Cysteines are critical determinants of spontaneous and seeded tau aggregation in cells

Cysteines are critical determinants of spontaneous and seeded tau aggregation in cells | 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 Cysteines are critical determinants of spontaneous and seeded tau aggregation in cells Lukasz Joachimiak, Parvathy Jayan, Simran Rastogi, Vaibhav Bommareddy, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9217102/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The frontotemporal dementia-linked S320F mutation in the microtubule-associated protein tau promotes spontaneous aggregation, yet the structural basis of its amyloidogenesis remains unclear. Using cryo-electron microscopy, we determined the structure of an S320F 295-330 tau fibril composed of parallel chains stabilized by the 306 VQIVYK 311 amyloid motif, with S320F buried in the fibril core and a C322-C322 disulfide linking two protofilaments. Although cysteines are dispensable for fibril formation by isolated peptide fragments in vitro, tau repeat domain constructs containing both C291 and C322 generate more potent seeds in cellular assays. In contrast, the C322S mutation suppresses spontaneous aggregation of S320F tau in cells, and combined C291S and C322S mutations inhibit seeded aggregation in both wild-type and S320F contexts. Systematic alanine mutagenesis coupled with seeding by tauopathy-derived material identifies cysteine residues as critical determinants of tau seeding, comparable in importance to core amyloid motifs. Together, these findings establish cysteines as central chemical regulators of tau aggregation and propagation. Biological sciences/Biophysics/Intrinsically disordered proteins Health sciences/Diseases/Neurological disorders/Neurodegenerative diseases/Alzheimer's disease Full Text Additional Declarations Yes there is potential Competing Interest. J.V.A. and M.I.D. are co-founders of Handshake Bio. L.S. is a co-founder of AmyGo Solutions. Handshake Bio or Amygo did not directly fund or influence the design, execution, or interpretation of the experiments presented in this manuscript. The remaining authors declare no competing interests. Supplementary Files SourceData.xlsx Related Manuscript File D1000297920valreportfullP1.pdf PDB Validation Report Cite Share Download PDF Status: Under Review 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. 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L.S. is a co-founder of AmyGo Solutions. Handshake Bio or Amygo did not directly fund or influence the design, execution, or interpretation of the experiments presented in this manuscript. The remaining authors declare no competing interests.","formattedTitle":"Cysteines are critical determinants of spontaneous and seeded tau aggregation in cells","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9217102/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9217102/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The frontotemporal dementia-linked S320F mutation in the microtubule-associated protein tau promotes spontaneous aggregation, yet the structural basis of its amyloidogenesis remains unclear. Using cryo-electron microscopy, we determined the structure of an S320F\u003csub\u003e295-330\u003c/sub\u003e tau fibril composed of parallel chains stabilized by the \u003csup\u003e306\u003c/sup\u003eVQIVYK\u003csup\u003e311\u003c/sup\u003e amyloid motif, with S320F buried in the fibril core and a C322-C322 disulfide linking two protofilaments. Although cysteines are dispensable for fibril formation by isolated peptide fragments in vitro, tau repeat domain constructs containing both C291 and C322 generate more potent seeds in cellular assays. In contrast, the C322S mutation suppresses spontaneous aggregation of S320F tau in cells, and combined C291S and C322S mutations inhibit seeded aggregation in both wild-type and S320F contexts. Systematic alanine mutagenesis coupled with seeding by tauopathy-derived material identifies cysteine residues as critical determinants of tau seeding, comparable in importance to core amyloid motifs. Together, these findings establish cysteines as central chemical regulators of tau aggregation and propagation.","manuscriptTitle":"Cysteines are critical determinants of spontaneous and seeded tau aggregation in cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-23 11:48:54","doi":"10.21203/rs.3.rs-9217102/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":"06cbb7a6-8130-4bf8-ac0d-1498c4aa76ef","owner":[],"postedDate":"April 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":66764657,"name":"Biological sciences/Biophysics/Intrinsically disordered proteins"},{"id":66764658,"name":"Health sciences/Diseases/Neurological disorders/Neurodegenerative diseases/Alzheimer's disease"}],"tags":[],"updatedAt":"2026-04-23T11:48:54+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-23 11:48:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9217102","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9217102","identity":"rs-9217102","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}