{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T21:54:05Z","timestamp":1789595645103,"version":"build-2803163510"},"posted":{"date-parts":[[2025,12,18]]},"group-title":"Biochemistry","reference-count":86,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,12,18]],"date-time":"2025-12-18T00:00:00Z","timestamp":1766016000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"name":"the National Key R&D Program of China","award":["2023YFC2306500 to Y.Y.; 2022YFC3401500 to L.L.; 2023YFA0915300 to M.P."],"award-info":[{"award-number":["2023YFC2306500 to Y.Y.; 2022YFC3401500 to L.L.; 2023YFA0915300 to M.P."]}]},{"DOI":"10.13039\/100000002","name":"the National Institutes of Health","doi-asserted-by":"crossref","award":["R35GM143052"],"award-info":[{"award-number":["R35GM143052"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100001809","name":"the National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["22207070, 22477076 to Y.Y.; 92253302, T2488301, 22137005, and 22227810 to L.L.; 22277073 and 92253302 to M.P.; 22407086 to Y.W.; 22407085 to Q.Z.; 22307071 to Q.Q"],"award-info":[{"award-number":["22207070, 22477076 to Y.Y.; 92253302, T2488301, 22137005, and 22227810 to L.L.; 22277073 and 92253302 to M.P.; 22407086 to Y.W.; 22407085 to Q.Z.; 22307071 to Q.Q"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"name":"the Foundation of the National Facility for Translational Medicine","award":["TMSK-2024-110 to Y.Y."],"award-info":[{"award-number":["TMSK-2024-110 to Y.Y."]}]},{"name":"New Cornerstone Science Foundation (to L.L.), Shanghai Pilot Program for Basic Research - Shanghai Jiao Tong University","award":["21TQ1400224 to M.P."],"award-info":[{"award-number":["21TQ1400224 to M.P."]}]},{"name":"Foundation of Muyuan Laboratory","award":["118602240 to M.P."],"award-info":[{"award-number":["118602240 to M.P."]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,12,18]]},"abstract":"<jats:title>Summary<\/jats:title>\n                <jats:p>Lysine 27 (K27)-linked polyubiquitination plays critical yet incompletely defined roles in proteostasis, innate immunity, and disease progression; however, investigations into this process have long been hindered by its extremely low abundance and the lack of conformation-specific enrichment tools. Herein, we describe the development of a long-sought conformation-specific antibody, K27-IgG, which can selectively recognize\u2014among all ubiquitin chain types\u2014the unique architecture of K27-linked polyubiquitin (K27-polyUb) characterized by a distinct buried K27-isopeptide bond, with high affinity (KD = 4.66 nM). This antibody was derived from synthetic antibodies initially generated via phage display, using chemically synthesized K27-linked diubiquitin (K27-diUb) as the antigen. High-resolution co-crystal structures uncovered the unique K27-diUb interface targeted by these sAbs. Subsequent reformatting of these sAbs into a full-length human immunoglobulin G (IgG) scaffold yielded K27-IgG, notably exhibiting markedly enhanced affinity without compromising selectivity. Using K27-IgG as a tool, we achieved sensitive detection and immunoprecipitation (IP) of endogenous K27-polyUb in cells, and delineated the intracellular interaction landscape of K27-polyUb through complementary proteomic approaches. Two key findings emerged: 1) The molecular chaperone DNAJB1 is a specific reader of K27-linked ubiquitin chains (but not other linkages) and that K27-polyUb chains themselves exhibit chaperone-like activity, suggesting a novel mechanism by which K27-polyUb regulates chaperone-mediated proteostasis; 2) The E2 enzyme UBE2Q1 assembles K27-diUb, identifying it as a potential writer for this ubiquitin chain topology. Collectively, this study establishes K27-IgG as a robust tool for deciphering the K27-linked ubiquitin code, thereby opening new avenues for investigating the biological functions of K27-linked polyubiquitination.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Highlights<\/jats:title>\n                  <jats:list list-type=\"bullet\">\n                    <jats:list-item>\n                      <jats:p>First K27-linkage conformation-specific antibody with nanomolar affinity overcomes a major barrier in the field.<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>K27-IgG unlocks functional mapping of the K27 ubiquitin landscape under proteotoxic stress.<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>Molecular chaperone DNAJB1 is a selective \u201creader\u201d of K27-linked ubiquitin chains.<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>K27 chains possess intrinsic chaperone activity, enabling protein refolding and suppressing aggregation.<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>E2 enzyme UBE2Q1 is a \u201cwriter\u201d that directly assembles K27-linked ubiquitin chains.<\/jats:p>\n                    <\/jats:list-item>\n                  <\/jats:list>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Graphical abstract<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" orientation=\"portrait\" fig-type=\"figure\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"695067v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.64898\/2025.12.18.695067","type":"posted-content","created":{"date-parts":[[2025,12,19]],"date-time":"2025-12-19T00:55:11Z","timestamp":1766105711000},"source":"Crossref","is-referenced-by-count":2,"title":["Conformation-specific Antibody Deciphers K27-linked Ubiquitination in Chaperone-Mediated Proteostasis"],"prefix":"10.64898","author":[{"given":"Chengxiao","family":"Han","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yicheng","family":"Weng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qingyun","family":"Zheng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qian","family":"Qu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Satchal K.","family":"Erramilli","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhen","family":"Su","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yujuan","family":"Duan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yunxi","family":"Han","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoguo","family":"Zhai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingxian","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anthony A.","family":"Kossiakoff","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7216-8193","authenticated-orcid":false,"given":"Man","family":"Pan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Minglei","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lei","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5379-5148","authenticated-orcid":false,"given":"Yuanyuan","family":"Yu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025122011500625000_2025.12.18.695067v1.1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-biochem-060310-170328"},{"key":"2025122011500625000_2025.12.18.695067v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/s41580-022-00543-1"},{"key":"2025122011500625000_2025.12.18.695067v1.3","doi-asserted-by":"publisher","DOI":"10.1038\/ncb3358"},{"key":"2025122011500625000_2025.12.18.695067v1.4","doi-asserted-by":"publisher","DOI":"10.1038\/cr.2016.39"},{"key":"2025122011500625000_2025.12.18.695067v1.5","doi-asserted-by":"publisher","DOI":"10.1126\/science.aav0725"},{"key":"2025122011500625000_2025.12.18.695067v1.6","doi-asserted-by":"publisher","DOI":"10.1038\/s41594-024-01221-w"},{"key":"2025122011500625000_2025.12.18.695067v1.7","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-025-60542-6"},{"key":"2025122011500625000_2025.12.18.695067v1.8","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2019.01.018"},{"key":"2025122011500625000_2025.12.18.695067v1.9","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-018-0736-4"},{"key":"2025122011500625000_2025.12.18.695067v1.10","doi-asserted-by":"publisher","DOI":"10.1038\/s41420-022-01204-0"},{"key":"2025122011500625000_2025.12.18.695067v1.11","doi-asserted-by":"publisher","DOI":"10.1016\/j.it.2021.12.005"},{"key":"2025122011500625000_2025.12.18.695067v1.12","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkaa053"},{"key":"2025122011500625000_2025.12.18.695067v1.13","doi-asserted-by":"publisher","DOI":"10.3389\/fonc.2012.00005"},{"key":"2025122011500625000_2025.12.18.695067v1.14","doi-asserted-by":"publisher","DOI":"10.1186\/s11658-020-00245-6"},{"key":"2025122011500625000_2025.12.18.695067v1.15","doi-asserted-by":"publisher","DOI":"10.1038\/s41589-021-00823-5"},{"key":"2025122011500625000_2025.12.18.695067v1.16","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms11792"},{"key":"2025122011500625000_2025.12.18.695067v1.17","doi-asserted-by":"publisher","DOI":"10.1002\/advs.202509817"},{"key":"2025122011500625000_2025.12.18.695067v1.18","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2015.01.042"},{"key":"2025122011500625000_2025.12.18.695067v1.19","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2024.05.018"},{"key":"2025122011500625000_2025.12.18.695067v1.20","doi-asserted-by":"publisher","DOI":"10.1038\/ni.2898"},{"key":"2025122011500625000_2025.12.18.695067v1.21","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00321-18"},{"key":"2025122011500625000_2025.12.18.695067v1.22","doi-asserted-by":"publisher","DOI":"10.1126\/science.aau0159"},{"key":"2025122011500625000_2025.12.18.695067v1.23","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms14274"},{"key":"2025122011500625000_2025.12.18.695067v1.24","doi-asserted-by":"publisher","DOI":"10.1038\/nrm3394"},{"key":"2025122011500625000_2025.12.18.695067v1.25","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.00305-20"},{"key":"2025122011500625000_2025.12.18.695067v1.26","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2015.01.041"},{"key":"2025122011500625000_2025.12.18.695067v1.27","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2024.03.024"},{"key":"2025122011500625000_2025.12.18.695067v1.28","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-018-02869-x"},{"key":"2025122011500625000_2025.12.18.695067v1.29","doi-asserted-by":"publisher","DOI":"10.1016\/j.str.2016.01.007"},{"key":"2025122011500625000_2025.12.18.695067v1.30","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.6b04031"},{"key":"2025122011500625000_2025.12.18.695067v1.31","doi-asserted-by":"publisher","DOI":"10.1080\/15548627.2017.1376160"},{"key":"2025122011500625000_2025.12.18.695067v1.32","doi-asserted-by":"publisher","DOI":"10.1038\/ncb2012"},{"key":"2025122011500625000_2025.12.18.695067v1.33","doi-asserted-by":"publisher","DOI":"10.1016\/j.ccr.2014.05.015"},{"key":"2025122011500625000_2025.12.18.695067v1.34","doi-asserted-by":"publisher","DOI":"10.1016\/j.cellsig.2015.09.001"},{"key":"2025122011500625000_2025.12.18.695067v1.35","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.E08-05-0496"},{"key":"2025122011500625000_2025.12.18.695067v1.36","doi-asserted-by":"publisher","DOI":"10.1080\/15548627.2025.2468140"},{"key":"2025122011500625000_2025.12.18.695067v1.37","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2017.09.005"},{"key":"2025122011500625000_2025.12.18.695067v1.38","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2015.10.005"},{"key":"2025122011500625000_2025.12.18.695067v1.39","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-017-0017-2"},{"key":"2025122011500625000_2025.12.18.695067v1.40","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2014.12.021"},{"key":"2025122011500625000_2025.12.18.695067v1.41","doi-asserted-by":"publisher","DOI":"10.1038\/s41418-018-0090-y"},{"key":"2025122011500625000_2025.12.18.695067v1.42","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-019-09844-0"},{"key":"2025122011500625000_2025.12.18.695067v1.43","doi-asserted-by":"publisher","DOI":"10.1038\/ncb3097"},{"key":"2025122011500625000_2025.12.18.695067v1.44","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-019-1482-y"},{"key":"2025122011500625000_2025.12.18.695067v1.45","doi-asserted-by":"publisher","DOI":"10.1038\/s41589-019-0230-9"},{"key":"2025122011500625000_2025.12.18.695067v1.46","doi-asserted-by":"publisher","DOI":"10.1038\/nchembio.2508"},{"key":"2025122011500625000_2025.12.18.695067v1.47","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2008.07.039"},{"key":"2025122011500625000_2025.12.18.695067v1.48","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2010.07.001"},{"key":"2025122011500625000_2025.12.18.695067v1.49","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2011.12.053"},{"key":"2025122011500625000_2025.12.18.695067v1.50","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2017.08.020"},{"key":"2025122011500625000_2025.12.18.695067v1.51","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2017.09.040"},{"key":"2025122011500625000_2025.12.18.695067v1.52","doi-asserted-by":"publisher","DOI":"10.1038\/s41594-024-01354-y"},{"key":"2025122011500625000_2025.12.18.695067v1.53","doi-asserted-by":"publisher","DOI":"10.1002\/anie.201810814"},{"key":"2025122011500625000_2025.12.18.695067v1.54","doi-asserted-by":"publisher","DOI":"10.1002\/anie.201100996"},{"key":"2025122011500625000_2025.12.18.695067v1.55","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2013.152"},{"key":"2025122011500625000_2025.12.18.695067v1.56","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0043746"},{"key":"2025122011500625000_2025.12.18.695067v1.57","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymeth.2012.12.010"},{"key":"2025122011500625000_2025.12.18.695067v1.58","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(18)45815-2"},{"key":"2025122011500625000_2025.12.18.695067v1.59","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)80575-9"},{"key":"2025122011500625000_2025.12.18.695067v1.60","doi-asserted-by":"publisher","DOI":"10.1002\/advs.201800234"},{"key":"2025122011500625000_2025.12.18.695067v1.61","doi-asserted-by":"publisher","DOI":"10.1002\/anie.201708067"},{"key":"2025122011500625000_2025.12.18.695067v1.62","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2010.109"},{"key":"2025122011500625000_2025.12.18.695067v1.63","doi-asserted-by":"publisher","DOI":"10.1016\/j.mcpro.2024.100852"},{"key":"2025122011500625000_2025.12.18.695067v1.64","doi-asserted-by":"publisher","DOI":"10.1038\/ncb0805-736"},{"key":"2025122011500625000_2025.12.18.695067v1.65","doi-asserted-by":"publisher","DOI":"10.1126\/science.286.5446.1888"},{"key":"2025122011500625000_2025.12.18.695067v1.66","doi-asserted-by":"publisher","DOI":"10.1038\/s41580-019-0133-3"},{"key":"2025122011500625000_2025.12.18.695067v1.67","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-022-32370-5"},{"key":"2025122011500625000_2025.12.18.695067v1.68","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2010.01.001"},{"key":"2025122011500625000_2025.12.18.695067v1.69","doi-asserted-by":"publisher","DOI":"10.1038\/s42003-025-07906-2"},{"key":"2025122011500625000_2025.12.18.695067v1.70","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-020-2904-6"},{"key":"2025122011500625000_2025.12.18.695067v1.71","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.adp3672"},{"key":"2025122011500625000_2025.12.18.695067v1.72","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-53002-0"},{"key":"2025122011500625000_2025.12.18.695067v1.73","doi-asserted-by":"publisher","DOI":"10.1080\/19420862.2018.1426421"},{"key":"2025122011500625000_2025.12.18.695067v1.74","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4939-8706-1_6"},{"key":"2025122011500625000_2025.12.18.695067v1.75","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444909047337"},{"key":"2025122011500625000_2025.12.18.695067v1.76","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444904019158"},{"key":"2025122011500625000_2025.12.18.695067v1.77","doi-asserted-by":"publisher","DOI":"10.1107\/S2059798323003595"},{"key":"2025122011500625000_2025.12.18.695067v1.78","doi-asserted-by":"publisher","DOI":"10.1002\/pro.3943"},{"key":"2025122011500625000_2025.12.18.695067v1.79","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.1511"},{"key":"2025122011500625000_2025.12.18.695067v1.80","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.3901"},{"key":"2025122011500625000_2025.12.18.695067v1.81","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkac1000"},{"key":"2025122011500625000_2025.12.18.695067v1.82","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkac194"},{"key":"2025122011500625000_2025.12.18.695067v1.83","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-021-03824-5"},{"key":"2025122011500625000_2025.12.18.695067v1.84","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-020-2906-4"},{"key":"2025122011500625000_2025.12.18.695067v1.85","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gky869"},{"key":"2025122011500625000_2025.12.18.695067v1.86","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkab1081"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2025.12.18.695067","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,20]],"date-time":"2025-12-20T19:50:14Z","timestamp":1766260214000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2025.12.18.695067"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,18]]},"references-count":86,"URL":"https:\/\/doi.org\/10.64898\/2025.12.18.695067","relation":{},"subject":[],"published":{"date-parts":[[2025,12,18]]},"subtype":"preprint"}}