Conformation-specific Antibody Deciphers K27-linked Ubiquitination in Chaperone-Mediated Proteostasis

Chengxiao Han, Yicheng Weng, Qingyun Zheng, Qian Qu, Satchal K. Erramilli, Zhen Su, Yujuan Duan, Yunxi Han, Xiaoguo Zhai, Jingxian Li, Anthony A. Kossiakoff, Man Pan, Minglei Zhao, Lei Liu, Yuanyuan Yu
2025 · doi:10.64898/2025.12.18.695067
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Summary 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—among all ubiquitin chain types—the 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. Highlights First K27-linkage conformation-specific antibody with nanomolar affinity overcomes a major barrier in the field. K27-IgG unlocks functional mapping of the K27 ubiquitin landscape under proteotoxic stress. Molecular chaperone DNAJB1 is a selective “reader” of K27-linked ubiquitin chains. K27 chains possess intrinsic chaperone activity, enabling protein refolding and suppressing aggregation. E2 enzyme UBE2Q1 is a “writer” that directly assembles K27-linked ubiquitin chains. Graphical abstract Full Text Availability The license terms selected by the author(s) for this preprint version do not permit archiving in PMC. The full text is available from the preprint server.

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[{'doi': None, 'name': 'the National Key R&D Program of China', 'awards': ['2023YFC2306500 to Y.Y.; 2022YFC3401500 to L.L.; 2023YFA0915300 to M.P.']}, {'doi': '10.13039/100000002', 'name': 'the National Institutes of Health', 'awards': ['R35GM143052']}, {'doi': '10.13039/501100001809', 'name': 'the National Natural Science Foundation of China', 'awards': ['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']}, {'doi': None, 'name': 'the Foundation of the National Facility for Translational Medicine', 'awards': ['TMSK-2024-110 to Y.Y.']}, {'doi': None, 'name': 'New Cornerstone Science Foundation (to L.L.), Shanghai Pilot Program for Basic Research - Shanghai Jiao Tong University', 'awards': ['21TQ1400224 to M.P.']}, {'doi': None, 'name': 'Foundation of Muyuan Laboratory', 'awards': ['118602240 to M.P.']}]

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