{"paper_id":"32592ebb-63c9-4ddb-8bb2-c7958d82e78b","body_text":"1 \n \nCathepsin C–Catalyzed Ligation Generates \nIntralysosomal Amyloid Fibrils from Dipeptide Esters \nRuben D. Elias1*, Robert T. O’Neill1, Idil I. Demiralp1, Seth Allen1, Daniel Serwas2, Hannah \nSiems2, Elizabeth A. Montabana2, Carl Ash1, Daniella A. Yacoubian1, Oren L. Lederberg1, \nBenjamin F. Cravatt1, David A. Agard2,3, Jeffery W. Kelly1* 5 \n  \n1Department of Chemistry, The Scripps Research Institute; La Jolla, CA, USA \n \n2Biohub, San Francisco; Redwood City, CA, USA \n 10 \n3Department of Biochemistry & Biophysics, University of California; San Francisco, CA, USA \n \n*Corresponding authors. Email: relias@scripps.edu, jkelly@scripps.edu \n \nAbstract: 15 \nAmyloid fibril-associated endolysosomal dysfunction is implicated in multiple \nneurodegenerative diseases. We report the rapid generation of intralysosomal amyloid fibrils by \nsimply treating cells with certain dipeptide methyl esters. Cathepsin C mediates the ligation of \ndipeptides into oligopeptides that, sequence-dependently, self-assemble into amyloid fibrils. \nProgressive fibril growth, not fibril deposition, mediates lysosomal membrane permeabilization. 20 \nCryo-electron tomography studies reveal intralysosomal fibrils and broken lysosomal \nmembranes upon dipeptide treatment. Certain oligopeptide fibril structures are competent to \ncross-seed the aggregation of neurodegeneration-associated Tau(P301S) at lysosomal sites. \nSimilarly, the degree of lysosomal membrane permeabilization and ESCRT-repair response can \nbe tuned with dipeptide sequence variation. The presented Cathepsin C-dependent amyloid fibril 25 \nformation approach lends itself toward the development of novel tools to further probe lysosomal \nbiology and pathobiology. \n \n \n  30 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 25, 2025. ; https://doi.org/10.64898/2025.12.23.696283doi: bioRxiv preprint \n\n \n2 \n \nThe causal relationship between autolysosome dysfunction and the onset and progression of \nneurodegenerative diseases has become a major research focus (1-6). A prion-like transmission \nof amyloid burden from affected to unaffected cells is proposed to drive pathology of certain \namyloidoses, including tauopathies and synucleopathies (7, 8), where endocytosis of \nextracellular amyloid seeds, followed by their eventual endolysosomal breakout, mediates 5 \nseeding (9-13). Accordingly, autolysosomal deacidification and markers for lysosomal \nmembrane damage are observed in murine and neuronal models of Alzheimer’s disease (14, 15). \nEndolysosomal amyloid fibril breakout is thought to be mediated by the membranolytic \nproperties of amyloid assemblies (16). Yet, cellular models of this endolysosomal breakout are \nimprecise, consisting of supplementing cell culture medium with preformed amyloid fibrils and 10 \nexposing cells for extended periods (typically 1-2 days) before observing a given effect (9, 12, \n17), resulting in heterogeneity in the number of affected cells and in subcellular localization of \nthe endocytosed amyloid at a given timepoint. \n \nL-leucyl-L-leucine methyl ester (LLOMe, Fig. 1A) has emerged as a routine biochemical reagent \n15 \nto induce mild to acute lysosomal membrane damage upon addition to cell culture media, \nfacilitating the investigation of cellular lysosomal repair mechanisms (18-25). The mechanism of \naction of LLOMe has previously been characterized to involve its ligation into membranolytic \n(Leu-Leu)n-OH and (Leu-Leu)n-OMe oligopeptides (hereafter designated (Leu-Leu)n, where \nn=2-3 under previous experimental conditions (26)). (Leu-Leu)n oligomer formation was 20 \nidentified to be catalyzed by the incompletely characterized ligase activity of the lysosomal \ndipeptidase cathepsin C (CTSC)(27-29). Despite widespread use of LLOMe as a lysosome \ndamaging agent, the mechanistic basis of (Leu-Leu)n membranolytic function has historically \nbeen unclear. \n 25 \n(Leu-Leu)n oligopeptides self-assemble into cross β-sheet amyloid fibrils \n \nDue to their high hydrophobicity, low solubility, and high β-strand propensity, we hypothesized \nthat (Leu-Leu)n oligopeptides would spontaneously self-assemble into amyloid fibrils. \nCombining CTSC and LLOMe in phosphate buffer (pH 6.5) resulted in the formation of a 30 \nprecipitate. Liquid chromatography–mass spectrometry (LC-MS) analysis of the solubilized \nprecipitate revealed the presence of (Leu-Leu)1-4 oligopeptides, primarily (Leu-Leu)3 (Fig. 1B), \nin agreement with a previous study (26).  To probe structural order within the precipitate, we \nconducted an X-ray diffraction study, revealing diffraction rings at 11.5 and 4.8 Å (Fig. 1C), \nhallmarks of a cross-β-sheet amyloid fibril structure (30). Additionally, negative stain electron 35 \nmicroscopy revealed rod-like assemblies in the reaction mixture (Fig. 1D). Collectively, these \nresults suggest that CTSC-catalyzed ligation of LLOMe affords (Leu-Leu)3-4 oligopeptides, that, \nwhen they exceed their critical concentration, rapidly self-assemble to form new or add onto \nexisting amyloid fibrils (Fig. 1E). \n 40 \nIn U-2 OS cells, LLOMe treatment resulted in visibly expanded LAMP1-positive endolysosomes \n(Fig. S1A), possibly due to the genesis of lumenal amyloid fibrils. To probe amyloid content \nwithin endolysosomes, cells were stained with the amyloid-binding dye AmyTracker (31), \nresulting in robust staining within endolysosomes (Fig. 1F). We observed similar results using \namyloid-binding fluorophores thioflavin T and Proteostat (Fig. S1B, C). Additionally, the \n45 \nnuclear stain Hoechst 33442, characterized as amyloid-sensitive (32, 33), faintly stained \nextranuclear puncta colocalizing with AmyTracker and Proteostat (Fig. S1D, E). The \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 25, 2025. ; https://doi.org/10.64898/2025.12.23.696283doi: bioRxiv preprint \n\n \n3 \n \nAmyTracker signal was not derived from LLOMe-induced lysosomal deacidification, as \nlysosome deacidification using bafilomycin A1 alone yielded no AmyTracker puncta (Fig S1F). \nPretreatment with the CTSC inhibitor AZD5248, which forms a covalent bond with the active \nsite Cys234 residue (34), ablated LLOMe-induced AmyTracker staining and endolysosomal \nexpansion (Fig. S1G), confirming CTSC activity is responsible for the generation of amyloid \n5 \nfibrils. AmyTracker puncta were predominantly encircled by CHMP4B, a marker for ESCRT-\nmediated lysosomal repair (Fig. 1G)(18, 19), suggesting that amyloid formation causes \nlysosomal damage. Overall, we provide in vitro and in-cell evidence for the spontaneous amyloid \nfibril formation of CTSC-derived (Leu-Leu)n oligopeptides, and that these intralysosomal \namyloid fibrils mediate lysosomal damage.  10 \n \nAfter amyloidogenesis, remnant (Leu-Leu)\nn amyloid fibrils are membranolytically inert \n \nLLOMe-induced lysosomal damage is commonly carried out in a ‘reversible’ manner by \nremoving LLOMe after a given treatment window (19, 35). Accordingly, we hypothesized that \n15 \n(Leu-Leu)n amyloid fibrils would be short lived. In agreement with previous results (18), we \nobserved that after brief LLOMe treatment, CHMP4B was rapidly recruited to endolysosomes \nand dissipated within 180 minutes after LLOMe washout (Fig. S2); however endolysosomes \nremained swollen at this timepoint. Strikingly, 6 hours after LLOMe washout, cells were still \nrobustly amyloid laden (AmyTracker), with puncta dimly present even after 12 hours (Fig. 1H, \n20 \nI). This suggests that the membranolytic activity of (Leu-Leu)n amyloid fibrils only occurs \nduring active fibril growth (amyloidogenesis), and that after LLOMe washout, previously formed \n(Leu-Leu)n amyloid fibrils are membranolytically inert and are cleared within endolysosomes \nover tens of hours. Scrutinizing this hypothesis, we treated cells with LLOMe followed by \nwashout, waited three hours for ESCRT-mediated repair and machinery dissociation, then 25 \nrepeated LLOMe treatment. After the second LLOMe treatment we observed two sets of \nAmyTracker puncta: one surrounded by CHMP4B rings and one not (Fig. 1J), suggesting the \npresence of both membranolytic and inert (Leu-Leu)\nn amyloid fibril deposits. Accordingly, we \nspeculate that (Leu-Leu)n amyloid fibril membranolytic activity arises from physical interactions \nbetween growing amyloid fibrils and the endolysosomal membrane (16). 30 \n \nCryo-electron tomography reveals fibril-laden, damaged lysosomes \n \nTo provide additional evidence for (Leu-Leu)\nn intralysosomal amyloid fibril formation and \ndeleterious membrane interactions, we conducted cryo-electron tomography studies employing a 35 \nstrategy to affinity isolate lysosomes. HEK293T cells stably expressing the lysosomal membrane \nprotein TMEM192-GFP were treated with LLOMe, lysed using mechanical disruption, and the \nlysate containing intact lysosomes was incubated on anti-GFP nanobody-functionalized grids \nbefore plunge freezing and tilt series collection (Fig. 2A). Using this strategy, we observed \nlumenal, fibrous cargo with observable fibril-membrane contacts in a subset of observed \n40 \nlysosomes (Fig. 2B-D). In many cases we additionally observed the accumulation of membrane \nassociated and membrane-spanning ‘fuzzy’ densities (Fig. 2B), likely indicative of the \nrecruitment of lysosomal repair machineries. We furthermore rarely observed flotillin-like \ncomplexes on the membrane surface (Fig. 2C), in agreement with previous studies (36, 37). \nLysosomal membranes were often discontinuous along a given region, with prominently 45 \nmisshapen membranes occurring concurrently with high lumenal fibril densities (Fig. 2 D). \nOverall these observations provide visible evidence that LLOMe treatment of cells results in \nlysosomal membrane disruption linked with the accumulation of lumenal amyloid fibrils. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 25, 2025. ; https://doi.org/10.64898/2025.12.23.696283doi: bioRxiv preprint \n\n \n4 \n \n \nLLOMe induces the aggregation of cytosolic Tau(P301S) \n \nA recent report indicated that endocytosed intralysosomal tau fibrils can seed aggregation of \ncytosolic tau at the lysosomal membrane-cytosol interface (10). Given that we observed \n5 \nintralysosomal fibril-membrane disruptions, we hypothesized that (Leu-Leu)n amyloid fibrils \ncould behave similarly to cross-seed the aggregation of Tau. Utilizing overexpression of \ncytosolic Tau-RD(P301S)-YFP as a reporter in HEK293T cells, we observed the formation of \nbright puncta within one hour of LLOMe exposure (Fig. 3A). Tau-RD(P301S) puncta formation \nwas not observed upon treatment with bafilomycin A1 or cotreatment of LLOMe with CTSC \n10 \ninhibitor AZD5248 (Fig. 3B, C), confirming that Tau-RD(P301S) aggregation was not the result \nof lysosomal deacidification, but instead required the LLOMe-CTSC interaction. Puncta were \neither surrounded by or overlayed with endolysosomal marker LAMP1 (Fig. 3D) or CHMP4B \n(Fig. 3E), suggesting Tau-RD(P301S) aggregation occurred at sites of lysosomal damage. We \nnext examined whether LLOMe could cross-seed another amyloidogenic reporter, GFP-\n15 \nαSynuclein(A53T). However, αSynuclein(A53T) remained dispersed upon LLOMe exposure \n(Fig. 3F), suggesting the (Leu-Leu)\nn amyloid structural match was lacking for αSynuclein \nseeding. Altogether we evidence that (Leu-Leu)n amyloid fibrils produced upon LLOMe \nexposure harbor relevant structure to seed the aggregation of Tau-RD(P301S) (Fig. 3G). \n 20 \nVarying dipeptide composition tunes the CTSC-mediated endolysosomal damage response \n \nWe anticipated that further exploration of the CTSC-dipeptide ligation platform through \ndipeptide sequence variation could reveal distinct aggregate/amyloid structures and cellular \nphenotypes. Seeking to design dipeptides with similar properties to LLOMe, we chose to \n25 \nincorporate phenylalanine, an aromatic, hydrophobic residue that is itself amyloidogenic (38). \nThe dipeptides LFOMe, FLOMe, and FFOMe were readily ligated into tetrapeptides by CTSC-\nmediated ligation in vitro (Fig. S3A-C). Unlike LLOMe, cell treatment with these dipeptides led \nto no or very poor AmyTracker fluorescence (Fig. 4A, vehicle and LLOMe examples in Fig. \nS4A); however, endolysosomal vesicles were visibly enlarged by all dipeptide-OMe treatments \n30 \n(Fig. S4B), suggestive of the intralysosomal buildup of oligopeptide products/aggregates. \nProteostat staining within endolysosomal vesicles was robust (Fig. 4B, vehicle and LLOMe  \nexamples in Fig. S4C), arguing that LFOMe-, FLOMe-, and FFOMe-derived oligopeptide \naggregates may differ structurally from LLOMe-derived cross-β-sheet structures. Recruitment of \nCHMP4B to endolysosomes was diminished using LFOMe and FLOMe relative to LLOMe; \n35 \nhowever, FFOMe invoked little to no CHMP4B puncta localization to endolysosomes (Fig. 4C, \nFig. S4D). In contrast, we observed LC3B-II accumulation, a marker of lysophagy (22) and \nATG8-mediated lysosomal damage response (23, 39), with all dipeptides studied (Fig. 4D).  \nLFOMe and FLOMe weakly induced Tau-RD(P301S) puncta formation in HEK293T cells, \nwhile FFOMe did not detectably do so (Fig. 4E).We posit that FFOMe is a lysosomal damaging \n40 \nagent that negligibly invokes the ESCRT repair response relative to LLOMe, apparently through \nthe CTSC-mediated formation of oligomers that form predominantly non-amyloid aggregates \nthat are incapable of Tau-RD(P301S)-seeding. \n \nLysosomal damage-mediated cell death (40) was characterized utilizing LLOMe, LFOMe, \n45 \nFLOMe, and FFOMe in iPSC-derived microglia, which exhibit high lysosomal function (41, 42) \nand are likely to be more susceptible to lysosomal damage-mediated cell death than HEK293T or \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 25, 2025. ; https://doi.org/10.64898/2025.12.23.696283doi: bioRxiv preprint \n\n \n5 \n \nU-2 OS cells (LLOMe was historically used to kill immune cells (26)). Consistent with this, a \nmajor loss in viability was observed after only a 6-hour exposure of 0.5 mM LLOMe, resulting \nin an IC50 of 0.38 mM (Fig. 4F). LFOMe, FLOMe, and FFOMe displayed slightly reduced \ntoxicities, with IC50’s of 0.66, 0.55, and 0.55 mM respectively, suggesting a link between \nmicroglial cell death and dipeptide-derived aggregate structures. Further exploration into the 5 \nrelationship between sequence-derived aggregate structure, resulting lysosomal damage, and \nmicroglial and neuronal death can further inform the roles of intralysosomal aggregates in \nneurodegenerative diseases. \n \nA reactive CTSC-dipeptide thioester intermediate mediates dipeptide ligation \n10 \n \nWe next investigated the peptide structural requirements for the CTSC-mediated amyloid fibril \ngeneration. Neither Leu-Leu (LLOH), LLOMe diastereomers at either residue, nor N,N-dimethyl \nLLOMe formed AmyTracker puncta or visibly expanded endolysosomes upon cell treatment \n(Fig. S5A), implying the necessity of the methyl ester, L stereochemistry, and an NH\n3+ termini–15 \nCTSC(Asp1) interaction unobstructed by additional methyl groups, an interpretation supported \nby a molecular docking study (Fig. S5B) (43). To probe whether CTSC-mediated ligation is \nrestricted to dipeptides, we employed LOMe, LLLOH, and LLLOMe in cells. Neither LOMe nor \nLLLOH resulted in the formation of AmyTracker puncta; however, LLLOMe resulted in robust \nAmyTracker puncta formation (Fig. S5C), implicating the combination of the methyl ester and a \n20 \nLeu-based peptide chain ≥2 as necessary for productive CTSC-mediated ligation and subsequent \namyloid fibril formation. \n \nDelving further into the substrate requirements of the CTSC peptide ligation reaction, we \nperformed three in vitro reactions with CTSC: first, using LOMe, second, using LOMe + \n25 \nLLOMe, and third, using the tripeptide GLLOMe (used in place of LLLOMe due to increased \nsolubility), and characterized reaction product masses using LC-MS. LOMe alone did not react \nwith CTSC (Fig. S6A); however, addition of equimolar amounts of LLOMe and LOMe resulted \nin the formation of L-LLOMe and L-LL-LLOMe (Fig. S6B). Intriguingly, GLLOMe + CTSC \nresulted in modest formation of LOMe and GL-GLLOMe (Fig. S6C), implying the hydrolysis of \n30 \nGL-LOMe to release LOMe, and ligation of GL- to the N-terminus of a new GLLOMe molecule. \nIn light of this and previous results using LLLOH and LLLOMe, we hypothesize that the methyl \nester drives the lysosomal accumulation of these peptides through a mechanism to be \ndetermined. \n \n35 \nAltogether, these observations, and those from pretreatment with the CTSC inhibitor AZD5248 \nablating AmyTracker staining, permit us to speculate on the nature of the CTSC-dipeptide \nmethyl ester interaction (Fig. 5A). We propose the initial reaction proceeds similar to the \ncharacterized subtiligase reaction mechanism (44): the R\n1R2OMe C-terminal carbonyl is attacked \nby a CTSC active site thiolate, ultimately releasing methanol and forming a metastable, 40 \npopulated thioester intermediate CTSC-R1R2 (45) (note here the C-terminal methyl ester \nfunctions as a leaving group, and can be functionally replaced with β-naphthylamide and O-\nbenzyl groups (46) or by a polypeptide chain as would be the case in a typical CTSC \naminopeptidase reaction). Dependent on reaction conditions, the thioester can either be \nhydrolyzed to release R1R2-OH, or CTSC can facilitate the nucleophilic attack on the thioester 45 \nby the amino terminus of a second peptide H2N-R3…R n  in a CTSC-catalyzed backreaction akin \nto native chemical ligation (47) to release the product R1R2-R3…R n. From this model, the \nconcentration of the nucleophilic peptide is important to affect ligation, however the nature of \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 25, 2025. ; https://doi.org/10.64898/2025.12.23.696283doi: bioRxiv preprint \n\n \n6 \n \nthe C-terminus of the nucleophilic peptide is inconsequential. Indeed for LLOMe we observe \nboth carboxylic acid and methyl ester oligopeptides (Fig. 1B). Additionally, the permissible \nlength of the attacking peptide appears unfixed, as in the case of LLOMe we observe up to (Leu-\nLeu)4 oligopeptides, implying a (Leu-Leu)3 nucleophilic peptide (Fig. 1B). Even a single amino \nacid methyl ester, i.e. LOMe can serve as the nucleophile (Fig. S6B). Results using the tripeptide 5 \nGLLOMe suggest the product length follows the trend 2+n, where n is the length of the attacking \npeptide. We essentially argue that aminopeptidase activity generates the ligase-competent CTSC-\ndipeptide thioester intermediate: CTSC initially acts as an aminopeptidase, removing two amino \nacids from a peptide N-terminus, forming a reactive thioester with said dipeptide, then either \nreleases the dipeptide by thioester hydrolysis or ligates the dipeptide onto an exogenously \n10 \nsupplied amino terminus-bearing amino acid/peptide present at a high local concentration. \n \nDiscussion \n \nTo our knowledge, LLOMe is the first characterized biochemical reagent that, upon addition to \n15 \ncell culture media, induces the rapid intra-endolysosomal genesis of amyloid fibrils, a discovery \nthat recontextualizes numerous previous studies carried out with this endolysosome damaging \nreagent. We present here a characterization of the rate of intralysosomal amyloid fibril clearance \nin U-2 OS cells (Fig. 1H,  I), which demonstrates that endolysosomal clearance of amyloid fibrils \nis generally slow, despite taking place in an acidic, denaturing, and protease-rich environment. 20 \nIndeed, in earlier investigations (48, 49), fluorescent Aβ42 fibrils were fed to neuronal cell \ncultures and remained within endolysosomal compartments for weeks. The (Leu-Leu)n amyloid \nfibril structure has fewer residues per monomer participating in the cross-β-sheet structure, likely \nenabling faster dissociation of individual peptides from the fibril, which is probably rate-limiting \nfor endoproteolysis. We suspect (Leu-Leu)n fibrils induce Tau aggregation through primary or 25 \nsecondary nucleation. Further investigation into the (Leu-Leu)n fibril structure may inform the \nmechanistic requirements for Tau self- and cross-seeding in neurodegenerative contexts. We \nadditionally highlight the potential to engineer novel dipeptides, tripeptides, and beyond, which \nmay afford more complex oligopeptide structures through CTSC ligation to more specifically \ninduce aggregation of specific disease-associated amyloidogenic proteins that (Leu-Leu)\nn 30 \namyloid cannot, such as αSynuclein(A53T). \n \nIn summary, dipeptide methyl ester treatment represents a simple method to generate \npathologically relevant amyloid fibrils and other aggregate structures directly within the \nlysosome, dependent on the endogenously expressed enzyme CTSC. The displayed modularity \n35 \nof this system presents a platform rife with opportunity to generate novel tools to explore \nlysosomal biology and, in particular, to study neurodegenerative pathobiology. \nReferences \n1. R. A. Nixon, D. C. Rubinsztein, Mechanisms of autophagy–lysosome dysfunction in \nneurodegenerative diseases. Nature Reviews Molecular Cell Biology 25, 926-946 (2024). \n40 \n2. J. Root, P. Merino, A. Nuckols, M. Johnson, T. Kukar, Lysosome dysfunction as a cause \nof neurodegenerative diseases: Lessons from frontotemporal dementia and amyotrophic \nlateral sclerosis. Neurobiology of Disease 154, 105360 (2021). \n3. R. A. Nixon, Amyloid precursor protein and endosomal- lysosomal dysfunction in \nAlzheimer's disease: inseparable partners in a multifactorial disease. Faseb j 31, 2729-45 \n2743 (2017). \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 25, 2025. ; https://doi.org/10.64898/2025.12.23.696283doi: bioRxiv preprint \n\n \n7 \n \n4. R. L. Wallings, S. W. Humble, M. E. Ward, R. Wade- Martins, Lysosomal Dysfunction at \nthe Centre of Parkinson’s Disease and Frontotemporal Dementia/Amyotrophic Lateral \nSclerosis. Trends in Neurosciences 42, 899-912 (2019). \n5. W. Zhang, C. Xu, J. Sun, H.-M. Shen, J. Wang, C. Yang, Impairment of the autophagy–\nlysosomal pathway in Alzheimer's diseases: Pathogenic mechanisms and therapeutic 5 \npotential. Acta Pharmaceutica Sinica B 12, 1019-1040 (2022). \n6. V . Udayar, Y . Chen, E. Sidransky, R. Jagasia, Lysosomal dysfunction in \nneurodegeneration: emerging concepts and methods. Trends in Neurosciences 45, 184-\n199 (2022). \n7. J. I. Ayers, B. I. Giasson, D. R. Borchelt, Prion-like Spreading in Tauopathies. Biol \n10 \nPsychiatry 83, 337-346 (2018). \n8. A. Jan, N. P. Gonçalves, C. B. Vaegter, P. H. Jensen, N. Ferreira, The Prion- Like \nSpreading of Alpha-Synuclein in Parkinson's Disease: Update on Models and \nHypotheses. Int J Mol Sci 22,  (2021). \n9. W. P. Flavin, L. Bousset, Z. C. Green, Y . Chu, S. Skarpathiotis, M. J. Chaney, J. H. 15 \nKordower, R. Melki, E. M. Campbell, Endocytic vesicle rupture is a conserved \nmechanism of cellular invasion by amyloid proteins. Acta Neuropathologica 134, 629-\n653 (2017). \n10. K. Rose, T. Jepson, S. Shukla, A. Maya-Romero, M. Kampmann, K. Xu, J. H. Hurley, \nTau fibrils induce nanoscale membrane damage and nucleate cytosolic tau at lysosomes. \n20 \nProceedings of the National Academy of Sciences 121, e2315690121 (2024). \n11. K. Kakuda, K. Ikenaka, A. Kuma, J. Doi, C. Aguirre, N. Wang, T. Ajiki, C.- J. Choong, Y . \nKimura, S. M. M. Badawy, T. Shima, S. Nakamura, K. Baba, S. Nagano, Y . Nagai, T. \nYoshimori, H. Mochizuki, Lysophagy protects against propagation of α-synuclein \naggregation through ruptured lysosomal vesicles. Proceedings of the National Academy \n25 \nof Sciences 121, e2312306120 (2024). \n12. P. Yuste-Checa, V . A. Trinkaus, I. Riera-Tur, R. Imamoglu, T. F. Schaller, H. Wang, I. \nDudanova, M. S. Hipp, A. Bracher, F. U. Hartl, The extracellular chaperone Clusterin \nenhances Tau aggregate seeding in a cellular model. Nature Communications 12, 4863 \n(2021). \n30 \n13. S. Kolay, A. R. Vega, D. A. Dodd, V . A. Perez, O. M. Kashmer, C. L. White, 3rd, M. I. \nDiamond, The dual fates of exogenous tau seeds: Lysosomal clearance versus \ncytoplasmic amplification. Journal of Biological Chemistry 298,  (2022). \n14. J.-H. Lee, D.-S. Yang, C. N. Goulbourne, E. Im, P. Stavrides, A. Pensalfini, H. Chan, C. \nBouchet-Marquis, C. Bleiwas, M. J. Berg, C. Huo, J. Peddy, M. Pawlik, E. Levy, M. Rao, \n35 \nM. Staufenbiel, R. A. Nixon, Faulty autolysosome acidification in Alzheimer’s disease \nmouse models induces autophagic build-up of Aβ in neurons, yielding senile plaques. \nNature Neuroscience 25, 688-701 (2022). \n15. C.-C. Chou, R. Vest, M. A. Prado, J. Wilson-Grady, J. A. Paulo, Y . Shibuya, P. Moran-\nLosada, T.-T. Lee, J. Luo, S. P. Gygi, J. W. Kelly, D. Finley, M. Wernig, T. Wyss-Coray, J. \n40 \nFrydman, Proteostasis and lysosomal repair deficits in transdifferentiated neurons of \nAlzheimer’s disease. Nature Cell Biology 27, 619-632 (2025). \n16. M. F. M. Sciacca, C. Tempra, F. Scollo, D. Milardi, C. La Rosa, Amyloid growth and \nmembrane damage: Current themes and emerging perspectives from theory and \nexperiments on Aβ and hIAPP. Biochimica et Biophysica Acta (BBA) - Biomembranes \n45 \n1860, 1625-1638 (2018). \n17. J. J. Chen, D. L. Nathaniel, P. Raghavan, M. Nelson, R. Tian, E. Tse, J. Y . Hong, S. K. \nSee, S.-A. Mok, M. Y . Hein, D. R. Southworth, L. T. Grinberg, J. E. Gestwicki, M. D. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 25, 2025. ; https://doi.org/10.64898/2025.12.23.696283doi: bioRxiv preprint \n\n \n8 \n \nLeonetti, M. Kampmann, Compromised function of the ESCRT pathway promotes \nendolysosomal escape of tau seeds and propagation of tau aggregation. Journal of \nBiological Chemistry 294, 18952-18966 (2019). \n18. M. Radulovic, K. O. Schink, E. M. Wenzel, V . Nähse, A. Bongiovanni, F. Lafont, H. \nStenmark, ESCRT-mediated lysosome repair precedes lysophagy and promotes cell 5 \nsurvival. The EMBO Journal 37, e99753 (2018). \n19. M. L. Skowyra, P. H. Schlesinger, T. V . Naismith, P. I. Hanson, Triggered recruitment of \nESCRT machinery promotes endolysosomal repair. Science 360, eaar5078 (2018). \n20. M. Radulovic, E. M. Wenzel, S. Gilani, L. K. Holland, A. H. Lystad, S. Phuyal, V . M. \nOlkkonen, A. Brech, M. Jäättelä, K. Maeda, C. Raiborg, H. Stenmark, Cholesterol 10 \ntransfer via endoplasmic reticulum contacts mediates lysosome damage repair. The \nEMBO Journal 41, e112677 (2022). \n21. J. X. Tan, T. Finkel, A phosphoinositide signalling pathway mediates rapid lysosomal \nrepair. Nature 609, 815-821 (2022). \n22. I. Maejima, A. Takahashi, H. Omori, T. Kimura, Y . Takabatake, T. Saitoh, A. Yamamoto, \n15 \nM. Hamasaki, T. Noda, Y . Isaka, T. Yoshimori, Autophagy sequesters damaged lysosomes \nto control lysosomal biogenesis and kidney injury. The EMBO Journal 32, 2336-2347 \n(2013). \n23. J. Cross, J. Durgan, D. G. McEwan, M. Tayler, K. M. Ryan, O. Florey, Lysosome damage \ntriggers direct ATG8 conjugation and ATG2 engagement via non-canonical autophagy. \n20 \nJournal of Cell Biology 222,  (2023). \n24. J. Jia, F. Wang, Z. Bhujabal, R. Peters, M. Mudd, T. Duque, L. Allers, R. Javed, M. \nSalemi, C. Behrends, B. Phinney, T. Johansen, V . Deretic, Stress granules and mTOR are \nregulated by membrane atg8ylation during lysosomal damage. Journal of Cell Biology \n221,  (2022). \n25 \n25. V . V . Eapen, S. Swarup, M. J. Hoyer, J. A. Paulo, J. W. Harper, Quantitative proteomics \nreveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of \ndamaged lysosomes by lysophagy. eLife 10, e72328 (2021). \n26. D. L. Thiele, P. E. Lipsky, Mechanism of L-leucyl-L- leucine methyl ester-mediated \nkilling of cytotoxic lymphocytes: dependence on a lysosomal thiol protease, dipeptidyl 30 \npeptidase I, that is enriched in these cells. Proc Natl Acad Sci U S A 87, 83-87 (1990). \n27. M. J. McGuire, P. E. Lipsky, D. L. Thiele, Purification and characterization of dipeptidyl \npeptidase I from human spleen. Arch Biochem Biophys 295, 280-288 (1992). \n28. T. Uchimoto, H. Nohara, R. Kamehara, M. Iwamura, N. Watanabe, Y . Kobayashi, \nMechanism of apoptosis induced by a lysosomotropic agent, L-Leucyl-L-Leucine methyl 35 \nester. Apoptosis 4, 357-362 (1999). \n29. H. Würz, A. Tanaka, J. S. Fruton, Polymerization of Dipeptide Amides by Cathepsin C*. \nBiochemistry 1, 19-29 (1962). \n30. H. Li, F. Rahimi, S. Sinha, P. Maiti, G. Bitan, K. Murakami, in Encyclopedia of \nAnalytical Chemistry. (2009). 40 \n31. E. Pretorius, M. J. Page, L. Hendricks, N. B. Nkosi, S. R. Benson, D. B. Kell, Both \nlipopolysaccharide and lipoteichoic acids potently induce anomalous fibrin amyloid \nformation: assessment with novel Amytracker™ stains. J R Soc Interface 15,  (2018). \n32. N. Q. Thai, N. H. Tseng, M. T. Vu, T. T. Nguyen, H. Q. Linh, C. K. Hu, Y . R. Chen, M. S. \nLi, Discovery of DNA dyes Hoechst 34580 and 33342 as good candidates for inhibiting \n45 \namyloid beta formation: in silico and in vitro study. J Comput Aided Mol Des 30, 639-650 \n(2016). \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 25, 2025. ; https://doi.org/10.64898/2025.12.23.696283doi: bioRxiv preprint \n\n \n9 \n \n33. Y . Uchida, H. Takahashi, Rapid detection of A β deposits in APP transgenic mice by \nHoechst 33342. Neuroscience Letters 448, 279-281 (2008). \n34. M. Furber, A.-K. Tiden, P. Gardiner, A. Mete, R. Ford, I. Millichip, L. Stein, A. Mather, \nE. Kinchin, C. Luckhurst, S. Barber, P. Cage, H. Sanganee, R. Austin, K. Chohan, R. \nBeri, B. Thong, A. Wallace, V . Oreffo, R. Hutchinson, S. Harper, J. Debreczeni, J. Breed, \n5 \nL. Wissler, K. Edman, Cathepsin C Inhibitors: Property Optimization and Identification \nof a Clinical Candidate. Journal of Medicinal Chemistry 57, 2357-2367 (2014). \n35. P. Gahlot, B. Kravic, G. Rota, J. van den Boom, S. Levantovsky, N. Schulze, E. Maspero, \nS. Polo, C. Behrends, H. Meyer, Lysosomal damage sensing and lysophagy initiation by \nSPG20-ITCH. Molecular Cell 84, 1556-1569.e1510 (2024). \n10 \n36. J. Singh, H. Elhabashy, P. Muthukottiappan, M. Stepath, M. Eisenacher, O. Kohlbacher, \nV . Gieselmann, D. Winter, Cross-linking of the endolysosomal system reveals potential \nflotillin structures and cargo. Nature Communications 13, 6212 (2022). \n37. B. M. McVeigh, J. J. De Jesús-Pérez, D. H. Siepe, P. Gogoi, S. K. Mageswaran, M. \nKalocsay, E. M. Mihelc, V . Y . Moiseenkova-Bell, Visualization of lysosomal membrane \n15 \nproteins by cryo electron tomography. Nature Communications 16, 9234 (2025). \n38. L. Adler-Abramovich, L. Vaks, O. Carny, D. Trudler, A. Magno, A. Caflisch, D. Frenkel, \nE. Gazit, Phenylalanine assembly into toxic fibrils suggests amyloid etiology in \nphenylketonuria. Nature Chemical Biology 8, 701-706 (2012). \n39. S. Nakamura, S. Shigeyama, S. Minami, T. Shima, S. Akayama, T. Matsuda, A. Esposito, \n20 \nG. Napolitano, A. Kuma, T. Namba-Hamano, J. Nakamura, K. Yamamoto, M. Sasai, A. \nTokumura, M. Miyamoto, Y . Oe, T. Fujita, S. Terawaki, A. Takahashi, M. Hamasaki, M. \nYamamoto, Y . Okada, M. Komatsu, T. Nagai, Y . Takabatake, H. Xu, Y . Isaka, A. Ballabio, \nT. Yoshimori, LC3 lipidation is essential for TFEB activation during the lysosomal \ndamage response to kidney injury. Nature Cell Biology 22, 1252-1263 (2020). \n25 \n40. A. Serrano-Puebla, P. Boya, Lysosomal membrane permeabilization in cell death: new \nevidence and implications for health and disease. Ann N Y Acad Sci 1371, 30-44 (2016). \n41. C. Gao, J. Jiang, Y . Tan, S. Chen, Microglia in neurodegenerative diseases: mechanism \nand potential therapeutic targets. Signal Transduction and Targeted Therapy 8, 359 \n(2023). 30 \n42. A. Majumdar, D. Cruz, N. Asamoah, A. Buxbaum, I. Sohar, P. Lobel, F. R. Maxfield, \nActivation of Microglia Acidifies Lysosomes and Leads to Degradation of Alzheimer \nAmyloid Fibrils. Molecular Biology of the Cell 18, 1490-1496 (2007). \n43. D. Turk, V . Janjić, I. Štern, M. Podobnik, D. Lamba, S. W. Dahl, C. Lauritzen, J. \nPedersen, V . Turk, B. Turk, Structure of human dipeptidyl peptidase I (cathepsin C): \n35 \nexclusion domain added to an endopeptidase framework creates the machine for \nactivation of granular serine proteases. The EMBO Journal 20, 6570-6582 (2001). \n44. A. M. Weeks, J. A. Wells, Engineering peptide ligase specificity by proteomic \nidentification of ligation sites. Nature Chemical Biology 14, 50-57 (2018). \n45. J. L. Schneck, J. P. Villa, P. McDevitt, M. S. McQueney, S. H. Thrall, T. D. Meek, \n40 \nChemical Mechanism of a Cysteine Protease, Cathepsin C, As Revealed by Integration of \nboth Steady-State and Pre-Steady-State Solvent Kinetic Isotope Effects. Biochemistry 47, \n8697-8710 (2008). \n46. D. L. Thiele, P. E. Lipsky, The action of leucyl-leucine methyl ester on cytotoxic \nlymphocytes requires uptake by a novel dipeptide-specific facilitated transport system \n45 \nand dipeptidyl peptidase I-mediated conversion to membranolytic products. Journal of \nExperimental Medicine 172, 183-194 (1990). \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 25, 2025. ; https://doi.org/10.64898/2025.12.23.696283doi: bioRxiv preprint \n\n \n10 \n \n47. P. A. Cistrone, M. J. Bird, D. T. Flood, A. P. Silvestri, J. C. J. Hintzen, D. A. Thompson, \nP. E. Dawson, Native Chemical Ligation of Peptides and Proteins. Curr Protoc Chem \nBiol 11, e61 (2019). \n48. D. M. Paresce, H. Chung, F. R. Maxfield, Slow Degradation of Aggregates of the \nAlzheimer's Disease Amyloid β-Protein by Microglial Cells*. Journal of Biological 5 \nChemistry 272, 29390-29397 (1997). \n49. S. Söllvander, E. Nikitidou, R. Brolin, L. Söderberg, D. Sehlin, L. Lannfelt, A. \nErlandsson, Accumulation of amyloid-β by astrocytes result in enlarged endosomes and \nmicrovesicle-induced apoptosis of neurons. Molecular Neurodegeneration 11, 38 (2016). \n50. D. Trudler, K. L. Nazor, Y . S. Eisele, T. Grabauskas, N. Dolatabadi, J. Parker, A. Sultan, 10 \nZ. Zhong, M. S. Goodwin, Y . Levites, T. E. Golde, J. W. Kelly, M. R. Sierks, N. J. \nSchork, M. Karin, R. Ambasudhan, S. A. Lipton, Soluble α-synuclein–antibody \ncomplexes activate the NLRP3 inflammasome in hiPSC-derived microglia. Proceedings \nof the National Academy of Sciences 118, e2025847118 (2021). \n51. F. Wang, Y . Liu, Z. Yu, S. Li, S. Feng, Y . Cheng, D. A. Agard, General and robust \n15 \ncovalently linked graphene oxide affinity grids for high-resolution cryo-EM. Proc Natl \nAcad Sci U S A 117, 24269-24273 (2020). \n52. F. Wang, Z. Yu, M. Betegon, M. G. Campbell, T. Aksel, J. Zhao, S. Li, S. M. Douglas, Y . \nCheng, D. A. Agard, Amino and PEG-amino graphene oxide grids enrich and protect \nsamples for high-resolution single particle cryo-electron microscopy. J Struct Biol 209, 20 \n107437 (2020). \n53. A. Peck, Y . Yu, M. Paraan, D. Kimanius, U. H. Ermel, J. Hutchings, D. Serwas, H. Siems, \nN. S. Hill, M. Ali, J. Peukes, G. A. Greenan, S.-H. Sheu, E. A. Montabana, B. Carragher, \nC. S. Potter, D. A. Agard, S. Zheng, AreTomoLive: Automated reconstruction of \ncomprehensively-corrected and denoised cryo-electron tomograms in real-time and at \n25 \nhigh throughput. bioRxiv, 2025.2003.2011.642690 (2025). \n54. J. Schindelin, I. Arganda-Carreras, E. Frise, V . Kaynig, M. Longair, T. Pietzsch, S. \nPreibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y . Tinevez, D. J. White, V . Hartenstein, \nK. Eliceiri, P. Tomancak, A. Cardona, Fiji: an open-source platform for biological-image \nanalysis. Nature Methods 9, 676-682 (2012). 30 \n \n \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 25, 2025. ; https://doi.org/10.64898/2025.12.23.696283doi: bioRxiv preprint \n\n \n11 \n \nAcknowledgments:  \nWe acknowledge the expert assistance of Scott Henderson, Kimberly Vanderpool, and Theresa \nFassel of The Core Microscopy Facility at The Scripps Research Institute. We acknowledge the \nexpert assistance of Jake Bailey at the UCSD X-ray Crystallography Facility. We thank Emily P. \nBentley for providing expert editorial assistance. 5 \nFunding:  \nResearch reported in this publication was supported by the National Institute on Aging of the \nNational Institutes of Health grant RF1AG073418 (JWK) and the National Center for Advancing \nTranslational Sciences of the National Institutes of Health grant T32TR004396 (RDE). The \ncontent is solely the responsibility of the authors and does not necessarily represent the official \n10 \nviews of the National Institutes of Health. Further support was provided by the Department of \nEducation under the Title V DHSI Program Grant #P031S240270 (DAY). Additional funding \nwas provided by the Freedom Together Foundation (JWK).  The CZ Imaging Institute is fully \nfunded by the Chan Zuckerberg Initiative (CZII-2023–327779). We are thankful for the financial \nsupport of the George E. Hewitt Foundation for Medical Research (RTO). \n15 \nAuthor contributions:  \nConceptualization: RDE and JWK \nMethodology: RDE, DS, and JWK \nInvestigation: RDE, RTO, IID, DS, SA, HS, EM, CA, DAY, OLL \nFunding acquisition: BFC, DAA, JWK 20 \nSupervision: BFC, DAA, JWK \nWriting – original draft: RDE \nWriting – review & editing: RDE, JWK \nCompeting interests:  \nAuthors declare that they have no competing interests. \n25 \nData and materials availability: The full Cryo-electron tomography dataset collected here is \navailable on the CZ CryoET Data Portal (deposition_number TBA). For a control comparison \ndataset see (https://cryoetdataportal.czscience.com/datasets/10444).  Relevant data from image \nanalysis is available upon request. LFOMe, and FLOMe materials are available from the Kelly \nlab under the terms of an MTA 30 \nSupplementary Materials \nMaterials and Methods \nFigs. S1 to S30 \nReferences (51–53) \nMovies S1 to S3 \n35 \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 25, 2025. ; https://doi.org/10.64898/2025.12.23.696283doi: bioRxiv preprint \n\n \n12 \n \n \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 25, 2025. ; https://doi.org/10.64898/2025.12.23.696283doi: bioRxiv preprint \n\n \n13 \n \nFig. 1. LLOMe is converted into (Leu-Leu)n oligopeptides that spontaneously form β-sheet \nrich amyloids within endolysosomes. (A) Structure of LLOMe. (B) LC-MS chromatogram of \nLLOMe (100 mM) + CTSC (500 nM) overnight reaction precipitate dissolved in DMSO (left); \nassigned peaks identified by mass (right). (C) X-ray diffraction pattern of LLOMe + CTSC \nreaction precipitate. (D) Negative-stain electron microscopy of LLOMe + CTSC reaction 5 \nmixture. (E) Schematic of CTSC-mediated LLOMe ligation and consequent fibril formation. (F) \nRepresentative image of U-2 OS cells treated with LLOMe (1 mM, 10 minutes) fixed and stained \nagainst endolysosomal marker LAMP1 and AmyTracker680 (2 μg/mL). (G) Representative \nimage of CHMP4B association about AmyTracker puncta after LLOMe exposure (1 mM, 10 \nminutes). (H) Representative images and (I) quantitation of AmyTracker puncta number and \n10 \nintensity of U-2 OS cells treated with LLOMe (1 mM, 10 minutes) followed by LLOMe washout \nand further incubation for the indicated amount of time; n≥149 cells per timepoint, standard error \nof the mean shown as error bars, comparisons between the denoted and previous timepoint \nanalyzed using one-way ANOVA, ****=P≤0.0001, *=P<0.05. (J) Representative images \nshowing CHMP4B and AmyTracker staining before and after LLOMe exposure (1 mM, 10 \n15 \nminutes), 3 hours after LLOMe washout, and after a second LLOMe exposure (1 mM, 10 \nminutes). Yellow arrows distinguish AmyTracker puncta surrounded by CHMP4B. All \nscalebars=5 μm. \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 25, 2025. ; https://doi.org/10.64898/2025.12.23.696283doi: bioRxiv preprint \n\n \n14 \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 25, 2025. ; https://doi.org/10.64898/2025.12.23.696283doi: bioRxiv preprint \n\n \n15 \n \nFig. 2. Cryo-electron tomography of isolated LLOMe-exposed lysosomes. (A) Schematic of \non-grid affinity isolation of lysosomes from TMEM192-GFP-expressingHEK293T cells treated \nwith LLOMe (1 mM, 10 minutes). (B-D) Representative 2D tomogram slices of isolated \nlysosomes with (B) low lumenal fibril density with peripheral, ‘fuzzy’ densities, (C) high \nlumenal fibril density with peripheral flotillin-like complexes, and (D) high lumenal fibril 5 \ndensity with pronounced, misshapen membrane morphology. (pixel size=1.51 Å, scalebar=50 \nnm). Z-axis distance between slices relative to the first slice in each series is denoted. \nContinuous membranes are highlighted, a dashed circle highlights fibril density, and * denotes \nflotillin-like structures. \n  10 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 25, 2025. ; https://doi.org/10.64898/2025.12.23.696283doi: bioRxiv preprint \n\n \n16 \n \n \nFig. 3. (Leu-Leu)n amyloid fibrils seed the aggregation of cytosolic aggregation-prone Tau. \n(A-C) Representative images of HEK293T cells expressing Tau-RD(P301S)-YFP showing (A) \npuncta formation upon LLOMe exposure (1 mM, 60 minutes) or lack of puncta formation upon \n(B) bafilomycin A1 treatment (250 nM, 180 minutes) or (C) AZD5248 pretreatment (10 μM, 90 5 \nminutes) before LLOMe exposure (1 mM, 60 minutes). (D-E) Representative images of \ncolocalization of small Tau-RD(P301S) puncta and encirclement of large aggregates by (D) \nLAMP1 or (E) CHMP4B. (F) Representative images of GFP-αSynuclein(A53T) before and after \nLLOMe exposure (1 mM, 60 minutes). (G) Schematic of proposed mechanism by which (Leu-\nLeu)n amyloids seed the aggregation of certain susceptible cytosolic amyloidogenic proteins. All 10 \nscalebars=5 μm. \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 25, 2025. ; https://doi.org/10.64898/2025.12.23.696283doi: bioRxiv preprint \n\n \n17 \n \n \n \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 25, 2025. ; https://doi.org/10.64898/2025.12.23.696283doi: bioRxiv preprint \n\n \n18 \n \nFig. 4. Implied structural heterogeneity of phenylalanine-containing dipeptide methyl \nesters imparts different phenotypes than LLOMe. (A-B) Representative images of U-2 OS \ncells treated with the indicated dipeptide methyl esters (1 mM, 60 minutes) stained for LAMP 1 \nand (A) AmyTracker or (B) Proteostat, with accompanying plots displaying mean puncta \nfluorescence intensity of the corresponding dye (n≥135 cells in (A) or n≥522 cells in (B), 5 \nstandard error of the mean shown as error bars),  (C) Percent CHMP4B-LAMP1 overlap are per \ncell area (n≥242 cells per treatment, standard error of the mean shown as error bars); indicated \ncomparisons analyzed using one-way ANOVA, ****=P≤0.0001, **=P<0.01. (D) Western blot \ndisplaying accumulation of LC3B-II upon treatment of the indicated dipeptide methyl esters (1 \nmM, 60 minutes). (E) Representative images of Tau-RD(P301S)-YFP puncta formation in \n10 \nHEK293T upon treatment of indicated dipeptide methyl esters (1 mM, 60 minutes).  (F) Plot of \nviability of iPSC-derived microglial cultures against 6-hour dipeptide methyl ester exposure of \nthe indicated concentration; n=3 wells per datapoint, standard deviation shown as error bars \nwhere possible, data fit to variable slope IC\n50 curves. All scalebars=5 μm. \n  15 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 25, 2025. ; https://doi.org/10.64898/2025.12.23.696283doi: bioRxiv preprint \n\n \n19 \n \n \n \nFig. 5. Proposed mechanism of CTSC-mediated dipeptide ligation. (A) Schematic of \nproposed mechanism of CTSC R1-R2-OMe ligation, where R1 and R2 belong to the set of natural \nand unnatural amino acids for which CTSC bears affinity. The formation of a thioester 5 \nintermediate (3) can resolve through hydrolysis, releasing the dipeptide carboxylic acid, or (4) \nundergo nucleophilic attack by the N-terminus of an incoming peptide of length n, harboring \nresidues R\n3…R n, which may belong to a distinct set of amino acids from R1-R2. This attack \naffords the release of a new peptide (5) of length 2+(1+n), composed of R1-R2-R3…R n.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 25, 2025. ; https://doi.org/10.64898/2025.12.23.696283doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}