells showed reduced contact between ubiquitinated p62 bodies/aggregates and SGs. Neurons from derived ALS-induced pluripotent stem cells (iPSCs) showed reduced NS1-BP levels, resulting in SG dysregulation. These findings underscore the important role of NS1-BP in p62- and GABARAP-mediated SG regulation, shedding light on the molecular mechanisms of selective autophagic degradation of SGs and the cellular pathogenesis of ALS associated with SGs dysregulation. Results NS1-BP, characterized as a GABARAP subfamily-binding protein, regulates SGs number, size, and dynamics Identifying and studying proteins that bind to the LC3 subfamily- and GABARAP subfamily is crucial for understanding selective autophagy, as many receptors and adaptors involved possess LIR motifs. To accomplish this, a search for proteins containing LIR motifs using the iLIR database was performed (A web resource for LIR motif-containing proteins in eukaryotes) (Supplementary Table 1) 29 . Several LIR motif-containing proteins were found to potentially bind to the GABARAP subfamily. Among these, NS1-BP, a Kelch family protein, was identified as having a potential LIR motif within its second Kelch motif (Supplementary Fig. 1a). NS1-BP is recognized for its interactions with the influenza virus non-structural protein 1 and regulates viral or host RNA processing/export 30 , cancer progression 31 , and regulation of neurite/dendritic spines 32 . To investigate the binding properties of the LIR motifs from NS1-BP for each mATG8 protein, a previously developed in vitro assay was utilized 27 . Since NS1-BP functions as a dimer through its BTB/POZ domain, 33 , duplicated LIR motif of NS1-BP (2xLIR[NS1-BP]) fused with mRFP-3x nuclear localization signal (2xLIR[NS1-BP]-mRFP-3xNLS), along with EGFP-tagged mATG8 mutants EGFP-mATG8(GA) in HeLa cells were expressed. In these mutants, the C-terminal glycine residue was replaced with alanine to impair phosphatidylethanolamine (PE) conjugation (lipidation), inhibiting cellular localization to autophagosomes. The assay revealed that when 2xLIR(NS1-BP)-mRFP-3xNLS interacted with specific EGFP-mATG8(GA), cytosolic EGFP-mATG8(GA) was sequestered into the nucleus based on its binding preference in live cells (Supplementary Fig. 1b, c). EGFP-GABARAP(GA), GABARAPL1(GA), and GABARAPL2(GA) were localized to the nucleus, indicating that the 2xLIR motifs from NS1-BP preferentially bind to GABARAP subfamily proteins rather than LC3 subfamily proteins. In contrast, the 2xLIR mutant(NS1-BP[AA]) was diffusely localized to the nucleus and cytoplasm, suggesting a LIR-dependent binding pattern (Supplementary Fig. 1b, c). Further, GST-pulldown assay using HeLa cell lysates supported the interaction between endogenous NS1-BP and GABARAP subfamily proteins (Supplementary Fig. 1d), suggesting its possible role in stress-induced responses such as autophagy. To elucidate NS1-BP functional roles in the stress response pathway associated with autophagy, co-IP and LC-MS/MS analyses were performed using the HEK293T cell lysates expressing FLAG-NS1-BP under oxidative stress induced by sodium arsenite. The silver stained SDS-PAGE analysis of elutes showed 35, 60, and 70 kDa in NS1-BP samples but not in control IgG samples (Fig. 1 a). The co-IP and LC-MS/MS analysis results showed major binding partners, including heat shock protein A12B (HSPA12B) from the HSP70 family, mitochondrial heat shock protein 75 (MTHSP75) belonging to the HSP90 family, and heterogeneous nuclear ribonucleoprotein K (hnRNPK) and hnRNPA2B1 from the heterogeneous nuclear ribonucleoproteins family (Fig. 1 b). These results are significant because all of the identified proteins are involved in the stress response pathway. Among them, hnRNPA2B1 or hnRNPK is known to localize to SGs, and mutations in these proteins have been related to neurodegenerative diseases including ALS and membrane scaffold protein (MSP) 34 or Au–Kline syndrome with multiple malformation syndrome 35 . Co-IP experiments confirmed that FLAG-NS1-BP interacts with MYC-hnRNPA2B1 in HEK293T cells under oxidative stress conditions (Fig. 1 c). Furthermore, additional co-IP experiments using HEK293T cell lysates expressing FLAG-NS1-BP revealed interactions with core SGs components, including ataxin2, PABP, and hnRNPA1, upon oxidative stress (Fig. 1 d). NS1-BP predominantly colocalizes with TIA-1-containing SGs under oxidative stress or endoplasmic reticulum stress conditions (Fig. 1 e, f). Proximity ligation assays (PLA) further demonstrated an increased association of NS1-BP with Ataxin2 under oxidative stress, suggesting its potential role in the stress response pathway associated with SGs regulation (Fig. 1 g, h). To investigate the physiological roles of NS1-BP in regulating SGs, NS1-BP KO HeLa cells were generated using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 technology, confirmed by genomic polymerase chain reaction (PCR) (Supplementary Fig. 2b). Immunoblot and immunocytochemistry analyses confirmed the absence of NS1-BP protein in the NS1-BP KO cells than in WT cells (Supplementary Fig. 2d, e). Subsequently, SG number and size in WT and NS1-BP KO HeLa cells were assessed. A significant reduction in SGs number in NS1-BP KO HeLa cells compared to WT HeLa cells was found, accompanied by increased size under oxidative stress (Fig. 2 a-c). Expression of FLAG-NS1-BP rescued the size increase in NS1-BP KO HeLa cells (Fig. 2 a-c). Furthermore, fluorescence recovery after photobleaching (FRAP) analysis showed significantly reduced dynamics of G3BP1-positive SGs in NS1-BP KO HeLa cells compared to WT HeLa cells, which was restored upon NS1-BP re-introduction (Fig. 2 d, e), indicating the regulatory role of NS1-BP in SG dynamics. Overall, these results show that NS1-BP, characterized as a GABARAP subfamily-binding protein, localizes to SGs and modulates their size, number, or dynamics during oxidative stress. NS1-BP is strongly associated with p62 via kelch motif in NS1-BP and UBA domain in p62 in an oxidative stress-dependent manner To understand how NS1-BP regulates the size, number, or dynamics of SGs during oxidative stress, it is essential to consider SG disassembly mechanisms. Typically, after stress cessation, SGs disassemble through various kinds of chaperons, VCP, and RNA helicase 36 . However, under prolonged stress, SGs can also be selectively degraded by autophagy. Recent studies indicate that autophagy components, including mATG8s or p62, localize to SGs, with p62 playing a role in SG clearance 15, 37 . To investigate the interaction between NS1-BP and GABARAP subfamily proteins, their association in HEK293T cells expressing GFP-GABARAP subfamily proteins under oxidative stress conditions was examined. Supplementary Fig. 3a shows that the association between NS1-BP and GABARAP subfamily proteins was oxidative stress-dependent. Additionally, under oxidative stress conditions, NS1-BP predominantly co-localized with GABARAP subfamily-containing SGs, suggesting its potential role in SGs regulation (Supplementary Fig. 3b, c). In selective autophagy, mATG8s binds to p62, a selective autophagy adaptor, via the LIR motif to recruit ubiquitinated substrates into the autophagosomal membrane 38 . Therefore, NS1-BP was investigated to determine if it could associate with p62 under oxidative stress using HEK293T cell lysates expressing FLAG-NS1-BP or FLAG-p62. Figure 3 a shows that NS1-BP is strongly associated with p62 in an oxidative stress-dependent manner. NS1-BP only interacts with p62 under oxidative stress conditions and not under other stressors such as thapsigargin, sorbitol, or heat shock (Supplementary Fig. 4a). The domain involved in the association between NS1-BP and p62 during oxidative stress was identified. NS1-BP contains a BTB/POZ domain and Kelch motif, which forms a β-propeller structure 33 . Conversely, p62 contains multiple binding domains, including the N-terminal Phox-Bem1p (PB1) and C-terminal UBA domains 20 . PB1 is known to facilitate p62 oligomerization, while UBA is crucial for p62 multimerization and ubiquitination, essential for its function in sequestering ubiquitinated substrates. Therefore, co-IP experiments with anti-FLAG antibodies were performed using deletion mutants of NS1-BP or p62 (FLAG-NS1-BP, FLAG-NS1-BP ΔKelch, FLAG-NS1-BP ΔBTB/POZ, FLAG-p62, FLAG-p62 ΔUBA, or FLAG-p62 ΔPB1) (Fig. 3 b). Figure 3 c-f shows that the interaction between endogenous p62 and FLAG-NS1-BP ΔKelch or between endogenous NS1-BP and FLAG-p62 ΔUBA was significantly reduced, indicating that the Kelch domain in NS1-BP and UBA domain in p62 are necessary for their association. Additionally, the association between FLAG-p62 ΔPB1 and NS1-BP was slightly reduced, suggesting that PB1 oligomerization might partially contribute to their association. The binding of NS1-BP to p62 may not occur in their monomeric state under naïve conditions; however, to get the structural insight of the association of NS1-BP with p62, their binding regions were predicted using ColabFold modeling 39 . The prediction results showed two possibilities (Fig. 4 a, b). The kelch motif in NS1-BP was predicted to directly interact with the Kelch-like ECH-associated protein 1 (Keap1)-interacting region (KIR) in p62 as shown in the Keap1-p62 structure 40 . Additionally, the UBA domain in p62 was also predicted to be in close proximity to the kelch motif in NS1-BP as a different model, suggesting a possible association of p62 with NS1-BP, probably facilitated by post-translational modification such as phosphorylation or ubiquitination. Based on the predicted association sites identified through the ColabFold modeling, it was clarified whether the KIR motif in p62 could interact with NS1-BP upon oxidative stress condition. However, the co-IP results using a FLAG-p62 KIR mutant showed that the interaction between the KIR mutant and NS1-BP was not significantly affected (Fig. 4 c, d). This suggests that the kelch domain of NS1-BP strongly associates with the UBA domain in p62 under oxidative stress rather than the Keap1-KIR type interaction, suggesting that NS1-BP may modulate p62 function via the UBA domain in p62 under oxidative stress. NS1-BP plays a negative regulatory role in p62 ubiquitination at lysine 13, 420, 435 in p62 and autophagic degradation under oxidative stress Subsequently, the role of NS1-BP on the function of p62 during oxidative stress was queried. Accumulating evidence showed that during oxidative stress, p62 interacts with Keap1-Cul3, activating nuclear factor erythroid 2-related factor 2 (Nrf2), which in turn induces the expression of antioxidant-response genes 41, 42 . Lysine 420 within the UBA domain of p62 is responsible for its ubiquitination by Keap1/Cul3, and ubiquitination of the UBA domain increases p62 degradation by facilitating its sequestration into autophagic vacuoles 43 . Additionally, the report shows that NS1-BP (called KLHL39) inhibits KLHL20-mediated substrate ubiquitination by Cul3 and substrate degradation 31 . Therefore, it was hypothesized that NS1-BP might regulate p62 ubiquitination by Keap1/Cul3 and its degradation. To test this hypothesis, the protein level of p62 was initially assessed in WT and NS1-BP KO cells. Figure 5 a-b shows that the protein level of p62 was significantly reduced in NS1-BP KO cells, which was rescued by inhibiting autophagy with bafilomycin A1, suggesting that NS1-BP negatively regulates the autophagic degradation of p62. Subsequently, the effect of NS1-BP on the ubiquitination of p62 was examined. NS1-BP KO cells showed increased p62 ubiquitination than the WT cells, while its overexpression in WT cells reduced p62 ubiquitination, further supporting the hypothesis that NS1-BP inhibits p62 ubiquitination and degradation during oxidative stress (Fig. 5 c, d). To pinpoint the critical residues within p62 responsible for NS1-BP-mediated inhibition of p62 ubiquitination among the lysine residues essential for p62 ubiquitination, site-specific mutations were introduced by substituting lysine with arginine at lysine 7, 13, 165, 264, 420, and 435 in p62 38, 43, 44 . Ubiquitination IP using HA-Ubiquitin was performed in FLAG-p62 WT, K7R, K13R, K165R, K189R, K264R, K420R, or K435R expressing HEK293T cells. Figure 6 shows that although the expression of p62 lysine mutants K7R, K165R, K189R, or K264R significantly reduced p62 ubiquitination, similar to the observation with p62 WT, K13R expression, a lysine mutant within the PB1 domain, and K420R or K435R, lysine mutants in the UBA domain, did not reduce p62 ubiquitination as observed with p62 WT. Subsequently, the effect of the lysine mutant on the association of NS1-BP with p62 was investigated. The co-IP showed that compared to p62 WT, the p62 lysine mutant K420R reduced its association with NS1-BP, highlighting the significance of p62 lysine 420 located within the UBA domain for its interaction with NS1-BP. However, the association of NS1-BP with other p62 lysine mutants did not show any significant change compared to p62 WT (Supplementary Fig. 4b). Collectively, these findings reveal that NS1-BP inhibits p62 ubiquitination and autophagic degradation, and lysine residues 13, 420, or 435 in p62 are crucial for its inhibitory effect on p62 ubiquitination under oxidative stress conditions. Reduced levels of p62 in NS1-BP KO HeLa cells impaired clearance of SGs To further examine whether the reduced protein level of p62 in NS1-BP KO cells is responsible for the dysregulation of SGs, the number and size of SGs in p62 KO mouse embryonic fibroblasts (MEFs) were assessed. Figure 7 a-c shows the observed increase in SGs size and a decrease in their number in p62 KO MEFs, resembling the cellular phenotype observed in NS1-BP KO HeLa cells. To ascertain whether the decreased level of p62 contributes to the altered dynamics of SGs in NS1-BP KO HeLa cells, p62 WT or p62 lysine mutants were reintroduced into NS1-BP KO HeLa cells. The expression of p62 WT, K13R, or K264R in NS1-BP KO HeLa cells significantly restored the number and size of SGs, while p62 K420R or K435R did not show any change (Fig. 7 d-f). These results show that p62 K420 or K435 play key roles in ubiquitination and subsequent degradation of p62 in NS1-BP KO HeLa cells, thereby contributing to SGs dysregulation. Given that NS1-BP interacts not only with p62 but also with GABARAP family proteins, it was further investigated whether NS1-BP modulates GABARAP-mediated autophagic SGs degradation. Therefore, the number and size of SGs were examined in GABARAP TKO HeLa or HEK293T cells expressing a selective deconjugase for GABARAP/L1/L2-PE 28 . Figure 7 g-i and Supplementary Fig. 5a-d show that GABARAP/L1/L2-deficient cells exhibited an increase in SG size and a decrease in the number of SGs compared to WT or other LC3A/B-PE deconjugase expressing cells, resembling the phenotype observed in NS1-BP KO cells. Additionally, it was found that HeLa cells expressing siRNA targeting ATG16L1 showed a similar increase in SG size and decrease in the number of SGs, akin to observation in NS1-BP KO cells, p62 KO MEFs, and GABARAP TKO HeLa cells (Supplementary Fig. 5e-g). This indicates that autophagic degradation of SGs may be impaired in NS1-BP KO cells owing to the reduced protein level of p62, potentially caused by increased p62 ubiquitination leading to its degradation. Finally, cellular localization of p62, ubiquitin, SGs, GABARAP in WT and NS1-BP KO cells were examined to determine whether ubiquitinated p62 are properly targeted to SGs and recruits autophagosomal membrane (Supplementary Fig. 6). The co-localization of ubiquitinated G3BP1-positive SGs with GABARAP-containing autophagosome was significantly decreased in NS1-BP KO cells compared to WT cells. This suggests that NS1-BP may play a role in the proper targeting of SGs into GABARAP-containing SG structures, possibly through the ubiquitination of p62. NS1-BP was reduced in patients with ALS (TDP43 A382T) iPSC-derived motor neurons, leading to SG dysregulation The dysregulation of SGs is increasingly recognized as being associated with various neurodegenerative diseases. In the case of ALS, sporadic and familial cases often involve motor neurons experiencing oxidative stress and exhibiting abnormal accumulation of protein aggregates, including TDP-43. Therefore, it was initially investigated whether deficiency of NS1-BP also causes SG dysregulation in post-mitotic neurons. Supplementary Fig. 7 shows that cultured cortical neurons expressing NS1-BP shRNA demonstrated increased size and decreased number of SGs. These findings support the potential role of NS1-BP in the cellular pathogenesis of ALS associated with SGs. To investigate the protein level of NS1-BP and its relationship with SGs dysregulation in ALS, patients with ALS-derived motor neurons generated from induced pluripotent stem cells (iPSCs) were examined (Fig. 8 a). The findings revealed a significant reduction in NS1-BP protein levels in patients with ALS derived motor neurons compared to control motor neurons (Fig. 8 b, c). Furthermore, an increase in SG size and a decrease in SG number was observed, indicating SG dysregulation in patients with ALS-derived motor neurons (Fig. 8 d-f). These results were consistent with the results obtained from NS1-BP KO HeLa cells and NS1-BP deficient post-mitotic cortical neurons. Generally, this study provides insights into the cellular mechanisms underlying SG dysregulation in ALS and highlights the potential pathogenic role of NS1-BP in regulating SGs in motor neurons derived from patients with ALS. Discussion SGs are dynamic cytoplasmic structures formed in response to various stressors such as oxidative stress, heat shock, or viral infection. They temporarily sequester mRNAs and RNA-binding proteins to halt normal translation 4 , playing a crucial role in modulating responses to cellular stress and being implicated in the pathogenesis of diverse conditions, such as neurodegenerative diseases, ageing, and cancer 14, 45, 46, 47 . Recent studies have shed light on the relationship between SG dynamics and autophagy 6, 19, 37 . However, the interplay between autophagy and SGs, particularly the modulation by post-translational modifications of significant proteins, remains a vital but partially understood aspect of cellular stress response and homeostasis. This study reveals that NS1-BP, a protein previously recognised for its interactions with the influenza A virus non-structural protein NS1, plays a crucial role in regulating SG dynamics by modulating the autophagic machinery. The interaction of NS1-BP with GABARAP and p62—key components in autophagic processes—suggests a novel pathway through which SG dynamics are controlled under oxidative stress. This study provides the first evidence that the interaction of NS1-BP with p62 significantly affects the ubiquitination and protein level of p62, influencing its ability to regulate SG clearance via autophagy. The reduction in NS1-BP levels observed in patients with ALS-derived motor neurons, along with corresponding changes in SG morphology and dynamics, suggests the potential involvement of NS1-BP in ALS pathogenesis. This aligns with emerging evidence suggesting that impaired autophagy and defective SG dynamics contribute to the accumulation of protein aggregates, a hallmark of ALS and other neurodegenerative disorders. These studies propose several potential mechanisms by which NS1-BP influences SG and autophagy dynamics. Interaction of NS1-BP with GABARAP subfamily proteins The presence of an LIR motif within the second Kelch motif suggests its potential interaction with autophagy-related proteins, including LC3 and GABARAP, a member of the ATG8 family involved in autophagosome formation and selective autophagy. This analysis indicates that NS1-BP selectively binds to GABARAP subfamily proteins but not to LC3 proteins, indicating its specific role in certain autophagic pathways. This selective binding suggests that NS1-BP may influence the choice of autophagic routes employed by cells during the stress response, potentially favouring those involving GABARAP proteins. GABARAP subfamily proteins are characterized in the SG interactome 7 . GABARAPs directly interact with NUFIP2 and G3BP1, with Atg8ylation being necessary for their recruitment to damaged lysosomes 48 . Furthermore, the interaction between GABARAP and G3BP1 promotes the recruitment of SGs into autophagosomes, resulting in their degradation. This indicates that GABARAP, through its involvement in autophagy, can indirectly influence SG dynamics and clearance. Additionally, p62 interacts with SG components such as TIA-1 or G3BP1 and regulates their assembly, disassembly, or clearance 37, 49 . This data suggests NS1-BP may influence the recruitment of cargo, including damaged organelles or protein aggregates, to autophagosomes by modulating GABARAP function or localization. Given that NS1-BP regulates the size, number, and dynamics of SGs, its interaction with GABARAP affects the autophagic degradation of SGs. This process seems crucial during cellular stress recovery, where SGs need to be efficiently cleared to restore cellular homeostasis. Structural analysis of NS1-BP interaction with p62 p62 comprises several domains, including a PB1 domain for oligomerization, zinc finger (ZZ) domain for N-degron recognition 50 , TRAF6-binding domain, LIR motif for interaction with LC3 and GABARAP subfamily proteins in autophagy, KIR, and UBA domain at its C-terminus, which is crucial for binding polyubiquitin chains 20 . NS1-BP, a Kelch family protein, features a BTB/POZ domain facilitating its dimerization, along with Kelch repeats that form a β-propeller structure 33 . It is well characterized that the β-propeller of Kelch repeats in the Keap1 recognize the KIR segments in the p62 40 and the high-ranked ColabFold models for 1:1 complex between NS1-BP and p62 were predicted (Fig. 4 b). However, their interaction was only restricted to an oxidative stress condition, which is an oligomeric state of p62 51 . Therefore, we are tempting to speculate another complex model that the UBA domain of p62 contributes to the interaction with NS1-BP protein (Fig. 4 a, rank 5) because the IP analysis indicates that the Kelch motifs of NS1-BP and UBA domain of p62 are crucial for this interaction (Fig. 3 c-f). These results imply a specific binding affinity between NS1-BP and p62 alters the p62 behaviour under oxidative stress conditions. Small molecule inhibitors to the Kelch domain of Keap1 have been developed to treat diseases involving oxidative stress and inflammation 52 . Therefore, the detailed structural analysis of NS1-BP interactions with p62 and GABARAP may elucidate the precise molecular interfaces and post-translational modifications influencing their interactions, providing insights for the design of targeted therapeutics. Negative regulation of p62 ubiquitination by NS1-BP on autophagy Ubiquitination plays a crucial role in regulating the dynamics and function of SGs under stress conditions. Various SG proteins, including G3BP1, PABP, or FUS, undergo ubiquitination, directly influencing their role and the stability of SGs 19, 53, 54, 55 . The clearance of SGs through autophagy, particularly granulophagy, relies on the ubiquitination of SG components 53 . Several studies have demonstrated that ubiquitination of p62 at specific lysine residues, including K7 and K420, serves as a signal for selective cargo recognition, resulting in its sequestration into autophagosomes for degradation 38, 43 . Moreover, p62 itself can be ubiquitinated and targeted for degradation through autophagy, contributing to SG clearance and cellular homeostasis. Despite its significance, the fine-tuning mechanism for regulating p62 ubiquitination remains poorly understood. In this study, a novel role of NS1-BP was characterised, which negatively regulates the ubiquitination of p62, a crucial factor in autophagic degradation. By modulating p62 ubiquitination, NS1-BP affects the level and availability of p62 protein for autophagy, thereby influencing SG dynamics. This regulation is pivotal, as excessive or insufficient p62 levels can disrupt cellular balance and contribute to disease pathology. NS1-BP, through its modulation of p62, plays a role in this pathway. The interaction between p62 and GABARAP, facilitated by NS1-BP, seems essential for targeting SGs to autophagosomes. In NS1-BP deficient scenarios, the impaired interaction between p62 and the autophagic machinery leads to inadequate SG clearance, contributing to cellular stress and potentially initiating neurodegenerative changes. These findings indicate that the interactions of NS1-BP with both GABARAP and p62 suggest a coordinated role in associating autophagy with SG dynamics. By interacting with these proteins, NS1-BP may help orchestrate the balance between SG formation as a protective mechanism under stress and their clearance through autophagy once the stress is resolved. Regulation of SG dynamics by NS1-BP Through its interaction with p62 and GABARAP, NS1-BP influences SG dynamics by modulating their assembly and disassembly processes. These findings demonstrate that NS1-BP deficiency results in increased SG size and decreased number, resembling the effects observed with alterations in p62 and GABARAP levels. These findings underscore the complex interplay between SG formation, autophagy, and the cellular stress response. However, the exact mechanisms by which NS1-BP influences SG dynamics remain incompletely understood. NS1-BP may influence SGs through its interactions with other SG-associated proteins or by participating in cellular signalling pathways. Exploring NS1-BP's interactome, especially with other autophagy and SG components, may reveal additional roles and regulatory mechanisms. Implications of the role of NS1-BP in neurodegenerative diseases Using motor neurons derived from iPSCs of patients with ALS, we a significant reduction in NS1-BP levels and observed alterations in SG dynamics (increased size, decreased number) compared to controls (Fig. 8 ). These findings corroborate observations from NS1-BP deficient models, strengthening the hypothesis that NS1-BP plays a regulatory role in SG dynamics. This study connects NS1-BP, a protein implicated in RNA processing and cellular stress response, to the pathological features of ALS, particularly through its involvement in SG regulation in neurons. The dysregulation of SG dynamics and autophagic pathways are common features in neurodegenerative diseases. NS1-BP interaction with p62, modulating SG dynamics and autophagy, is significant for understanding diseases, including ALS. Targeting the NS1-BP-p62 interaction and its downstream effects on SG dynamics and autophagy offers a novel therapeutic avenue for conditions with SG accumulation and autophagy dysfunction. Modulating NS1-BP levels or imitating its action may restore SG and autophagic function, potentially alleviating disease symptoms or progression. This study enhances understanding of ALS pathogenesis, shedding light on targeting cellular stress responses to mitigate disease progression. Dysregulation in autophagy and SG dynamics is associated with neurodegenerative disorders and cancers. Understanding how NS1-BP modulates these processes through its interactions with GABARAP and p62 may reveal molecular mechanisms and therapeutic targets. Further research into the mechanisms and interactions of NS1-BP is crucial for developing targeted therapies for these conditions. Methods Cell lines and growth conditions HEK293T and HeLa cells were obtained from ATCC. To generate NS1-BP KO HeLa cells, cluster regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing was utilized to disrupt the genes encoding human NS1-BP in HeLa cells. The CRISPR/Cas9 system, consisting of CMV-Cas9-EF1a-puromycin/GFP-U6 optimized for cell transfection, was sourced from YSY Biotech Company Ltd (Nanjing, China). To develop the double-nicking NS1-BP-sgRNA-guided CRISPR/Cas9 plasmids, a pair of oligos were designed and subcloned into AflII-digested gRNA_Cloning Vector (Addgene 41824). These plasmids were co-transfected with pCas9-GFP (Addgene 44719) into HeLa cells using lipofectamine 2000 (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA). HeLa cells transfected with pCas9-GFP/NS1-BP two gRNAs were selected with puromycin, and single colonies were isolated. To generate cells stably expressing either 3xFlag-p62 WT or K420R, NS1-BP KO HeLa cells were infected with lentiviral particles and grown in a medium containing 10 µg/ml polybrene (Sigma Aldrich H9268) for 48 h. Stably expressing cells were selected with puromycin, and single colonies were isolated. All cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin in a humidified atmosphere containing 5% (v/v) of CO 2 at 37°C. DNA constructs NS1-BP was PCR-amplified from a human cDNA library and inserted into the pEGFP-C1 vector using BglII and BamHI restriction enzymes or into the 3xFlag-CMV7.1 vector through EcoRI and BamHI restriction enzymes. GST-LC3A, GST-LC3B, GST-LC3C, GST-GABARAP, GST-GABARAL1, and GST-GABARAPL2 were obtained from Addgene (Cambridge, USA). Additionally, p62 was PCR-amplified from an HA-p62 construct from Addgene and inserted into the 3xFlag-CMV7.1 vector through EcoRI and KpnI restriction enzymes. To generate domain deletion or mutant constructs, p62 (ΔPB1), p62 (ΔUBA), p62 (KIR mutant), p62 (LIR mutant), p62 K7R, p62 K13R, p62 K157R, p62 K165R, p62 K189R, p62 K264R, p62 K420R, p62 K435R, p62 K420Q, p62 K435Q, p62 K420/435Q, NS1-BP (ΔKelch), and NS1-BP (ΔBTB/POZ) were PCR-amplified with specific primers (Supplementary Table 2) and inserted into the 3xFlag-CMV7.1 vector through EcoRI or BglII (for 5’ region) and KpnI or BamHI for (3’ region) restriction enzymes. Additionally, p62 K13R, p62 K165R, and p62 K435R were amplified by PCR with specific primers and inserted into the 3xFlag-CMV7.1 vector using the infusion cloning method (Takara Bio 639648). To further generate p62 lentiviral constructs, 3xFlag-p62 WT and 3xFlag-p62 K420R were amplified by PCR using specific primers and inserted into the pLVX-EF1a-IRES-Puro (Takara Bio 631988). Lentivirus production To produce lentiviruses for infection, Lenti-X 293T (Takara Bio 632180) cells were co-transfected with pLVX-EF1a-3xFlag-NS1-BP, p62 WT or K420R, psPAX2, and pMD2.G using the CalPhos Mammalian Transfection Kit (Takara Bio 631312). Culture supernatant was collected at 48 h and 72 h post-transfection and passed through a 0.45-µm filter. The viral particles were concentrated by ultracentrifugation (22,600 x g for 3 h) and resuspended in Dulbecco’s phosphate-buffered saline (DPBS). Transfection, gene silencing, and drug treatment To transiently express DNA constructs, cells were transfected with plasmid DNA using either the calcium phosphate method or FuGENE HD (Promega E2311) following the instruction of the manufacturer. Knockdown experiments in mouse cortical neurons were performed using Lipofectamine 2000 (Thermo Fisher Scientific 11668-019) and the shRNA construct following the instructions of the manufacturer. To induce SG formation, cells were treated with sodium arsenite (S.A 0.5 mM, 1 h), thapsigargin (T.G 20 uM, 1 h), sorbitol (0.4 M, 2 h) or heat shock (42 ℃, 2 h). Immunoblot analysis Protein samples were separated through SDS-PAGE, transferred to PVDF membranes (Millipore IPVH00010) and incubated with primary antibodies overnight at 4°C. After washing with TBST (150 mM NaCl, 20 mM Tris-HCl (pH 7.4), 0.05% Tween 20), membranes were exposed to secondary antibodies conjugated with horseradish peroxidase for 1 h. Signals were detected with ECL solution (Millipore, WBKLS0500, USA). Antibodies used included NS1-BP (Santa Cruz sc-373909, 1:100), Flag (Sigma F1804, 1:10,000), p62 (Abnova H00008878-M01, 1:10,000), GABARAPL1 (Cell Signalling 26632, 1:1,000), G3BP1 (Proteintech 13057-2-AP, 1:1,000), Ataxin2 (Proteintech 21776-1-AP, 1:5,000), hnRNPA1(Abcam ab137780, 1:2000), PABP (Abcam ab21060, 1:1,000), HA (Cell Signalling 3724, 1:1000), GFP (Neuromab 75–131, 1:10,000), and Beta-actin (Sigma A5441, 1:10,000). Quantification was performed using ImageJ (NIH) software. Immunocytochemistry and confocal microscopy For immunostaining, transfected cells were washed with PBS, then fixed and permeabilized with methanol (Samchun) for 30 min at -20℃. After blocking with 3% bovine serum albumin for 1 h at room temperature, cells were incubated overnight at 4°C with anti-TIA-1 (Santa Cruz sc-1751), anti-G3BP1 (Proteintech 13057-2-AP), and anti- Flag (Sigma F1804). Subsequently, they were treated with anti-mouse or anti-rabbit secondary antibodies (Jackson Laboratory) for 2 h at room temperature. Following three washes with 1X PBS, the cells were mounted on glass slides and analysed using an LSM 880 microscope (Cal Zeiss, Germany). Silver staining and LC-MS/MS analysis Silver staining was performed using PageSilver™ Silver staining Kit (Thermo Fisher Scientific K0681) following the instruction of the manufacturer. LC-MS/MS analysis was performed at the Yonsei Proteomic Research Centre (Seoul, Korea). GST affinity isolation assay For the GST affinity-isolation assay, HeLa cells were washed with PBS, harvested, and lysed in GST affinity isolation buffer solution (50 mM Tris-HCl pH7.6, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, supplemented with protease inhibitor cocktail). After centrifugation at 13,000 x g for 20 min, the cleared cell lysates were incubated overnight with purified GST-mATG8 proteins and glutathione-conjugated agarose beads (Sigma Aldrich G4510) at 4℃. The following day, the samples were washed three to five times with the same GST affinity isolation buffer solution at 4℃, and the remaining supernatant was removed. The samples were resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, boiled immediately, and subjected to SDS-PAGE with Coomassie Brilliant Blue (Sigma Aldrich 1154440025) staining. Co-IP Cells were collected and lysed with 1% NP40 lysis buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 2 mM EDTA, 1% NP40), supplemented with protease inhibitor (Sigma P8340) and phosphatase inhibitor (Thermo Fisher Scientific 78426) on ice for 30 min. After centrifugation at 13,000 rpm at 4℃ for 15 min, the supernatant fractions were collected and incubated with protein A/G-agarose beads (Santa Cruz sc-2002) for 2 h at 4℃. This step served as a pre-clearing step, with the supernatants containing 1 mg of protein incubated overnight at 4℃ with appropriate antibodies. To precipitate the target protein, the supernatants were incubated with protein A/G agarose bead for 7 h at 4℃. Afterwards, the beads were washed with lysis buffer three times by centrifugation at 3,000 rpm at 4℃. The immunoprecipitated proteins were separated via SDS-PAGE, and western blotting was performed as previously described. PLA The in situ PLA was performed using a Duolink in situ kit (Sigma DUO92101) following the instruction of the manufacturer. Initially, cells were cultured on Matrigel-coated glass. After sodium arsenite treatment, cells were fixed and permeabilized using 100% methanol and then blocked with a blocking solution. Subsequently, cells were incubated with primary antibodies (NS1-BP, Santa Cruz sc-373909, 1:50; Ataxin-2, Proteintech 21776-1-AP, 1:100; p62, Abnova H00008878-M01, 1:100; Flag, Sigma F1804, 1:100), diluted in antibody diluent overnight at 4℃. Following this, secondary antibodies (PLA probe anti-mouse MINUS and PLA probe anti-rabbit PLUS) were applied for 1 h at 37℃. Cells were treated with ligase for 30 min at 37℃ and amplified with polymerase diluted in amplification buffer for 100 min at 37℃. In vivo p62 ubiquitination IP Cells were transfected with either 3xFlag-p62 WT or ubiquitination mutant (K7R, K13R, K157R, K165R, K189R, K264R, K420R, or K435R) along with HA-ub constructs, and either EGFP or EGFP-NS1-BP. After 24 h, cells were treated with MG132 (1 uM, 16 h) to inhibit proteasome. Cells were lysed with 1% SDS denaturing buffer (1% SDS, 50 mM Tris-HCl pH7.6, 150 mM NaCl) and supplemented with protease inhibitors. Lysates were boiled for 10 min and disrupted with gentle sonication. Additionally, these samples were diluted 1:20 with 1% Triton X-100 lysis buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100). Subsequent steps were consistent with the co-IP method described above. FRAP analysis HeLa cells expressing EGFP-G3BP1 were initially activated by imaging every 4 s for 2.5 min using a 100% 488-nm laser power and 100-ms exposure time to SGs. Subsequently, the EGFP-G3BP1-positive SGs were photobleached, and the EGFP signal intensity was measured before and after photobleaching. Structure prediction The predicted complex model between NS1-BP and p62/SQSTM1 was obtained by using ColabFold 39 . Five ranked models were obtained for the NS1-BP and p62 complex, and the binding modes of these complexes were classified into two categories. PyMOL software was utilized to visualize the models ( https://pymol.org/ ). iPSC generation from control and patients with ALS-derived primary fibroblast Control and ALS iPSC lines were reprogrammed from fibroblasts obtained from the Coriell Institute for Medical Research (NINDS collection) using a non-integrating induction method with OSKM factors. The fibroblasts were cultured at 37°C in Dulbecco’s modified Eagle’s medium (Corning) supplemented with 15% FBS (Corning) and 1% penicillin-streptomycin (Gibco). For gene delivery, electroporation (Invitrogen™ Neon™ Transfection System) was performed. Seven days after electroporation, the cells were transferred onto MEF feeder cells with iPSC maintenance medium containing 20% KO serum (Gibco 10828028), β-mercaptoethanol (Gibco 21985023), Gluta-MAX (Gibco 35050-061), MEM-Non Essential Amino Acids solution (Gibco 11140050), penicillin/streptomycin (Hyclone SV30010), and DMEM-F12 (Gibco 11320033). After 4 weeks, iPSC-like colonies were picked and cultured on new feeder cells. Furthermore, at an iPSC passage number of 5–7, the culture system was transitioned to a feeder-free system using a truncated recombinant human vitronectin (Gibco A14700) coated plate with Essential 8 Medium (Gibco A1517001). Motor neuron differentiation To generate highly pure motor neurons, a previous method was slightly modified 56 . To induce differentiation into neuronal precursor cells (NPC), iPSCs were dissociated using Accutase (Innovative Cell Technologies AT104) and seeded at a 1:5 ratio on Matrigel-coated plates. On the following day, the iPSC medium was replaced with a chemically defined neural medium (NEP media), including KO DMEM-F12 (Gibco 12660-012) and Neurobasal medium (Gibco 21103-049) at 1:1, 0.5x N2 (Gibco 17502-048), 0.5x B-27 (Gibco 17504-044), 1x GlutaMax supplements (Gibco 35050-061), 1x penicillin–streptomycin (Hyclone SV30010), 100µM Ascorbic acid (Sigma A4403), 3µM CHIR99021 (Stem Cell 72054), 2µM DMH-1 (Stem Cell 73634), and 2µM SB4315429 (Stem cell 72234). The culture medium was altered every other day. To induce motor neuron patterning (MNP), NPC cells were dissociated with Accutase and seeded at a 1:3 on Matrigel-coated plates with NEP medium supplemented with 0.1-µM Retionic acid (Sigma R2625) and 0.5 µM Puromorphamine (Sigma 540220) 7 days after seeding. The culture medium was altered every other day. For motor neuron differentiation, MNP cells were dissociated with Accutase and cultured in suspension in the NEP medium supplemented with 0.5 µM Retionic acid and 0.1µM Puromorphamine 7 days after MNP induction. The culture medium was altered every other day. To mature the motor neuron, MND cells were dissociated into single cells with Accutase and plated on mouse glial cells in a motor neuron mature medium. The mature medium include Neurobasal medium, 1x B-27 supplement, 1x GlutaMax, 1x penicillin-streptomycin (Gibco), 100 µM Ascorbic acid (Sigma), 2.6 µg/ml Adenosine 3',5'-cyclic monophoaphate (Sigma A9501), 0.5 µM Compound E (Stem cell 73954), 10 µg/ml Laminin (Sigma L2020), 10 µg/ml BDNF (Thermo Fisher Scientific 450-02-50UG), and 10 µg/ml GDNF (Thermo Fisher Scientific PHC7041). Medium refreshment with motor neuron mature medium occurred weekly for a total maturation period of 5 weeks. Declarations Data availability The data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper. Competing interests The author(s) declare no competing interests Author contributions P.J. conducted the majority of the experiments, analyzed the data, and contributed to manuscript drafting. H.H., H.C., S.P., J-W.J., S-W.P. conducted and assisted with IP experiments. D-H.C. contributed to the interpretation of results and edited the manuscript. H.-J.L. analyzed protein interactions using ColabFold modeling. H.K.S. designed the experiment and wrote the manuscript. K.M. contributed to the interpretation of results and edited the manuscript. D-J.J. & J-A.L. conceptualized the study, designed the experiments, and wrote the manuscript. All authors reviewed the manuscript, made necessary corrections, and approved the final version for publication. Consent for publication was obtained from each author. Acknowledgements The work was supported by the Science Research Center Program of the National Research Foundation NRF (Grant No. 2020R1A5A1019023, J.-A L.); Neurological Disorder Research Program of the NRF (Grant No. 2020M3E5D9079911, J.-A L.); Basic research program of the NRF (Grant No. 2023R1A2C2008092, J.-A L.). This work was also supported by JSPS KAKENHI (Grant No. JP19H05706; JP21H004771; 23K20044; 24H00060, M.K.); AMED (Grant No. JP22gm1410004h0003, M.K.) and by the Takeda Science Foundation to M.K. References Glick D, Barth S, Macleod KF. Autophagy: cellular and molecular mechanisms. J Pathol 221 , 3-12 (2010). Murrow L, Debnath J. Autophagy as a stress-response and quality-control mechanism: implications for cell injury and human disease. Annu Rev Pathol 8 , 105-137 (2013). Buchan JR, Yoon JH, Parker R. Stress-specific composition, assembly and kinetics of stress granules in Saccharomyces cerevisiae. J Cell Sci 124 , 228-239 (2011). Anderson P, Kedersha N. Stress granules. Curr Biol 19 , R397-398 (2009). Wheeler JR, Matheny T, Jain S, Abrisch R, Parker R. Distinct stages in stress granule assembly and disassembly. Elife 5 , (2016). Buchan JR, Kolaitis RM, Taylor JP, Parker R. Eukaryotic stress granules are cleared by autophagy and Cdc48/VCP function. Cell 153 , 1461-1474 (2013). Markmiller S , et al. Context-Dependent and Disease-Specific Diversity in Protein Interactions within Stress Granules. Cell 172 , 590-604 e513 (2018). Isabelle M, Gagne JP, Gallouzi IE, Poirier GG. Quantitative proteomics and dynamic imaging reveal that G3BP-mediated stress granule assembly is poly(ADP-ribose)-dependent following exposure to MNNG-induced DNA alkylation. J Cell Sci 125 , 4555-4566 (2012). Amen T, Kaganovich D. Stress granules inhibit fatty acid oxidation by modulating mitochondrial permeability. Cell Rep 35 , 109237 (2021). Molliex A , et al. Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 163 , 123-133 (2015). Kedersha N, Ivanov P, Anderson P. Stress granules and cell signaling: more than just a passing phase? Trends Biochem Sci 38 , 494-506 (2013). Portz B, Lee BL, Shorter J. FUS and TDP-43 Phases in Health and Disease. Trends Biochem Sci 46 , 550-563 (2021). Baron DM , et al. Amyotrophic lateral sclerosis-linked FUS/TLS alters stress granule assembly and dynamics. Mol Neurodegener 8 , 30 (2013). Jeon P, Lee JA. Dr. Jekyll and Mr. Hyde? Physiology and Pathology of Neuronal Stress Granules. Front Cell Dev Biol 9 , 609698 (2021). Chitiprolu M , et al. A complex of C9ORF72 and p62 uses arginine methylation to eliminate stress granules by autophagy. Nat Commun 9 , 2794 (2018). Ryu HH , et al. Autophagy regulates amyotrophic lateral sclerosis-linked fused in sarcoma-positive stress granules in neurons. Neurobiol Aging 35 , 2822-2831 (2014). Seguin SJ , et al. Inhibition of autophagy, lysosome and VCP function impairs stress granule assembly. Cell Death Differ 21 , 1838-1851 (2014). Wang B , et al. ULK1 and ULK2 Regulate Stress Granule Disassembly Through Phosphorylation and Activation of VCP/p97. Mol Cell 74 , 742-757 e748 (2019). Yang C , et al. Stress granule homeostasis is modulated by TRIM21-mediated ubiquitination of G3BP1 and autophagy-dependent elimination of stress granules. Autophagy , 1-18 (2023). Berkamp S, Mostafavi S, Sachse C. Structure and function of p62/SQSTM1 in the emerging framework of phase separation. FEBS J 288 , 6927-6941 (2021). Bjorkoy G , et al. p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J Cell Biol 171 , 603-614 (2005). Komatsu M , et al. Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice. Cell 131 , 1149-1163 (2007). Pankiv S , et al. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem 282 , 24131-24145 (2007). Kageyama S , et al. p62/SQSTM1-droplet serves as a platform for autophagosome formation and anti-oxidative stress response. Nat Commun 12 , 16 (2021). Lippai M, Low P. The role of the selective adaptor p62 and ubiquitin-like proteins in autophagy. Biomed Res Int 2014 , 832704 (2014). Liu WJ , et al. p62 links the autophagy pathway and the ubiqutin-proteasome system upon ubiquitinated protein degradation. Cell Mol Biol Lett 21 , 29 (2016). Park SW , et al. Monitoring LC3- or GABARAP-positive autophagic membranes using modified RavZ-based probes. Sci Rep 9 , 16593 (2019). Park SW , et al. Development of new tools to study membrane-anchored mammalian Atg8 proteins. Autophagy , 1-20 (2022). Jacomin A-C, Samavedam S, Promponas V, Nezis IP. iLIR database: A web resource for LIR motif-containing proteins in eukaryotes. Autophagy 12 , 1945-1953 (2016). Wolff T, O'Neill RE, Palese P. NS1-Binding protein (NS1-BP): a novel human protein that interacts with the influenza A virus nonstructural NS1 protein is relocalized in the nuclei of infected cells. J Virol 72 , 7170-7180 (1998). Chen HY , et al. KLHL39 suppresses colon cancer metastasis by blocking KLHL20-mediated PML and DAPK ubiquitination. Oncogene 34 , 5141-5151 (2015). Fujii R, Takumi T. TLS facilitates transport of mRNA encoding an actin-stabilizing protein to dendritic spines. J Cell Sci 118 , 5755-5765 (2005). Sasagawa K , et al. Identification of Nd1, a novel murine kelch family protein, involved in stabilization of actin filaments. J Biol Chem 277 , 44140-44146 (2002). Kim HJ , et al. Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS. Nature 495 , 467-473 (2013). Au PYB , et al. Phenotypic spectrum of Au-Kline syndrome: a report of six new cases and review of the literature. Eur J Hum Genet 26 , 1272-1281 (2018). Protter DSW, Parker R. Principles and Properties of Stress Granules. Trends Cell Biol 26 , 668-679 (2016). Zheng Y , et al. HDAC6, A Novel Cargo for Autophagic Clearance of Stress Granules, Mediates the Repression of the Type I Interferon Response During Coxsackievirus A16 Infection. Front Microbiol 11 , 78 (2020). Peng H , et al. Ubiquitylation of p62/sequestosome1 activates its autophagy receptor function and controls selective autophagy upon ubiquitin stress. Cell Res 27 , 657-674 (2017). Mirdita M, Schutze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M. ColabFold: making protein folding accessible to all. Nat Methods 19 , 679-682 (2022). Komatsu M , et al. The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1. Nature cell biology 12 , 213-223 (2010). Jain A , et al. p62/SQSTM1 is a target gene for transcription factor NRF2 and creates a positive feedback loop by inducing antioxidant response element-driven gene transcription. J Biol Chem 285 , 22576-22591 (2010). Ichimura Y , et al. Phosphorylation of p62 activates the Keap1-Nrf2 pathway during selective autophagy. Mol Cell 51 , 618-631 (2013). Lee Y, Chou TF, Pittman SK, Keith AL, Razani B, Weihl CC. Keap1/Cullin3 Modulates p62/SQSTM1 Activity via UBA Domain Ubiquitination. Cell Rep 19 , 188-202 (2017). Feng L , et al. Sirt1 deacetylates and stabilizes p62 to promote hepato-carcinogenesis. Cell Death Dis 12 , 405 (2021). Gao B , et al. Inhibition of anti-viral stress granule formation by coronavirus endoribonuclease nsp15 ensures efficient virus replication. PLoS Pathog 17 , e1008690 (2021). Jayabalan AK , et al. Stress granule formation, disassembly, and composition are regulated by alphavirus ADP-ribosylhydrolase activity. Proc Natl Acad Sci U S A 118 , (2021). Bentmann E, Haass C, Dormann D. Stress granules in neurodegeneration--lessons learnt from TAR DNA binding protein of 43 kDa and fused in sarcoma. Febs j 280 , 4348-4370 (2013). Jia J , et al. Stress granules and mTOR are regulated by membrane atg8ylation during lysosomal damage. J Cell Biol 221 , (2022). Anisimov S , et al. G3BP1 inhibits ubiquitinated protein aggregations induced by p62 and USP10. Sci Rep 9 , 12896 (2019). Kwon DH , et al. Insights into degradation mechanism of N-end rule substrates by p62/SQSTM1 autophagy adapter. Nat Commun 9 , 3291 (2018). Carroll B , et al. Oxidation of SQSTM1/p62 mediates the link between redox state and protein homeostasis. Nat Commun 9 , 256 (2018). Narayanan D , et al. Development of Noncovalent Small-Molecule Keap1-Nrf2 Inhibitors by Fragment-Based Drug Discovery. Journal of medicinal chemistry 65 , 14481-14526 (2022). Gwon Y, Maxwell BA, Kolaitis RM, Zhang P, Kim HJ, Taylor JP. Ubiquitination of G3BP1 mediates stress granule disassembly in a context-specific manner. Science (New York, NY) 372 , eabf6548 (2021). Yang Y, Luo Y, Yang C, Hu R, Qin X, Li C. TRIM25-mediated ubiquitination of G3BP1 regulates the proliferation and migration of human neuroblastoma cells. Biochimica et biophysica acta Gene regulatory mechanisms 1866 , 194954 (2023). Maxwell BA , et al. Ubiquitination is essential for recovery of cellular activities after heat shock. Science (New York, NY) 372 , eabc3593 (2021). Du ZW , et al. Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells. Nat Commun 6 , 6626 (2015). Additional Declarations There is NO Competing Interest. Supplementary Files Sourcedata.xlsx Source data Supplementaryinformation.pdf Cite Share Download PDF Status: Published Journal Publication published 30 Dec, 2024 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4380078","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":300567738,"identity":"6771e040-b136-472d-9137-b5b2645f973f","order_by":0,"name":"Jin-A Lee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAoElEQVRIiWNgGAWjYBACPiA2YKg4AKTYiNTCBtZyhlQtDIxtJGlhbz5Q8HPenTyDA2xpH4jTwnMswbB327NioJbDM4jTIpFjYMC77XDihgPszUQ6TP79B8O/c0jSIsHDYMzbANLCdphILTxpBsYyxw4nzjzMlkycFn72w88M39QcTuw73mZMnBaQRQZgiploDUC1D0hQPApGwSgYBSMRAAABCC4lXBIxmwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-6757-8371","institution":"Hannam University","correspondingAuthor":true,"prefix":"","firstName":"Jin-A","middleName":"","lastName":"Lee","suffix":""},{"id":300567742,"identity":"7aba16b4-48a3-4e75-817b-ba2e17e3507f","order_by":1,"name":"Pureum Jeon","email":"","orcid":"","institution":"Hannam University","correspondingAuthor":false,"prefix":"","firstName":"Pureum","middleName":"","lastName":"Jeon","suffix":""},{"id":300567744,"identity":"b0860dd9-2685-4b89-80d0-8dfb8e0e0963","order_by":2,"name":"Hyunji Ham","email":"","orcid":"","institution":"Hannam University","correspondingAuthor":false,"prefix":"","firstName":"Hyunji","middleName":"","lastName":"Ham","suffix":""},{"id":300567746,"identity":"998f4d7c-56b7-4ad9-8a02-67e882b0ff52","order_by":3,"name":"Haneul Choi","email":"","orcid":"","institution":"Hannam University","correspondingAuthor":false,"prefix":"","firstName":"Haneul","middleName":"","lastName":"Choi","suffix":""},{"id":300567748,"identity":"e9306a5e-19c5-48ae-8eee-23748e8b10f8","order_by":4,"name":"Semin Park","email":"","orcid":"","institution":"Hannam University","correspondingAuthor":false,"prefix":"","firstName":"Semin","middleName":"","lastName":"Park","suffix":""},{"id":300567750,"identity":"b3163ffd-e242-451e-91d2-4d10cb33944e","order_by":5,"name":"Jae-Woo Jang","email":"","orcid":"","institution":"Hannam University","correspondingAuthor":false,"prefix":"","firstName":"Jae-Woo","middleName":"","lastName":"Jang","suffix":""},{"id":300567752,"identity":"c2dceeec-b6ca-4f77-85e7-98a40ecc5cd1","order_by":6,"name":"Sang-Won Park","email":"","orcid":"","institution":"Kyungpook National University","correspondingAuthor":false,"prefix":"","firstName":"Sang-Won","middleName":"","lastName":"Park","suffix":""},{"id":300567754,"identity":"580501a5-b6d9-4ea1-ab70-9afe9a3bd09a","order_by":7,"name":"Dong-Hyung Cho","email":"","orcid":"","institution":"Kyungpook National University","correspondingAuthor":false,"prefix":"","firstName":"Dong-Hyung","middleName":"","lastName":"Cho","suffix":""},{"id":300567756,"identity":"83b5811d-4470-4eca-8fe6-57ae4767ed44","order_by":8,"name":"Hyun-Jung Lee","email":"","orcid":"","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Hyun-Jung","middleName":"","lastName":"Lee","suffix":""},{"id":300567758,"identity":"7517b49e-5700-4a24-894a-159c06eb1d0c","order_by":9,"name":"Hyun Kyu Song","email":"","orcid":"https://orcid.org/0000-0001-5684-4059","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Hyun","middleName":"Kyu","lastName":"Song","suffix":""},{"id":300567761,"identity":"09357f42-5474-4413-b971-edff52700bec","order_by":10,"name":"Masaaki Komatsu","email":"","orcid":"https://orcid.org/0000-0001-7672-7722","institution":"Juntendo University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Masaaki","middleName":"","lastName":"Komatsu","suffix":""},{"id":300567763,"identity":"865be528-9f10-468c-99ee-4389bdc7797b","order_by":11,"name":"Deok-Jin Jang","email":"","orcid":"","institution":"Kyungpook National University","correspondingAuthor":false,"prefix":"","firstName":"Deok-Jin","middleName":"","lastName":"Jang","suffix":""}],"badges":[],"createdAt":"2024-05-07 04:00:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4380078/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4380078/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-024-55446-w","type":"published","date":"2024-12-30T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":56225796,"identity":"4abb5598-5936-4fa0-98c3-10311367f852","added_by":"auto","created_at":"2024-05-10 06:04:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3333682,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction of NS1-BP with stress granule (SG) components. \u003cstrong\u003ea\u003c/strong\u003e Silver-stained SDS-PAGE gels depict immunoprecipitates from HEK293T cell lysates expressing Flag-NS1-BP. \u003cstrong\u003eb\u003c/strong\u003eThe table shows the interactors with Flag-NS1-BP analyzed using LC-MS/MS. \u003cstrong\u003ec\u003c/strong\u003eImmunoprecipitation and western blot analysis using HEK293T cell lysates expressing Flag-NS1-BP and Myc-hnRNPA2B1 with anti-Flag (or IgG) and anti-Myc antibodies. \u003cstrong\u003ed\u003c/strong\u003e Immunoprecipitation and western blot analysis using HEK293T cell lysates expressing Flag-NS1-BP with anti-Flag (or IgG), anti-Ataxin2, anti-PABP, and anti-hnRNPA1 antibodies. \u003cstrong\u003ee\u003c/strong\u003e Representative images illustrating the cellular localization of NS1-BP with TIA-1 in HeLa cells under sodium arsenite (S.A) (0.5 mM, 1 h) or thapsigargin (T.G) (50 μM, 1 h) treatment conditions. Scale bar, 10 μm. \u003cstrong\u003ef\u003c/strong\u003e Line scan graphs depicting the co-localization of NS1-BP with TIA-1. \u003cstrong\u003eg\u003c/strong\u003e Representative images displaying the results of the proximity ligation assay (PLA) for Ataxin2 with NS1-BP under sodium arsenite (0.5 mM, 1 h) condition. Scale bar, 10 μm. \u003cstrong\u003eh\u003c/strong\u003eBar graph showing the proximity ligation assay dots. Data were quantified with a two-tailed t-test and presented as mean ± SEM (*** p\u0026lt;0.001); Control, n = 18; S.A, n = 17.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4380078/v1/81e292a55a1988c9d97637f8.png"},{"id":56225791,"identity":"84f2630a-5518-4b75-833c-d8ea714af46e","added_by":"auto","created_at":"2024-05-10 06:04:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1750186,"visible":true,"origin":"","legend":"\u003cp\u003eRegulation of stress granule (SG) dynamics by NS1-BP. \u003cstrong\u003ea\u003c/strong\u003e Representative images depicting G3BP1-positive SGs in NS1-BP WT and KO HeLa cells expressing either Flag-vector or Flag-NS1-BP. Scale bar, 10 μm. \u003cstrong\u003eb, c\u003c/strong\u003e Bar graph illustrating the number(b) and size(c) of SGs. Each dots represent the average number or size of SGs per cell. Data were quantified by one-way ANOVA and presented as mean ± SEM (**** p\u0026lt;0.0001); WT, n = 44; WT+NS1-BP, n = 50; KO, n = 71; KO+NS1-BP, n = 61. \u003cstrong\u003ed\u003c/strong\u003e Representative images showing the GFP-G3BP1-containing SGs before and after photobleaching in NS1-BP WT, NS1-BP KO, or Flag-NS1-BP expressing NS1-BP KO HeLa cells. Scale bar, 5 μm. \u003cstrong\u003ee\u003c/strong\u003e The graph displaying normalized GFP-G3BP1 intensity after photobleaching. Data were quantified with a two-tailed t-test and presented as mean ± SEM (*** p\u0026lt;0.001); WT, n = 65; KO, n = 66; KO+NS1-BP, n = 11.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4380078/v1/802b5ecb56fed89af92eec77.png"},{"id":56225800,"identity":"05618988-597f-4690-8898-460a019787f0","added_by":"auto","created_at":"2024-05-10 06:04:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":607108,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction of\u003cstrong\u003e \u003c/strong\u003eNS1-BP with p62 via its kelch motif and UBA domain in p62 in an oxidative stress-dependent manner. \u003cstrong\u003ea\u003c/strong\u003eImmunoprecipitation and western blot using HEK293T cell lysates expressing Flag-NS1-BP or Flag-p62, treated with sodium arsenite (S.A 0.5 mM, 1 h) with anti-Flag (or IgG), anti-p62, and anti-NS1-BP antibodies. \u003cstrong\u003eb\u003c/strong\u003e Schematic model illustrating the constructs of 3xFlag-NS1-BP, 3xFlag-p62, and their deletion mutants (3xFlag-NS1-BP ΔKelch, 3xFlag-NS1-BP ΔBTB/POZ, 3xFlag-p62 ΔPB1, 3xFlag-p62 ΔUBA). \u003cstrong\u003ec\u003c/strong\u003e Immunoprecipitation and western blot using HEK293T cell lysates expressing Flag-NS1-BP WT, Flag-NS1-BP ΔKelch, or Flag-NS1-BP ΔBTB/POZ with anti-Flag (or IgG) and anti-p62 antibodies. \u003cstrong\u003ed\u003c/strong\u003eBar graph showing the normalization of p62. Data were quantified using one-way ANOVA and presented as mean ± SEM (* p\u0026lt;0.05). \u003cstrong\u003ee\u003c/strong\u003e Immunoprecipitation and western blot using HEK293T cell lysates expressing Flag-p62 WT, Flag-p62 ΔUBA, or Flag-p62 ΔPB1 with anti-Flag (or IgG) and anti-NS1-BP antibodies. \u003cstrong\u003ef\u003c/strong\u003e Bar graph showing the normalization of NS1-BP. Data were quantified using one-way ANOVA and presented as mean ± SEM (** p\u0026lt;0.005, * p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4380078/v1/aa7921210ec292d750763606.png"},{"id":56225794,"identity":"158ff1cb-3505-4c80-9c8c-91ad6bc833af","added_by":"auto","created_at":"2024-05-10 06:04:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1599562,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction between p62 and NS1-BP mediated through its UBA domain and KIR motif. \u003cstrong\u003ea\u003c/strong\u003e ColabFold prediction models of the complex depicting NS1-BP (magenta) and p62/SQSTM1 (cyan). Close-up view of the interacting boxed regions in both models is on the right. Rank 1-4 models are shown in the upper model. The NS1-BP interacts with the KIR of p62 (red) whereas the UBA domain (green) in p62 does not interact with NS1-BP in this complex model. Another predicted model of NS1-BP (magenta) and p62 (cyan) ranked fifth in ColabFold prediction (lower in this panel). The UBA domain (green) in p62/SQSTM1 involved in the interaction with NS1-BP whereas the KIR motif (black oval) locate at far from the NS1-BP. \u003cstrong\u003eb\u003c/strong\u003e Predicted local distance difference test (pLDDT) of the ColabFold prediction top5 ranked models. The predicted model of NS1-BP is more accurate than that of p62. \u0026nbsp;\u003cstrong\u003ec\u003c/strong\u003e Immunoprecipitation and western blot using HEK293T cell lysates expressing Flag-p62 WT, Flag-p62 LIR mutant, or Flag-p62 KIR mutant, treated with S.A (0.5 mM, 1 h) with anti-Flag (or IgG) and anti-NS1-BP antibodies. \u003cstrong\u003ed\u003c/strong\u003e Bar graph showing the normalization of NS1-BP. Data were quantified using one-way ANOVA and presented as mean ± SEM. n.s.; nonsignificant.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4380078/v1/ca4f935b64f2cdb775267640.png"},{"id":56225798,"identity":"a8e7c90c-2942-4d3e-848a-864235bbd60b","added_by":"auto","created_at":"2024-05-10 06:04:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":440391,"visible":true,"origin":"","legend":"\u003cp\u003eNegative regulation of p62 ubiquitination by NS1-BP. \u003cstrong\u003ea\u003c/strong\u003eNS1-BP WT and KO HeLa cells were treated with MG132 (10 μM, 6 h) or BafilomycinA1 (100 nM, 6 h) and assayed for western blot using anti-NS1-BP, anti-p62, and anti-β-actin antibodies \u003cstrong\u003eb\u003c/strong\u003e The bar graph represents the normalization of p62 protein levels. \u003cstrong\u003ec\u003c/strong\u003eUbiquitination immunoprecipitation (Ub-IP) and western blot using HEK293T cell lysates expressing GFP or GFP-NS1-BP, treated with sodium arsenite (0.5 mM, 1 h) with anti-Flag (or IgG) and anti-HA antibodies. \u003cstrong\u003ed\u003c/strong\u003e Bar graph showing the normalization of HA-ub. Data were quantified with a two-tailed t-test and presented as mean ± SEM. (* p\u0026lt;0.05); n = 5.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4380078/v1/f143666fcd1982e42fd1469f.png"},{"id":56225795,"identity":"6c8f0504-9986-49cf-b98b-95831a9750da","added_by":"auto","created_at":"2024-05-10 06:04:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1338250,"visible":true,"origin":"","legend":"\u003cp\u003eRegulation of p62 ubiquitination by NS1-BP on Lysine 13, Lysine 420, and Lysine 435. \u003cstrong\u003ea\u003c/strong\u003e Schematic model illustrating p62 ubiquitination sites. \u003cstrong\u003eb, c, d, e, j, k, i, n\u003c/strong\u003eUbiquitination immunoprecipitation (Ub-IP) and western blot using HEK293T cells lysates expressing EGFP or EGFP-NS1-BP and Flag-p62 K7R(b), Flag-p62 K13R(c), Flag-p62 K157R(d), Flag-p62 K165R(e), Flag-p62 K189R(j), Flag-p62 K264R(k), Flag-p62 K420R(i), or Flag-p62 K435R(n) treated with sodium arsenite (0.5 mM, 1 h) with anti-Flag (or IgG), anti-GFP, and anti-HA antibodies. \u003cstrong\u003ef, g, h, I, m, o, p, q.\u003c/strong\u003e Graphs showing the normalization of HA-ub. Data were quantified with a two-tailed t-test and presented as mean ± SEM (**** p\u0026lt;0.0001); n = 5.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4380078/v1/2d216c7d77c9b11fe592d2f8.png"},{"id":56225797,"identity":"763697de-43cc-4c85-b786-8194bc92015d","added_by":"auto","created_at":"2024-05-10 06:04:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4011509,"visible":true,"origin":"","legend":"\u003cp\u003eRegulation of stress granule (SG) dynamics by P62 and GABARAP family proteins. \u003cstrong\u003ea\u003c/strong\u003e Representative images showing the cellular localization of EGFP-G3BP1 in p62 WT and KO MEFs. Cells were transfected with EGFP-G3PB1 and treated with sodium arsenite (0.5 mM, 1 h). Scale bar, 10 μm. \u003cstrong\u003eb, c\u003c/strong\u003e Graphs showing the number (b) and size (c) of SGs per cell. Data were quantified using a two-tailed t-test and presented as mean ± SEM (*** p\u0026lt;0.0001). \u003cstrong\u003ed\u003c/strong\u003eRepresentative images showing G3BP1 positive SGs in NS1-BP KO HeLa cells expressing Flag vector, Flag-p62 WT, Flag-p62 K13R, Flag-p62 K264R, Flag-p62 K420R, or Flag-p62 K435R treated with sodium arsenite (0.5 mM, 1 h). \u003cstrong\u003ee, f\u003c/strong\u003eBar graphs showing the number (e) and size (f) SG per cell. Data were quantified using a one-way ANOVA and presented as mean ± SEM. (***\u0026lt;0.0001). \u003cstrong\u003eg\u003c/strong\u003e Representative images showing G3BP1-positive SGs in GABARAP triple knockout HeLa cells expressing or not expressing Flag-NS1-BP upon sodium arsenite treatment condition. \u003cstrong\u003eh, i\u003c/strong\u003e Bar graphs showing the number(h) and size(i) of SG per cell. Data were quantified using a two-tailed t-test and presented as mean ± SEM (**** p\u0026lt;0.0001); None, n = 81; Flag-NS1-BP, n = 71.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4380078/v1/094a9c95e6d00cfca17acce5.png"},{"id":56225792,"identity":"2f107be4-9722-43c5-888b-8efdb7e9fa65","added_by":"auto","created_at":"2024-05-10 06:04:51","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":5425930,"visible":true,"origin":"","legend":"\u003cp\u003eDysregulation of stress granules (SGs)due to reduced NS1-BP levels in ALS-patient iPSC-derived motor neurons. \u003cstrong\u003ea\u003c/strong\u003eSchematic model illustrating iPSC differentiation to motor neurons. \u003cstrong\u003eb\u003c/strong\u003e Analysis of NS1-BP protein levels through western blot using anti-NS1-BP, anti-beta-actin in CTL-MN and ALS-MN. \u003cstrong\u003ec\u003c/strong\u003e Bar graph indicating the normalization of NS1-BP protein levels. Data were quantified using a two-tailed t-test and presented as mean ± SEM (** p\u0026lt;0.005); n = 5. \u003cstrong\u003ed\u003c/strong\u003eRepresentative images showing NS1-BP and G3BP1-positive SGs in CTL-MN and ALS-MN upon sodium arsenite (0.5 mM, 90 min.) treatment or not. Scale bar, 10 μm. \u003cstrong\u003ee, f\u003c/strong\u003e Bar graphs showing the SG size(e) and number(f). White arrows indicate NS1-BP and G3BP1-positive SGs. Data were quantified using a two-tailed t-test and presented as mean ± SEM (**** p\u0026lt;0.0001); CTL-MN, n = 60; ALS-MN, n =48.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4380078/v1/57d5493d31570bb5804c7870.png"},{"id":72685728,"identity":"c1686a17-519b-42b8-9740-bcbb670cab4d","added_by":"auto","created_at":"2024-12-31 08:15:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":23899325,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4380078/v1/2fabc98f-afdd-488f-8a6e-d5dd6e81ee98.pdf"},{"id":56225799,"identity":"c64ea810-2864-453e-b435-797c6100617f","added_by":"auto","created_at":"2024-05-10 06:04:52","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":24987097,"visible":true,"origin":"","legend":"Source data","description":"","filename":"Sourcedata.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4380078/v1/bd28b956031ffac23f782521.xlsx"},{"id":56225793,"identity":"d3181747-0eb5-497c-9fab-5510c5115533","added_by":"auto","created_at":"2024-05-10 06:04:51","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1855710,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Supplementaryinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4380078/v1/7579d2eaf425d4462a56d88f.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"NS1 binding protein regulates stress granule dynamics and clearance by inhibiting p62 ubiquitination","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAutophagy is cellular recycling, breaking down damaged proteins and organelles for energy and cellular repair\u003csup\u003e1\u003c/sup\u003e. It aids adaptation to stress conditions, including nutrient shortage, oxidative stress, and infection\u003csup\u003e2, 3\u003c/sup\u003e. Regulated by different signals, autophagy maintains cell health, preventing toxic build-up.\u003c/p\u003e \u003cp\u003eConversely, stress granules (SGs) are dynamic structures that form in cell cytoplasm in response to stress, including heat shock, viral infection, or oxidative stress\u003csup\u003e4\u003c/sup\u003e. Comprising RNA and RNA-binding proteins, they store mRNA temporarily, shielding it from degradation. When stress subsides, SGs aid mRNA translation and regulate gene expression and stress response. SGs are rapidly assembled and disassembled by various chaperons, valosin-containing protein (VCP), or RNA helicase\u003csup\u003e5, 6\u003c/sup\u003e. Some specific or prolonged stress conditions also facilitate SG degradation.\u003c/p\u003e \u003cp\u003eOver the past two decades, approximately 100 proteins have been identified in the SG interactome\u003csup\u003e7, 8, 9\u003c/sup\u003e. Some with low complexity regions enable multiple interactions with various binding partners\u003csup\u003e10\u003c/sup\u003e. SGs serve as signalling platforms, aiding the coordination of cellular processes during stress, including apoptosis, cell growth, and metabolic control\u003csup\u003e11\u003c/sup\u003e. Dysregulated SG dynamics can impair RNA metabolism and protein homeostasis\u003csup\u003e12\u003c/sup\u003e, potentially inducing neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS)\u003csup\u003e13, 14\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAutophagy and SGs interact closely. Autophagy, activated during stress, clears damaged or aggregated proteins, including those in SGs\u003csup\u003e2\u003c/sup\u003e, regulating their assembly and disassembly\u003csup\u003e6, 15, 16\u003c/sup\u003e. This interplay enables cells to adapt to stress and maintain cellular homeostasis.\u003c/p\u003e \u003cp\u003eRecent evidence associates SGs with autophagy, suggesting a molecular connection\u003csup\u003e7, 17, 18\u003c/sup\u003e. Autophagy components, including VCP, p62/SQSTM1 (from hereon p62), mammalian ATG8s (mATG8s), and Unc-51-like kinase 1/2 (ULK1/2), indicating their role in SG regulation or clearance. For example, VCP regulates selective autophagy involved in SGs degradation, and mutations in VCP impair autophagy-related functions\u003csup\u003e6, 17\u003c/sup\u003e. Additionally, ULK1/2, associated with autophagy, regulate SG disassembly through phosphorylation and activation of VCP/p97\u003csup\u003e18\u003c/sup\u003e. Moreover, SG homeostasis is influenced by tripartite motif-containing protein 21 (TRIM21)-mediated ubiquitination of Ras-GTPase-activating protein (GAP)-binding protein 1 (G3BP1) and autophagy-dependent SG elimination\u003csup\u003e19\u003c/sup\u003e. Among autophagy regulators of SGs, p62 stands out as a multifunctional protein. It aids in degrading ubiquitinated proteins via autophagy and participates in various signalling pathways such as nutrient sensing, oxidative stress, infection, immunity, and inflammation\u003csup\u003e20\u003c/sup\u003e. Acting as an autophagic receptor/adaptor, p62 interacts with ubiquitinated cargo via its ubiquitin association (UBA) domain and recruits them to the growing autophagosome membrane through its LC3-interacting (LIR) motif\u003csup\u003e21, 22, 23\u003c/sup\u003e. Recent research indicates that p62-droplets also facilitate autophagosome formation and counter oxidative stress\u003csup\u003e24\u003c/sup\u003e. Additionally, the C9ORF72-p62 complex aids in the autophagic clearance of SG via arginine methylation\u003csup\u003e15\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eProteomic analyses have revealed a network of proteins associated with SGs, including mATG8 proteins, such as LC3B and GABARAP, key constituents of these granules\u003csup\u003e7\u003c/sup\u003e. These proteins interact with LIR motif-containing proteins, including p62, acting as adaptors/receptors, and recruit selective cargos or autophagic machinery proteins to autophagosomes during selective autophagy\u003csup\u003e25, 26\u003c/sup\u003e. However, the specific mechanisms by which p62 and mATG8 proteins selectively recognize SGs and regulate autophagic SG clearance remain unclear. Identifying the components in SGs that interact with p62 and mATG8 proteins and how they recruit autophagic machinery for autophagosome formation under various stress conditions is essential. Recent findings indicate that various types of selective autophagy employ receptors/adaptors with LIR motifs to recognize and tether specific substrates to phagophores. Recently, LC3 subfamily- or GABARAP subfamily-selective LIR motifs have been identified, along with several proteins that bind to either the LC3 subfamily or GABARAP subfamily, aiming to characterize their function in selective autophagy\u003csup\u003e27, 28\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTherefore, this study aims to investigate NS1-BP, a protein characterized as containing an LIR motif that specifically interacts with GABARAP subfamily proteins; however, not with LC3 subfamily proteins. Co-immunoprecipitation (co-IP) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis revealed NS1-BP interaction with SG components, including Ataxin2, Poly(A)-binding protein (PABP), T-cell-restricted intracellular antigen-1 (TIA-1), and G3BP1. NS1-BP was observed to localize to GABARAP-containing SGs during oxidative stress. Furthermore, it strongly associates with p62, acting as an adaptor/receptor for selective autophagy in an oxidative stress-dependent manner. NS1-BP KO cells demonstrated increased SG size and decreased SG numbers, akin to observation in p62 KO cells or GABARAP triple KO (TKO) cells, suggesting impaired autophagic clearance. Additionally, NS1-BP KO cells showed increased p62 ubiquitination, leading to p62 autophagic degradation, while NS1-BP overexpression reduced p62 ubiquitination. Additionally, wild-type (WT) p62 expression, not p62 mutants (K420R or K435R), rescued p62 ubiquitination and the cellular phenotype in NS1-BP KO cells. NS1-BP KO cells showed reduced contact between ubiquitinated p62 bodies/aggregates and SGs. Neurons from derived ALS-induced pluripotent stem cells (iPSCs) showed reduced NS1-BP levels, resulting in SG dysregulation. These findings underscore the important role of NS1-BP in p62- and GABARAP-mediated SG regulation, shedding light on the molecular mechanisms of selective autophagic degradation of SGs and the cellular pathogenesis of ALS associated with SGs dysregulation.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eNS1-BP, characterized as a GABARAP subfamily-binding protein, regulates SGs number, size, and dynamics\u003c/h2\u003e \u003cp\u003eIdentifying and studying proteins that bind to the LC3 subfamily- and GABARAP subfamily is crucial for understanding selective autophagy, as many receptors and adaptors involved possess LIR motifs. To accomplish this, a search for proteins containing LIR motifs using the iLIR database was performed (A web resource for LIR motif-containing proteins in eukaryotes) (Supplementary Table\u0026nbsp;1)\u003csup\u003e29\u003c/sup\u003e. Several LIR motif-containing proteins were found to potentially bind to the GABARAP subfamily. Among these, NS1-BP, a Kelch family protein, was identified as having a potential LIR motif within its second Kelch motif (Supplementary Fig.\u0026nbsp;1a). NS1-BP is recognized for its interactions with the influenza virus non-structural protein 1 and regulates viral or host RNA processing/export\u003csup\u003e30\u003c/sup\u003e, cancer progression\u003csup\u003e31\u003c/sup\u003e, and regulation of neurite/dendritic spines\u003csup\u003e32\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo investigate the binding properties of the LIR motifs from NS1-BP for each mATG8 protein, a previously developed \u003cem\u003ein vitro\u003c/em\u003e assay was utilized\u003csup\u003e27\u003c/sup\u003e. Since NS1-BP functions as a dimer through its BTB/POZ domain,\u003csup\u003e33\u003c/sup\u003e, duplicated LIR motif of NS1-BP (2xLIR[NS1-BP]) fused with mRFP-3x nuclear localization signal (2xLIR[NS1-BP]-mRFP-3xNLS), along with EGFP-tagged mATG8 mutants EGFP-mATG8(GA) in HeLa cells were expressed. In these mutants, the C-terminal glycine residue was replaced with alanine to impair phosphatidylethanolamine (PE) conjugation (lipidation), inhibiting cellular localization to autophagosomes. The assay revealed that when 2xLIR(NS1-BP)-mRFP-3xNLS interacted with specific EGFP-mATG8(GA), cytosolic EGFP-mATG8(GA) was sequestered into the nucleus based on its binding preference in live cells (Supplementary Fig.\u0026nbsp;1b, c). EGFP-GABARAP(GA), GABARAPL1(GA), and GABARAPL2(GA) were localized to the nucleus, indicating that the 2xLIR motifs from NS1-BP preferentially bind to GABARAP subfamily proteins rather than LC3 subfamily proteins. In contrast, the 2xLIR mutant(NS1-BP[AA]) was diffusely localized to the nucleus and cytoplasm, suggesting a LIR-dependent binding pattern (Supplementary Fig.\u0026nbsp;1b, c). Further, GST-pulldown assay using HeLa cell lysates supported the interaction between endogenous NS1-BP and GABARAP subfamily proteins (Supplementary Fig.\u0026nbsp;1d), suggesting its possible role in stress-induced responses such as autophagy.\u003c/p\u003e \u003cp\u003eTo elucidate NS1-BP functional roles in the stress response pathway associated with autophagy, co-IP and LC-MS/MS analyses were performed using the HEK293T cell lysates expressing FLAG-NS1-BP under oxidative stress induced by sodium arsenite. The silver stained SDS-PAGE analysis of elutes showed 35, 60, and 70 kDa in NS1-BP samples but not in control IgG samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The co-IP and LC-MS/MS analysis results showed major binding partners, including heat shock protein A12B (HSPA12B) from the HSP70 family, mitochondrial heat shock protein 75 (MTHSP75) belonging to the HSP90 family, and heterogeneous nuclear ribonucleoprotein K (hnRNPK) and hnRNPA2B1 from the heterogeneous nuclear ribonucleoproteins family (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). These results are significant because all of the identified proteins are involved in the stress response pathway. Among them, hnRNPA2B1 or hnRNPK is known to localize to SGs, and mutations in these proteins have been related to neurodegenerative diseases including ALS and membrane scaffold protein (MSP)\u003csup\u003e34\u003c/sup\u003e or Au\u0026ndash;Kline syndrome with multiple malformation syndrome\u003csup\u003e35\u003c/sup\u003e. Co-IP experiments confirmed that FLAG-NS1-BP interacts with MYC-hnRNPA2B1 in HEK293T cells under oxidative stress conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Furthermore, additional co-IP experiments using HEK293T cell lysates expressing FLAG-NS1-BP revealed interactions with core SGs components, including ataxin2, PABP, and hnRNPA1, upon oxidative stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). NS1-BP predominantly colocalizes with TIA-1-containing SGs under oxidative stress or endoplasmic reticulum stress conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, f). Proximity ligation assays (PLA) further demonstrated an increased association of NS1-BP with Ataxin2 under oxidative stress, suggesting its potential role in the stress response pathway associated with SGs regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, h).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the physiological roles of NS1-BP in regulating SGs, NS1-BP KO HeLa cells were generated using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 technology, confirmed by genomic polymerase chain reaction (PCR) (Supplementary Fig.\u0026nbsp;2b). Immunoblot and immunocytochemistry analyses confirmed the absence of NS1-BP protein in the NS1-BP KO cells than in WT cells (Supplementary Fig.\u0026nbsp;2d, e). Subsequently, SG number and size in WT and NS1-BP KO HeLa cells were assessed. A significant reduction in SGs number in NS1-BP KO HeLa cells compared to WT HeLa cells was found, accompanied by increased size under oxidative stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c). Expression of FLAG-NS1-BP rescued the size increase in NS1-BP KO HeLa cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c). Furthermore, fluorescence recovery after photobleaching (FRAP) analysis showed significantly reduced dynamics of G3BP1-positive SGs in NS1-BP KO HeLa cells compared to WT HeLa cells, which was restored upon NS1-BP re-introduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, e), indicating the regulatory role of NS1-BP in SG dynamics. Overall, these results show that NS1-BP, characterized as a GABARAP subfamily-binding protein, localizes to SGs and modulates their size, number, or dynamics during oxidative stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eNS1-BP is strongly associated with p62 via kelch motif in NS1-BP and UBA domain in p62 in an oxidative stress-dependent manner\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo understand how NS1-BP regulates the size, number, or dynamics of SGs during oxidative stress, it is essential to consider SG disassembly mechanisms. Typically, after stress cessation, SGs disassemble through various kinds of chaperons, VCP, and RNA helicase\u003csup\u003e36\u003c/sup\u003e. However, under prolonged stress, SGs can also be selectively degraded by autophagy. Recent studies indicate that autophagy components, including mATG8s or p62, localize to SGs, with p62 playing a role in SG clearance\u003csup\u003e15, 37\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo investigate the interaction between NS1-BP and GABARAP subfamily proteins, their association in HEK293T cells expressing GFP-GABARAP subfamily proteins under oxidative stress conditions was examined. Supplementary Fig.\u0026nbsp;3a shows that the association between NS1-BP and GABARAP subfamily proteins was oxidative stress-dependent. Additionally, under oxidative stress conditions, NS1-BP predominantly co-localized with GABARAP subfamily-containing SGs, suggesting its potential role in SGs regulation (Supplementary Fig.\u0026nbsp;3b, c). In selective autophagy, mATG8s binds to p62, a selective autophagy adaptor, via the LIR motif to recruit ubiquitinated substrates into the autophagosomal membrane\u003csup\u003e38\u003c/sup\u003e. Therefore, NS1-BP was investigated to determine if it could associate with p62 under oxidative stress using HEK293T cell lysates expressing FLAG-NS1-BP or FLAG-p62. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea shows that NS1-BP is strongly associated with p62 in an oxidative stress-dependent manner. NS1-BP only interacts with p62 under oxidative stress conditions and not under other stressors such as thapsigargin, sorbitol, or heat shock (Supplementary Fig.\u0026nbsp;4a).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe domain involved in the association between NS1-BP and p62 during oxidative stress was identified. NS1-BP contains a BTB/POZ domain and Kelch motif, which forms a β-propeller structure\u003csup\u003e33\u003c/sup\u003e. Conversely, p62 contains multiple binding domains, including the N-terminal Phox-Bem1p (PB1) and C-terminal UBA domains\u003csup\u003e20\u003c/sup\u003e. PB1 is known to facilitate p62 oligomerization, while UBA is crucial for p62 multimerization and ubiquitination, essential for its function in sequestering ubiquitinated substrates. Therefore, co-IP experiments with anti-FLAG antibodies were performed using deletion mutants of NS1-BP or p62 (FLAG-NS1-BP, FLAG-NS1-BP ΔKelch, FLAG-NS1-BP ΔBTB/POZ, FLAG-p62, FLAG-p62 ΔUBA, or FLAG-p62 ΔPB1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-f shows that the interaction between endogenous p62 and FLAG-NS1-BP ΔKelch or between endogenous NS1-BP and FLAG-p62 ΔUBA was significantly reduced, indicating that the Kelch domain in NS1-BP and UBA domain in p62 are necessary for their association. Additionally, the association between FLAG-p62 ΔPB1 and NS1-BP was slightly reduced, suggesting that PB1 oligomerization might partially contribute to their association.\u003c/p\u003e \u003cp\u003eThe binding of NS1-BP to p62 may not occur in their monomeric state under na\u0026iuml;ve conditions; however, to get the structural insight of the association of NS1-BP with p62, their binding regions were predicted using ColabFold modeling\u003csup\u003e39\u003c/sup\u003e. The prediction results showed two possibilities (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). The kelch motif in NS1-BP was predicted to directly interact with the Kelch-like ECH-associated protein 1 (Keap1)-interacting region (KIR) in p62 as shown in the Keap1-p62 structure\u003csup\u003e40\u003c/sup\u003e. Additionally, the UBA domain in p62 was also predicted to be in close proximity to the kelch motif in NS1-BP as a different model, suggesting a possible association of p62 with NS1-BP, probably facilitated by post-translational modification such as phosphorylation or ubiquitination.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the predicted association sites identified through the ColabFold modeling, it was clarified whether the KIR motif in p62 could interact with NS1-BP upon oxidative stress condition. However, the co-IP results using a FLAG-p62 KIR mutant showed that the interaction between the KIR mutant and NS1-BP was not significantly affected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d). This suggests that the kelch domain of NS1-BP strongly associates with the UBA domain in p62 under oxidative stress rather than the Keap1-KIR type interaction, suggesting that NS1-BP may modulate p62 function via the UBA domain in p62 under oxidative stress.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNS1-BP plays a negative regulatory role in p62 ubiquitination at lysine 13, 420, 435 in p62 and autophagic degradation under oxidative stress\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSubsequently, the role of NS1-BP on the function of p62 during oxidative stress was queried. Accumulating evidence showed that during oxidative stress, p62 interacts with Keap1-Cul3, activating nuclear factor erythroid 2-related factor 2 (Nrf2), which in turn induces the expression of antioxidant-response genes\u003csup\u003e41, 42\u003c/sup\u003e. Lysine 420 within the UBA domain of p62 is responsible for its ubiquitination by Keap1/Cul3, and ubiquitination of the UBA domain increases p62 degradation by facilitating its sequestration into autophagic vacuoles\u003csup\u003e43\u003c/sup\u003e. Additionally, the report shows that NS1-BP (called KLHL39) inhibits KLHL20-mediated substrate ubiquitination by Cul3 and substrate degradation\u003csup\u003e31\u003c/sup\u003e. Therefore, it was hypothesized that NS1-BP might regulate p62 ubiquitination by Keap1/Cul3 and its degradation. To test this hypothesis, the protein level of p62 was initially assessed in WT and NS1-BP KO cells. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-b shows that the protein level of p62 was significantly reduced in NS1-BP KO cells, which was rescued by inhibiting autophagy with bafilomycin A1, suggesting that NS1-BP negatively regulates the autophagic degradation of p62. Subsequently, the effect of NS1-BP on the ubiquitination of p62 was examined. NS1-BP KO cells showed increased p62 ubiquitination than the WT cells, while its overexpression in WT cells reduced p62 ubiquitination, further supporting the hypothesis that NS1-BP inhibits p62 ubiquitination and degradation during oxidative stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo pinpoint the critical residues within p62 responsible for NS1-BP-mediated inhibition of p62 ubiquitination among the lysine residues essential for p62 ubiquitination, site-specific mutations were introduced by substituting lysine with arginine at lysine 7, 13, 165, 264, 420, and 435 in p62\u003csup\u003e38, 43, 44\u003c/sup\u003e. Ubiquitination IP using HA-Ubiquitin was performed in FLAG-p62 WT, K7R, K13R, K165R, K189R, K264R, K420R, or K435R expressing HEK293T cells. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows that although the expression of p62 lysine mutants K7R, K165R, K189R, or K264R significantly reduced p62 ubiquitination, similar to the observation with p62 WT, K13R expression, a lysine mutant within the PB1 domain, and K420R or K435R, lysine mutants in the UBA domain, did not reduce p62 ubiquitination as observed with p62 WT.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, the effect of the lysine mutant on the association of NS1-BP with p62 was investigated. The co-IP showed that compared to p62 WT, the p62 lysine mutant K420R reduced its association with NS1-BP, highlighting the significance of p62 lysine 420 located within the UBA domain for its interaction with NS1-BP. However, the association of NS1-BP with other p62 lysine mutants did not show any significant change compared to p62 WT (Supplementary Fig.\u0026nbsp;4b). Collectively, these findings reveal that NS1-BP inhibits p62 ubiquitination and autophagic degradation, and lysine residues 13, 420, or 435 in p62 are crucial for its inhibitory effect on p62 ubiquitination under oxidative stress conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eReduced levels of p62 in NS1-BP KO HeLa cells impaired clearance of SGs\u003c/h2\u003e \u003cp\u003eTo further examine whether the reduced protein level of p62 in NS1-BP KO cells is responsible for the dysregulation of SGs, the number and size of SGs in p62 KO mouse embryonic fibroblasts (MEFs) were assessed. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-c shows the observed increase in SGs size and a decrease in their number in p62 KO MEFs, resembling the cellular phenotype observed in NS1-BP KO HeLa cells. To ascertain whether the decreased level of p62 contributes to the altered dynamics of SGs in NS1-BP KO HeLa cells, p62 WT or p62 lysine mutants were reintroduced into NS1-BP KO HeLa cells. The expression of p62 WT, K13R, or K264R in NS1-BP KO HeLa cells significantly restored the number and size of SGs, while p62 K420R or K435R did not show any change (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed-f). These results show that p62 K420 or K435 play key roles in ubiquitination and subsequent degradation of p62 in NS1-BP KO HeLa cells, thereby contributing to SGs dysregulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven that NS1-BP interacts not only with p62 but also with GABARAP family proteins, it was further investigated whether NS1-BP modulates GABARAP-mediated autophagic SGs degradation. Therefore, the number and size of SGs were examined in GABARAP TKO HeLa or HEK293T cells expressing a selective deconjugase for GABARAP/L1/L2-PE\u003csup\u003e28\u003c/sup\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg-i and Supplementary Fig.\u0026nbsp;5a-d show that GABARAP/L1/L2-deficient cells exhibited an increase in SG size and a decrease in the number of SGs compared to WT or other LC3A/B-PE deconjugase expressing cells, resembling the phenotype observed in NS1-BP KO cells. Additionally, it was found that HeLa cells expressing siRNA targeting ATG16L1 showed a similar increase in SG size and decrease in the number of SGs, akin to observation in NS1-BP KO cells, p62 KO MEFs, and GABARAP TKO HeLa cells (Supplementary Fig.\u0026nbsp;5e-g). This indicates that autophagic degradation of SGs may be impaired in NS1-BP KO cells owing to the reduced protein level of p62, potentially caused by increased p62 ubiquitination leading to its degradation.\u003c/p\u003e \u003cp\u003eFinally, cellular localization of p62, ubiquitin, SGs, GABARAP in WT and NS1-BP KO cells were examined to determine whether ubiquitinated p62 are properly targeted to SGs and recruits autophagosomal membrane (Supplementary Fig.\u0026nbsp;6). The co-localization of ubiquitinated G3BP1-positive SGs with GABARAP-containing autophagosome was significantly decreased in NS1-BP KO cells compared to WT cells. This suggests that NS1-BP may play a role in the proper targeting of SGs into GABARAP-containing SG structures, possibly through the ubiquitination of p62.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNS1-BP was reduced in patients with ALS (TDP43 A382T) iPSC-derived motor neurons, leading to SG dysregulation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe dysregulation of SGs is increasingly recognized as being associated with various neurodegenerative diseases. In the case of ALS, sporadic and familial cases often involve motor neurons experiencing oxidative stress and exhibiting abnormal accumulation of protein aggregates, including TDP-43. Therefore, it was initially investigated whether deficiency of NS1-BP also causes SG dysregulation in post-mitotic neurons. Supplementary Fig.\u0026nbsp;7 shows that cultured cortical neurons expressing NS1-BP shRNA demonstrated increased size and decreased number of SGs. These findings support the potential role of NS1-BP in the cellular pathogenesis of ALS associated with SGs.\u003c/p\u003e \u003cp\u003eTo investigate the protein level of NS1-BP and its relationship with SGs dysregulation in ALS, patients with ALS-derived motor neurons generated from induced pluripotent stem cells (iPSCs) were examined (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). The findings revealed a significant reduction in NS1-BP protein levels in patients with ALS derived motor neurons compared to control motor neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb, c). Furthermore, an increase in SG size and a decrease in SG number was observed, indicating SG dysregulation in patients with ALS-derived motor neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed-f). These results were consistent with the results obtained from NS1-BP KO HeLa cells and NS1-BP deficient post-mitotic cortical neurons. Generally, this study provides insights into the cellular mechanisms underlying SG dysregulation in ALS and highlights the potential pathogenic role of NS1-BP in regulating SGs in motor neurons derived from patients with ALS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSGs are dynamic cytoplasmic structures formed in response to various stressors such as oxidative stress, heat shock, or viral infection. They temporarily sequester mRNAs and RNA-binding proteins to halt normal translation\u003csup\u003e4\u003c/sup\u003e, playing a crucial role in modulating responses to cellular stress and being implicated in the pathogenesis of diverse conditions, such as neurodegenerative diseases, ageing, and cancer\u003csup\u003e14, 45, 46, 47\u003c/sup\u003e. Recent studies have shed light on the relationship between SG dynamics and autophagy\u003csup\u003e6, 19, 37\u003c/sup\u003e. However, the interplay between autophagy and SGs, particularly the modulation by post-translational modifications of significant proteins, remains a vital but partially understood aspect of cellular stress response and homeostasis.\u003c/p\u003e \u003cp\u003eThis study reveals that NS1-BP, a protein previously recognised for its interactions with the influenza A virus non-structural protein NS1, plays a crucial role in regulating SG dynamics by modulating the autophagic machinery. The interaction of NS1-BP with GABARAP and p62\u0026mdash;key components in autophagic processes\u0026mdash;suggests a novel pathway through which SG dynamics are controlled under oxidative stress. This study provides the first evidence that the interaction of NS1-BP with p62 significantly affects the ubiquitination and protein level of p62, influencing its ability to regulate SG clearance via autophagy. The reduction in NS1-BP levels observed in patients with ALS-derived motor neurons, along with corresponding changes in SG morphology and dynamics, suggests the potential involvement of NS1-BP in ALS pathogenesis. This aligns with emerging evidence suggesting that impaired autophagy and defective SG dynamics contribute to the accumulation of protein aggregates, a hallmark of ALS and other neurodegenerative disorders. These studies propose several potential mechanisms by which NS1-BP influences SG and autophagy dynamics.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eInteraction of NS1-BP with GABARAP subfamily proteins\u003c/h2\u003e \u003cp\u003eThe presence of an LIR motif within the second Kelch motif suggests its potential interaction with autophagy-related proteins, including LC3 and GABARAP, a member of the ATG8 family involved in autophagosome formation and selective autophagy. This analysis indicates that NS1-BP selectively binds to GABARAP subfamily proteins but not to LC3 proteins, indicating its specific role in certain autophagic pathways. This selective binding suggests that NS1-BP may influence the choice of autophagic routes employed by cells during the stress response, potentially favouring those involving GABARAP proteins. GABARAP subfamily proteins are characterized in the SG interactome\u003csup\u003e7\u003c/sup\u003e. GABARAPs directly interact with NUFIP2 and G3BP1, with Atg8ylation being necessary for their recruitment to damaged lysosomes\u003csup\u003e48\u003c/sup\u003e. Furthermore, the interaction between GABARAP and G3BP1 promotes the recruitment of SGs into autophagosomes, resulting in their degradation. This indicates that GABARAP, through its involvement in autophagy, can indirectly influence SG dynamics and clearance. Additionally, p62 interacts with SG components such as TIA-1 or G3BP1 and regulates their assembly, disassembly, or clearance\u003csup\u003e37, 49\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis data suggests NS1-BP may influence the recruitment of cargo, including damaged organelles or protein aggregates, to autophagosomes by modulating GABARAP function or localization. Given that NS1-BP regulates the size, number, and dynamics of SGs, its interaction with GABARAP affects the autophagic degradation of SGs. This process seems crucial during cellular stress recovery, where SGs need to be efficiently cleared to restore cellular homeostasis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStructural analysis of NS1-BP interaction with p62\u003c/h2\u003e \u003cp\u003ep62 comprises several domains, including a PB1 domain for oligomerization, zinc finger (ZZ) domain for N-degron recognition\u003csup\u003e50\u003c/sup\u003e, TRAF6-binding domain, LIR motif for interaction with LC3 and GABARAP subfamily proteins in autophagy, KIR, and UBA domain at its C-terminus, which is crucial for binding polyubiquitin chains\u003csup\u003e20\u003c/sup\u003e. NS1-BP, a Kelch family protein, features a BTB/POZ domain facilitating its dimerization, along with Kelch repeats that form a β-propeller structure\u003csup\u003e33\u003c/sup\u003e. It is well characterized that the β-propeller of Kelch repeats in the Keap1 recognize the KIR segments in the p62\u003csup\u003e40\u003c/sup\u003e and the high-ranked ColabFold models for 1:1 complex between NS1-BP and p62 were predicted (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). However, their interaction was only restricted to an oxidative stress condition, which is an oligomeric state of p62\u003csup\u003e51\u003c/sup\u003e. Therefore, we are tempting to speculate another complex model that the UBA domain of p62 contributes to the interaction with NS1-BP protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, rank 5) because the IP analysis indicates that the Kelch motifs of NS1-BP and UBA domain of p62 are crucial for this interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-f). These results imply a specific binding affinity between NS1-BP and p62 alters the p62 behaviour under oxidative stress conditions. Small molecule inhibitors to the Kelch domain of Keap1 have been developed to treat diseases involving oxidative stress and inflammation\u003csup\u003e52\u003c/sup\u003e. Therefore, the detailed structural analysis of NS1-BP interactions with p62 and GABARAP may elucidate the precise molecular interfaces and post-translational modifications influencing their interactions, providing insights for the design of targeted therapeutics.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eNegative regulation of p62 ubiquitination by NS1-BP on autophagy\u003c/h2\u003e \u003cp\u003eUbiquitination plays a crucial role in regulating the dynamics and function of SGs under stress conditions. Various SG proteins, including G3BP1, PABP, or FUS, undergo ubiquitination, directly influencing their role and the stability of SGs\u003csup\u003e19, 53, 54, 55\u003c/sup\u003e. The clearance of SGs through autophagy, particularly granulophagy, relies on the ubiquitination of SG components\u003csup\u003e53\u003c/sup\u003e. Several studies have demonstrated that ubiquitination of p62 at specific lysine residues, including K7 and K420, serves as a signal for selective cargo recognition, resulting in its sequestration into autophagosomes for degradation\u003csup\u003e38, 43\u003c/sup\u003e. Moreover, p62 itself can be ubiquitinated and targeted for degradation through autophagy, contributing to SG clearance and cellular homeostasis. Despite its significance, the fine-tuning mechanism for regulating p62 ubiquitination remains poorly understood.\u003c/p\u003e \u003cp\u003eIn this study, a novel role of NS1-BP was characterised, which negatively regulates the ubiquitination of p62, a crucial factor in autophagic degradation. By modulating p62 ubiquitination, NS1-BP affects the level and availability of p62 protein for autophagy, thereby influencing SG dynamics. This regulation is pivotal, as excessive or insufficient p62 levels can disrupt cellular balance and contribute to disease pathology. NS1-BP, through its modulation of p62, plays a role in this pathway. The interaction between p62 and GABARAP, facilitated by NS1-BP, seems essential for targeting SGs to autophagosomes. In NS1-BP deficient scenarios, the impaired interaction between p62 and the autophagic machinery leads to inadequate SG clearance, contributing to cellular stress and potentially initiating neurodegenerative changes. These findings indicate that the interactions of NS1-BP with both GABARAP and p62 suggest a coordinated role in associating autophagy with SG dynamics. By interacting with these proteins, NS1-BP may help orchestrate the balance between SG formation as a protective mechanism under stress and their clearance through autophagy once the stress is resolved.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRegulation of SG dynamics by NS1-BP\u003c/h3\u003e\n\u003cp\u003eThrough its interaction with p62 and GABARAP, NS1-BP influences SG dynamics by modulating their assembly and disassembly processes. These findings demonstrate that NS1-BP deficiency results in increased SG size and decreased number, resembling the effects observed with alterations in p62 and GABARAP levels. These findings underscore the complex interplay between SG formation, autophagy, and the cellular stress response. However, the exact mechanisms by which NS1-BP influences SG dynamics remain incompletely understood. NS1-BP may influence SGs through its interactions with other SG-associated proteins or by participating in cellular signalling pathways. Exploring NS1-BP's interactome, especially with other autophagy and SG components, may reveal additional roles and regulatory mechanisms.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eImplications of the role of NS1-BP in neurodegenerative diseases\u003c/h2\u003e \u003cp\u003eUsing motor neurons derived from iPSCs of patients with ALS, we a significant reduction in NS1-BP levels and observed alterations in SG dynamics (increased size, decreased number) compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These findings corroborate observations from NS1-BP deficient models, strengthening the hypothesis that NS1-BP plays a regulatory role in SG dynamics. This study connects NS1-BP, a protein implicated in RNA processing and cellular stress response, to the pathological features of ALS, particularly through its involvement in SG regulation in neurons.\u003c/p\u003e \u003cp\u003eThe dysregulation of SG dynamics and autophagic pathways are common features in neurodegenerative diseases. NS1-BP interaction with p62, modulating SG dynamics and autophagy, is significant for understanding diseases, including ALS. Targeting the NS1-BP-p62 interaction and its downstream effects on SG dynamics and autophagy offers a novel therapeutic avenue for conditions with SG accumulation and autophagy dysfunction. Modulating NS1-BP levels or imitating its action may restore SG and autophagic function, potentially alleviating disease symptoms or progression. This study enhances understanding of ALS pathogenesis, shedding light on targeting cellular stress responses to mitigate disease progression.\u003c/p\u003e \u003cp\u003eDysregulation in autophagy and SG dynamics is associated with neurodegenerative disorders and cancers. Understanding how NS1-BP modulates these processes through its interactions with GABARAP and p62 may reveal molecular mechanisms and therapeutic targets. Further research into the mechanisms and interactions of NS1-BP is crucial for developing targeted therapies for these conditions.\u003c/p\u003e \u003c/div\u003e "},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eCell lines and growth conditions\u003c/h2\u003e \u003cp\u003eHEK293T and HeLa cells were obtained from ATCC. To generate NS1-BP KO HeLa cells, cluster regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing was utilized to disrupt the genes encoding human NS1-BP in HeLa cells. The CRISPR/Cas9 system, consisting of CMV-Cas9-EF1a-puromycin/GFP-U6 optimized for cell transfection, was sourced from YSY Biotech Company Ltd (Nanjing, China). To develop the double-nicking NS1-BP-sgRNA-guided CRISPR/Cas9 plasmids, a pair of oligos were designed and subcloned into AflII-digested gRNA_Cloning Vector (Addgene 41824). These plasmids were co-transfected with pCas9-GFP (Addgene 44719) into HeLa cells using lipofectamine 2000 (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA). HeLa cells transfected with pCas9-GFP/NS1-BP two gRNAs were selected with puromycin, and single colonies were isolated. To generate cells stably expressing either 3xFlag-p62 WT or K420R, NS1-BP KO HeLa cells were infected with lentiviral particles and grown in a medium containing 10 \u0026micro;g/ml polybrene (Sigma Aldrich H9268) for 48 h. Stably expressing cells were selected with puromycin, and single colonies were isolated.\u003c/p\u003e \u003cp\u003eAll cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin in a humidified atmosphere containing 5% (v/v) of CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDNA constructs\u003c/h2\u003e \u003cp\u003eNS1-BP was PCR-amplified from a human cDNA library and inserted into the pEGFP-C1 vector using BglII and BamHI restriction enzymes or into the 3xFlag-CMV7.1 vector through EcoRI and BamHI restriction enzymes. GST-LC3A, GST-LC3B, GST-LC3C, GST-GABARAP, GST-GABARAL1, and GST-GABARAPL2 were obtained from Addgene (Cambridge, USA). Additionally, p62 was PCR-amplified from an HA-p62 construct from Addgene and inserted into the 3xFlag-CMV7.1 vector through EcoRI and KpnI restriction enzymes.\u003c/p\u003e \u003cp\u003eTo generate domain deletion or mutant constructs, p62 (ΔPB1), p62 (ΔUBA), p62 (KIR mutant), p62 (LIR mutant), p62 K7R, p62 K13R, p62 K157R, p62 K165R, p62 K189R, p62 K264R, p62 K420R, p62 K435R, p62 K420Q, p62 K435Q, p62 K420/435Q, NS1-BP (ΔKelch), and NS1-BP (ΔBTB/POZ) were PCR-amplified with specific primers (Supplementary Table\u0026nbsp;2) and inserted into the 3xFlag-CMV7.1 vector through EcoRI or BglII (for 5\u0026rsquo; region) and KpnI or BamHI for (3\u0026rsquo; region) restriction enzymes. Additionally, p62 K13R, p62 K165R, and p62 K435R were amplified by PCR with specific primers and inserted into the 3xFlag-CMV7.1 vector using the infusion cloning method (Takara Bio 639648). To further generate p62 lentiviral constructs, 3xFlag-p62 WT and 3xFlag-p62 K420R were amplified by PCR using specific primers and inserted into the pLVX-EF1a-IRES-Puro (Takara Bio 631988).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLentivirus production\u003c/h2\u003e \u003cp\u003eTo produce lentiviruses for infection, Lenti-X 293T (Takara Bio 632180) cells were co-transfected with pLVX-EF1a-3xFlag-NS1-BP, p62 WT or K420R, psPAX2, and pMD2.G using the CalPhos Mammalian Transfection Kit (Takara Bio 631312). Culture supernatant was collected at 48 h and 72 h post-transfection and passed through a 0.45-\u0026micro;m filter. The viral particles were concentrated by ultracentrifugation (22,600 x g for 3 h) and resuspended in Dulbecco\u0026rsquo;s phosphate-buffered saline (DPBS).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTransfection, gene silencing, and drug treatment\u003c/h2\u003e \u003cp\u003eTo transiently express DNA constructs, cells were transfected with plasmid DNA using either the calcium phosphate method or FuGENE HD (Promega E2311) following the instruction of the manufacturer. Knockdown experiments in mouse cortical neurons were performed using Lipofectamine 2000 (Thermo Fisher Scientific 11668-019) and the shRNA construct following the instructions of the manufacturer. To induce SG formation, cells were treated with sodium arsenite (S.A 0.5 mM, 1 h), thapsigargin (T.G 20 uM, 1 h), sorbitol (0.4 M, 2 h) or heat shock (42 ℃, 2 h).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblot analysis\u003c/h2\u003e \u003cp\u003eProtein samples were separated through SDS-PAGE, transferred to PVDF membranes (Millipore IPVH00010) and incubated with primary antibodies overnight at 4\u0026deg;C. After washing with TBST (150 mM NaCl, 20 mM Tris-HCl (pH 7.4), 0.05% Tween 20), membranes were exposed to secondary antibodies conjugated with horseradish peroxidase for 1 h. Signals were detected with ECL solution (Millipore, WBKLS0500, USA). Antibodies used included NS1-BP (Santa Cruz sc-373909, 1:100), Flag (Sigma F1804, 1:10,000), p62 (Abnova H00008878-M01, 1:10,000), GABARAPL1 (Cell Signalling 26632, 1:1,000), G3BP1 (Proteintech 13057-2-AP, 1:1,000), Ataxin2 (Proteintech 21776-1-AP, 1:5,000), hnRNPA1(Abcam ab137780, 1:2000), PABP (Abcam ab21060, 1:1,000), HA (Cell Signalling 3724, 1:1000), GFP (Neuromab 75\u0026ndash;131, 1:10,000), and Beta-actin (Sigma A5441, 1:10,000). Quantification was performed using ImageJ (NIH) software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eImmunocytochemistry and confocal microscopy\u003c/h2\u003e \u003cp\u003eFor immunostaining, transfected cells were washed with PBS, then fixed and permeabilized with methanol (Samchun) for 30 min at -20℃. After blocking with 3% bovine serum albumin for 1 h at room temperature, cells were incubated overnight at 4\u0026deg;C with anti-TIA-1 (Santa Cruz sc-1751), anti-G3BP1 (Proteintech 13057-2-AP), and anti- Flag (Sigma F1804). Subsequently, they were treated with anti-mouse or anti-rabbit secondary antibodies (Jackson Laboratory) for 2 h at room temperature. Following three washes with 1X PBS, the cells were mounted on glass slides and analysed using an LSM 880 microscope (Cal Zeiss, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSilver staining and LC-MS/MS analysis\u003c/h2\u003e \u003cp\u003eSilver staining was performed using PageSilver\u0026trade; Silver staining Kit (Thermo Fisher Scientific K0681) following the instruction of the manufacturer. LC-MS/MS analysis was performed at the Yonsei Proteomic Research Centre (Seoul, Korea).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eGST affinity isolation assay\u003c/h2\u003e \u003cp\u003eFor the GST affinity-isolation assay, HeLa cells were washed with PBS, harvested, and lysed in GST affinity isolation buffer solution (50 mM Tris-HCl pH7.6, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, supplemented with protease inhibitor cocktail). After centrifugation at 13,000 x g for 20 min, the cleared cell lysates were incubated overnight with purified GST-mATG8 proteins and glutathione-conjugated agarose beads (Sigma Aldrich G4510) at 4℃. The following day, the samples were washed three to five times with the same GST affinity isolation buffer solution at 4℃, and the remaining supernatant was removed. The samples were resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, boiled immediately, and subjected to SDS-PAGE with Coomassie Brilliant Blue (Sigma Aldrich 1154440025) staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eCo-IP\u003c/h2\u003e \u003cp\u003eCells were collected and lysed with 1% NP40 lysis buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 2 mM EDTA, 1% NP40), supplemented with protease inhibitor (Sigma P8340) and phosphatase inhibitor (Thermo Fisher Scientific 78426) on ice for 30 min. After centrifugation at 13,000 rpm at 4℃ for 15 min, the supernatant fractions were collected and incubated with protein A/G-agarose beads (Santa Cruz sc-2002) for 2 h at 4℃. This step served as a pre-clearing step, with the supernatants containing 1 mg of protein incubated overnight at 4℃ with appropriate antibodies. To precipitate the target protein, the supernatants were incubated with protein A/G agarose bead for 7 h at 4℃. Afterwards, the beads were washed with lysis buffer three times by centrifugation at 3,000 rpm at 4℃. The immunoprecipitated proteins were separated via SDS-PAGE, and western blotting was performed as previously described.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003ePLA\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ein situ\u003c/em\u003e PLA was performed using a Duolink \u003cem\u003ein situ\u003c/em\u003e kit (Sigma DUO92101) following the instruction of the manufacturer. Initially, cells were cultured on Matrigel-coated glass. After sodium arsenite treatment, cells were fixed and permeabilized using 100% methanol and then blocked with a blocking solution. Subsequently, cells were incubated with primary antibodies (NS1-BP, Santa Cruz sc-373909, 1:50; Ataxin-2, Proteintech 21776-1-AP, 1:100; p62, Abnova H00008878-M01, 1:100; Flag, Sigma F1804, 1:100), diluted in antibody diluent overnight at 4℃. Following this, secondary antibodies (PLA probe anti-mouse MINUS and PLA probe anti-rabbit PLUS) were applied for 1 h at 37℃. Cells were treated with ligase for 30 min at 37℃ and amplified with polymerase diluted in amplification buffer for 100 min at 37℃.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003ep62 ubiquitination IP\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCells were transfected with either 3xFlag-p62 WT or ubiquitination mutant (K7R, K13R, K157R, K165R, K189R, K264R, K420R, or K435R) along with HA-ub constructs, and either EGFP or EGFP-NS1-BP. After 24 h, cells were treated with MG132 (1 uM, 16 h) to inhibit proteasome. Cells were lysed with 1% SDS denaturing buffer (1% SDS, 50 mM Tris-HCl pH7.6, 150 mM NaCl) and supplemented with protease inhibitors. Lysates were boiled for 10 min and disrupted with gentle sonication. Additionally, these samples were diluted 1:20 with 1% Triton X-100 lysis buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100). Subsequent steps were consistent with the co-IP method described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eFRAP analysis\u003c/h2\u003e \u003cp\u003eHeLa cells expressing EGFP-G3BP1 were initially activated by imaging every 4 s for 2.5 min using a 100% 488-nm laser power and 100-ms exposure time to SGs. Subsequently, the EGFP-G3BP1-positive SGs were photobleached, and the EGFP signal intensity was measured before and after photobleaching.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eStructure prediction\u003c/h2\u003e \u003cp\u003eThe predicted complex model between NS1-BP and p62/SQSTM1 was obtained by using ColabFold\u003csup\u003e39\u003c/sup\u003e. Five ranked models were obtained for the NS1-BP and p62 complex, and the binding modes of these complexes were classified into two categories. PyMOL software was utilized to visualize the models (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pymol.org/\u003c/span\u003e\u003cspan address=\"https://pymol.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eiPSC generation from control and patients with ALS-derived primary fibroblast\u003c/h2\u003e \u003cp\u003eControl and ALS iPSC lines were reprogrammed from fibroblasts obtained from the Coriell Institute for Medical Research (NINDS collection) using a non-integrating induction method with OSKM factors. The fibroblasts were cultured at 37\u0026deg;C in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (Corning) supplemented with 15% FBS (Corning) and 1% penicillin-streptomycin (Gibco).\u003c/p\u003e \u003cp\u003eFor gene delivery, electroporation (Invitrogen\u0026trade; Neon\u0026trade; Transfection System) was performed. Seven days after electroporation, the cells were transferred onto MEF feeder cells with iPSC maintenance medium containing 20% KO serum (Gibco 10828028), β-mercaptoethanol (Gibco 21985023), Gluta-MAX (Gibco 35050-061), MEM-Non Essential Amino Acids solution (Gibco 11140050), penicillin/streptomycin (Hyclone SV30010), and DMEM-F12 (Gibco 11320033). After 4 weeks, iPSC-like colonies were picked and cultured on new feeder cells. Furthermore, at an iPSC passage number of 5\u0026ndash;7, the culture system was transitioned to a feeder-free system using a truncated recombinant human vitronectin (Gibco A14700) coated plate with Essential 8 Medium (Gibco A1517001).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eMotor neuron differentiation\u003c/h2\u003e \u003cp\u003eTo generate highly pure motor neurons, a previous method was slightly modified\u003csup\u003e56\u003c/sup\u003e. To induce differentiation into neuronal precursor cells (NPC), iPSCs were dissociated using Accutase (Innovative Cell Technologies AT104) and seeded at a 1:5 ratio on Matrigel-coated plates. On the following day, the iPSC medium was replaced with a chemically defined neural medium (NEP media), including KO DMEM-F12 (Gibco 12660-012) and Neurobasal medium (Gibco 21103-049) at 1:1, 0.5x N2 (Gibco 17502-048), 0.5x B-27 (Gibco 17504-044), 1x GlutaMax supplements (Gibco 35050-061), 1x penicillin\u0026ndash;streptomycin (Hyclone SV30010), 100\u0026micro;M Ascorbic acid (Sigma A4403), 3\u0026micro;M CHIR99021 (Stem Cell 72054), 2\u0026micro;M DMH-1 (Stem Cell 73634), and 2\u0026micro;M SB4315429 (Stem cell 72234). The culture medium was altered every other day. To induce motor neuron patterning (MNP), NPC cells were dissociated with Accutase and seeded at a 1:3 on Matrigel-coated plates with NEP medium supplemented with 0.1-\u0026micro;M Retionic acid (Sigma R2625) and 0.5 \u0026micro;M Puromorphamine (Sigma 540220) 7 days after seeding. The culture medium was altered every other day. For motor neuron differentiation, MNP cells were dissociated with Accutase and cultured in suspension in the NEP medium supplemented with 0.5 \u0026micro;M Retionic acid and 0.1\u0026micro;M Puromorphamine 7 days after MNP induction. The culture medium was altered every other day. To mature the motor neuron, MND cells were dissociated into single cells with Accutase and plated on mouse glial cells in a motor neuron mature medium. The mature medium include Neurobasal medium, 1x B-27 supplement, 1x GlutaMax, 1x penicillin-streptomycin (Gibco), 100 \u0026micro;M Ascorbic acid (Sigma), 2.6 \u0026micro;g/ml Adenosine 3',5'-cyclic monophoaphate (Sigma A9501), 0.5 \u0026micro;M Compound E (Stem cell 73954), 10 \u0026micro;g/ml Laminin (Sigma L2020), 10 \u0026micro;g/ml BDNF (Thermo Fisher Scientific 450-02-50UG), and 10 \u0026micro;g/ml GDNF (Thermo Fisher Scientific PHC7041). Medium refreshment with motor neuron mature medium occurred weekly for a total maturation period of 5 weeks.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.\u003c/p\u003e \u003c/div\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe author(s) declare no competing interests\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eP.J. conducted the majority of the experiments, analyzed the data, and contributed to manuscript drafting. H.H., H.C., S.P., J-W.J., S-W.P. conducted and assisted with IP experiments. D-H.C.\u0026nbsp;contributed to the interpretation of results and edited the manuscript.\u003c/p\u003e\n\u003cp\u003eH.-J.L. analyzed protein interactions using ColabFold modeling. H.K.S. designed the experiment and wrote the manuscript. K.M. contributed to the interpretation of results and edited the manuscript. D-J.J. \u0026amp; J-A.L. conceptualized the study, designed the experiments, and wrote the manuscript. All authors reviewed the manuscript, made necessary corrections, and approved the final version for publication. Consent for publication was obtained from each author.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe work was supported by the Science Research Center Program of the National Research Foundation NRF (Grant No. 2020R1A5A1019023, J.-A L.); Neurological Disorder Research Program of the NRF (Grant No. 2020M3E5D9079911, J.-A L.); Basic research program of the NRF (Grant No. 2023R1A2C2008092, J.-A L.). This work was also supported by JSPS KAKENHI (Grant No. JP19H05706; JP21H004771; 23K20044; 24H00060, M.K.); AMED (Grant No. JP22gm1410004h0003, M.K.) and by the Takeda Science Foundation to M.K.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGlick D, Barth S, Macleod KF. Autophagy: cellular and molecular mechanisms. \u003cem\u003eJ Pathol\u003c/em\u003e \u003cstrong\u003e221\u003c/strong\u003e, 3-12 (2010).\u003c/li\u003e\n\u003cli\u003eMurrow L, Debnath J. Autophagy as a stress-response and quality-control mechanism: implications for cell injury and human disease. \u003cem\u003eAnnu Rev Pathol\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 105-137 (2013).\u003c/li\u003e\n\u003cli\u003eBuchan JR, Yoon JH, Parker R. Stress-specific composition, assembly and kinetics of stress granules in Saccharomyces cerevisiae. \u003cem\u003eJ Cell Sci\u003c/em\u003e \u003cstrong\u003e124\u003c/strong\u003e, 228-239 (2011).\u003c/li\u003e\n\u003cli\u003eAnderson P, Kedersha N. Stress granules. \u003cem\u003eCurr Biol\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, R397-398 (2009).\u003c/li\u003e\n\u003cli\u003eWheeler JR, Matheny T, Jain S, Abrisch R, Parker R. Distinct stages in stress granule assembly and disassembly. \u003cem\u003eElife\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, (2016).\u003c/li\u003e\n\u003cli\u003eBuchan JR, Kolaitis RM, Taylor JP, Parker R. Eukaryotic stress granules are cleared by autophagy and Cdc48/VCP function. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e153\u003c/strong\u003e, 1461-1474 (2013).\u003c/li\u003e\n\u003cli\u003eMarkmiller S\u003cem\u003e, et al.\u003c/em\u003e Context-Dependent and Disease-Specific Diversity in Protein Interactions within Stress Granules. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e172\u003c/strong\u003e, 590-604 e513 (2018).\u003c/li\u003e\n\u003cli\u003eIsabelle M, Gagne JP, Gallouzi IE, Poirier GG. Quantitative proteomics and dynamic imaging reveal that G3BP-mediated stress granule assembly is poly(ADP-ribose)-dependent following exposure to MNNG-induced DNA alkylation. \u003cem\u003eJ Cell Sci\u003c/em\u003e \u003cstrong\u003e125\u003c/strong\u003e, 4555-4566 (2012).\u003c/li\u003e\n\u003cli\u003eAmen T, Kaganovich D. Stress granules inhibit fatty acid oxidation by modulating mitochondrial permeability. \u003cem\u003eCell Rep\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 109237 (2021).\u003c/li\u003e\n\u003cli\u003eMolliex A\u003cem\u003e, et al.\u003c/em\u003e Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e163\u003c/strong\u003e, 123-133 (2015).\u003c/li\u003e\n\u003cli\u003eKedersha N, Ivanov P, Anderson P. Stress granules and cell signaling: more than just a passing phase? \u003cem\u003eTrends Biochem Sci\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 494-506 (2013).\u003c/li\u003e\n\u003cli\u003ePortz B, Lee BL, Shorter J. FUS and TDP-43 Phases in Health and Disease. \u003cem\u003eTrends Biochem Sci\u003c/em\u003e \u003cstrong\u003e46\u003c/strong\u003e, 550-563 (2021).\u003c/li\u003e\n\u003cli\u003eBaron DM\u003cem\u003e, et al.\u003c/em\u003e Amyotrophic lateral sclerosis-linked FUS/TLS alters stress granule assembly and dynamics. \u003cem\u003eMol Neurodegener\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 30 (2013).\u003c/li\u003e\n\u003cli\u003eJeon P, Lee JA. Dr. Jekyll and Mr. Hyde? Physiology and Pathology of Neuronal Stress Granules. \u003cem\u003eFront Cell Dev Biol\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 609698 (2021).\u003c/li\u003e\n\u003cli\u003eChitiprolu M\u003cem\u003e, et al.\u003c/em\u003e A complex of C9ORF72 and p62 uses arginine methylation to eliminate stress granules by autophagy. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 2794 (2018).\u003c/li\u003e\n\u003cli\u003eRyu HH\u003cem\u003e, et al.\u003c/em\u003e Autophagy regulates amyotrophic lateral sclerosis-linked fused in sarcoma-positive stress granules in neurons. \u003cem\u003eNeurobiol Aging\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 2822-2831 (2014).\u003c/li\u003e\n\u003cli\u003eSeguin SJ\u003cem\u003e, et al.\u003c/em\u003e Inhibition of autophagy, lysosome and VCP function impairs stress granule assembly. \u003cem\u003eCell Death Differ\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 1838-1851 (2014).\u003c/li\u003e\n\u003cli\u003eWang B\u003cem\u003e, et al.\u003c/em\u003e ULK1 and ULK2 Regulate Stress Granule Disassembly Through Phosphorylation and Activation of VCP/p97. \u003cem\u003eMol Cell\u003c/em\u003e \u003cstrong\u003e74\u003c/strong\u003e, 742-757 e748 (2019).\u003c/li\u003e\n\u003cli\u003eYang C\u003cem\u003e, et al.\u003c/em\u003e Stress granule homeostasis is modulated by TRIM21-mediated ubiquitination of G3BP1 and autophagy-dependent elimination of stress granules. \u003cem\u003eAutophagy\u003c/em\u003e, 1-18 (2023).\u003c/li\u003e\n\u003cli\u003eBerkamp S, Mostafavi S, Sachse C. Structure and function of p62/SQSTM1 in the emerging framework of phase separation. \u003cem\u003eFEBS J\u003c/em\u003e \u003cstrong\u003e288\u003c/strong\u003e, 6927-6941 (2021).\u003c/li\u003e\n\u003cli\u003eBjorkoy G\u003cem\u003e, et al.\u003c/em\u003e p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. \u003cem\u003eJ Cell Biol\u003c/em\u003e \u003cstrong\u003e171\u003c/strong\u003e, 603-614 (2005).\u003c/li\u003e\n\u003cli\u003eKomatsu M\u003cem\u003e, et al.\u003c/em\u003e Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e131\u003c/strong\u003e, 1149-1163 (2007).\u003c/li\u003e\n\u003cli\u003ePankiv S\u003cem\u003e, et al.\u003c/em\u003e p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e282\u003c/strong\u003e, 24131-24145 (2007).\u003c/li\u003e\n\u003cli\u003eKageyama S\u003cem\u003e, et al.\u003c/em\u003e p62/SQSTM1-droplet serves as a platform for autophagosome formation and anti-oxidative stress response. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 16 (2021).\u003c/li\u003e\n\u003cli\u003eLippai M, Low P. The role of the selective adaptor p62 and ubiquitin-like proteins in autophagy. \u003cem\u003eBiomed Res Int\u003c/em\u003e \u003cstrong\u003e2014\u003c/strong\u003e, 832704 (2014).\u003c/li\u003e\n\u003cli\u003eLiu WJ\u003cem\u003e, et al.\u003c/em\u003e p62 links the autophagy pathway and the ubiqutin-proteasome system upon ubiquitinated protein degradation. \u003cem\u003eCell Mol Biol Lett\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 29 (2016).\u003c/li\u003e\n\u003cli\u003ePark SW\u003cem\u003e, et al.\u003c/em\u003e Monitoring LC3- or GABARAP-positive autophagic membranes using modified RavZ-based probes. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 16593 (2019).\u003c/li\u003e\n\u003cli\u003ePark SW\u003cem\u003e, et al.\u003c/em\u003e Development of new tools to study membrane-anchored mammalian Atg8 proteins. \u003cem\u003eAutophagy\u003c/em\u003e, 1-20 (2022).\u003c/li\u003e\n\u003cli\u003eJacomin A-C, Samavedam S, Promponas V, Nezis IP. iLIR database: A web resource for LIR motif-containing proteins in eukaryotes. \u003cem\u003eAutophagy\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 1945-1953 (2016).\u003c/li\u003e\n\u003cli\u003eWolff T, O\u0026apos;Neill RE, Palese P. NS1-Binding protein (NS1-BP): a novel human protein that interacts with the influenza A virus nonstructural NS1 protein is relocalized in the nuclei of infected cells. \u003cem\u003eJ Virol\u003c/em\u003e \u003cstrong\u003e72\u003c/strong\u003e, 7170-7180 (1998).\u003c/li\u003e\n\u003cli\u003eChen HY\u003cem\u003e, et al.\u003c/em\u003e KLHL39 suppresses colon cancer metastasis by blocking KLHL20-mediated PML and DAPK ubiquitination. \u003cem\u003eOncogene\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 5141-5151 (2015).\u003c/li\u003e\n\u003cli\u003eFujii R, Takumi T. TLS facilitates transport of mRNA encoding an actin-stabilizing protein to dendritic spines. \u003cem\u003eJ Cell Sci\u003c/em\u003e \u003cstrong\u003e118\u003c/strong\u003e, 5755-5765 (2005).\u003c/li\u003e\n\u003cli\u003eSasagawa K\u003cem\u003e, et al.\u003c/em\u003e Identification of Nd1, a novel murine kelch family protein, involved in stabilization of actin filaments. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e277\u003c/strong\u003e, 44140-44146 (2002).\u003c/li\u003e\n\u003cli\u003eKim HJ\u003cem\u003e, et al.\u003c/em\u003e Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e495\u003c/strong\u003e, 467-473 (2013).\u003c/li\u003e\n\u003cli\u003eAu PYB\u003cem\u003e, et al.\u003c/em\u003e Phenotypic spectrum of Au-Kline syndrome: a report of six new cases and review of the literature. \u003cem\u003eEur J Hum Genet\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 1272-1281 (2018).\u003c/li\u003e\n\u003cli\u003eProtter DSW, Parker R. Principles and Properties of Stress Granules. \u003cem\u003eTrends Cell Biol\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 668-679 (2016).\u003c/li\u003e\n\u003cli\u003eZheng Y\u003cem\u003e, et al.\u003c/em\u003e HDAC6, A Novel Cargo for Autophagic Clearance of Stress Granules, Mediates the Repression of the Type I Interferon Response During Coxsackievirus A16 Infection. \u003cem\u003eFront Microbiol\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 78 (2020).\u003c/li\u003e\n\u003cli\u003ePeng H\u003cem\u003e, et al.\u003c/em\u003e Ubiquitylation of p62/sequestosome1 activates its autophagy receptor function and controls selective autophagy upon ubiquitin stress. \u003cem\u003eCell Res\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 657-674 (2017).\u003c/li\u003e\n\u003cli\u003eMirdita M, Schutze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M. ColabFold: making protein folding accessible to all. \u003cem\u003eNat Methods\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 679-682 (2022).\u003c/li\u003e\n\u003cli\u003eKomatsu M\u003cem\u003e, et al.\u003c/em\u003e The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1. \u003cem\u003eNature cell biology\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 213-223 (2010).\u003c/li\u003e\n\u003cli\u003eJain A\u003cem\u003e, et al.\u003c/em\u003e p62/SQSTM1 is a target gene for transcription factor NRF2 and creates a positive feedback loop by inducing antioxidant response element-driven gene transcription. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e285\u003c/strong\u003e, 22576-22591 (2010).\u003c/li\u003e\n\u003cli\u003eIchimura Y\u003cem\u003e, et al.\u003c/em\u003e Phosphorylation of p62 activates the Keap1-Nrf2 pathway during selective autophagy. \u003cem\u003eMol Cell\u003c/em\u003e \u003cstrong\u003e51\u003c/strong\u003e, 618-631 (2013).\u003c/li\u003e\n\u003cli\u003eLee Y, Chou TF, Pittman SK, Keith AL, Razani B, Weihl CC. Keap1/Cullin3 Modulates p62/SQSTM1 Activity via UBA Domain Ubiquitination. \u003cem\u003eCell Rep\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 188-202 (2017).\u003c/li\u003e\n\u003cli\u003eFeng L\u003cem\u003e, et al.\u003c/em\u003e Sirt1 deacetylates and stabilizes p62 to promote hepato-carcinogenesis. \u003cem\u003eCell Death Dis\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 405 (2021).\u003c/li\u003e\n\u003cli\u003eGao B\u003cem\u003e, et al.\u003c/em\u003e Inhibition of anti-viral stress granule formation by coronavirus endoribonuclease nsp15 ensures efficient virus replication. \u003cem\u003ePLoS Pathog\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, e1008690 (2021).\u003c/li\u003e\n\u003cli\u003eJayabalan AK\u003cem\u003e, et al.\u003c/em\u003e Stress granule formation, disassembly, and composition are regulated by alphavirus ADP-ribosylhydrolase activity. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e118\u003c/strong\u003e, (2021).\u003c/li\u003e\n\u003cli\u003eBentmann E, Haass C, Dormann D. Stress granules in neurodegeneration--lessons learnt from TAR DNA binding protein of 43 kDa and fused in sarcoma. \u003cem\u003eFebs j\u003c/em\u003e \u003cstrong\u003e280\u003c/strong\u003e, 4348-4370 (2013).\u003c/li\u003e\n\u003cli\u003eJia J\u003cem\u003e, et al.\u003c/em\u003e Stress granules and mTOR are regulated by membrane atg8ylation during lysosomal damage. \u003cem\u003eJ Cell Biol\u003c/em\u003e \u003cstrong\u003e221\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003eAnisimov S\u003cem\u003e, et al.\u003c/em\u003e G3BP1 inhibits ubiquitinated protein aggregations induced by p62 and USP10. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 12896 (2019).\u003c/li\u003e\n\u003cli\u003eKwon DH\u003cem\u003e, et al.\u003c/em\u003e Insights into degradation mechanism of N-end rule substrates by p62/SQSTM1 autophagy adapter. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 3291 (2018).\u003c/li\u003e\n\u003cli\u003eCarroll B\u003cem\u003e, et al.\u003c/em\u003e Oxidation of SQSTM1/p62 mediates the link between redox state and protein homeostasis. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 256 (2018).\u003c/li\u003e\n\u003cli\u003eNarayanan D\u003cem\u003e, et al.\u003c/em\u003e Development of Noncovalent Small-Molecule Keap1-Nrf2 Inhibitors by Fragment-Based Drug Discovery. \u003cem\u003eJournal of medicinal chemistry\u003c/em\u003e \u003cstrong\u003e65\u003c/strong\u003e, 14481-14526 (2022).\u003c/li\u003e\n\u003cli\u003eGwon Y, Maxwell BA, Kolaitis RM, Zhang P, Kim HJ, Taylor JP. Ubiquitination of G3BP1 mediates stress granule disassembly in a context-specific manner. \u003cem\u003eScience (New York, NY)\u003c/em\u003e \u003cstrong\u003e372\u003c/strong\u003e, eabf6548 (2021).\u003c/li\u003e\n\u003cli\u003eYang Y, Luo Y, Yang C, Hu R, Qin X, Li C. TRIM25-mediated ubiquitination of G3BP1 regulates the proliferation and migration of human neuroblastoma cells. \u003cem\u003eBiochimica et biophysica acta Gene regulatory mechanisms\u003c/em\u003e \u003cstrong\u003e1866\u003c/strong\u003e, 194954 (2023).\u003c/li\u003e\n\u003cli\u003eMaxwell BA\u003cem\u003e, et al.\u003c/em\u003e Ubiquitination is essential for recovery of cellular activities after heat shock. \u003cem\u003eScience (New York, NY)\u003c/em\u003e \u003cstrong\u003e372\u003c/strong\u003e, eabc3593 (2021).\u003c/li\u003e\n\u003cli\u003eDu ZW\u003cem\u003e, et al.\u003c/em\u003e Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 6626 (2015).\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":"Stress granules, ubiquitin, p62/SQSTM1, GABARAP, autophagy, amyotrophic lateral sclerosis","lastPublishedDoi":"10.21203/rs.3.rs-4380078/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4380078/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNS1 binding protein (NS1-BP), a non-structural NS1-binding protein of influenza A virus, regulates viral or host RNA processing/export, cancer progression, or neurite/dendritic spine regulation. However, its precise roles in stress-induced responses without viral infection are largely unknown. Therefore, this study aims to investigate the novel roles of NS1-BP, which interact with GABARAP subfamily proteins, including LC3-interacting region-containing proteins, in regulating stress granules (SGs) during oxidative stress. NS1-BP interacts with core SG components and localizes to GABARAP-containing SGs during oxidative stress. Moreover, it associates with p62, acting as an adaptor for selective autophagy via its Kelch-motif and ubiquitin-associated domain in p62 in a stress-dependent manner. NS1-BP knockout (KO) HeLa cells demonstrated altered SG dynamics, mirroring observation in p62 KO or GABARAP triple KO cells, indicating impaired autophagic SG degradation. NS1-BP KO cells, compared to wild-type (WT) cells, showed increased p62 ubiquitination, leading to autophagic p62 degradation, while NS1-BP overexpression reduces p62 ubiquitination. In NS1-BP KO cells, overexpression of p62 WT, not p62 K420R or K435R, restored SGs size and number. Additionally, amyotrophic lateral sclerosis (ALS)-induced pluripotent stem cell-derived motor neurons showed reduced NS1-BP levels, resulting in SG morphology dysregulation. Our findings reveal the novel role of NS1-BP in negatively regulating p62 ubiquitination, influencing SG dynamics and clearance during oxidative stress. This highlights its relevance to ALS pathogenesis associated with SGs.\u003c/p\u003e","manuscriptTitle":"NS1 binding protein regulates stress granule dynamics and clearance by inhibiting p62 ubiquitination","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-10 06:04:45","doi":"10.21203/rs.3.rs-4380078/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":"2c37646e-437f-4f94-9a5e-9f559bbfce5b","owner":[],"postedDate":"May 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":31713439,"name":"Biological sciences/Cell biology"},{"id":31713441,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2024-12-31T08:15:06+00:00","versionOfRecord":{"articleIdentity":"rs-4380078","link":"https://doi.org/10.1038/s41467-024-55446-w","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2024-12-30 05:00:00","publishedOnDateReadable":"December 30th, 2024"},"versionCreatedAt":"2024-05-10 06:04:45","video":"","vorDoi":"10.1038/s41467-024-55446-w","vorDoiUrl":"https://doi.org/10.1038/s41467-024-55446-w","workflowStages":[]},"version":"v1","identity":"rs-4380078","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4380078","identity":"rs-4380078","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}