{"doi":"10.64898/2025.12.18.695067","title":"Conformation-specific Antibody Deciphers K27-linked Ubiquitination in Chaperone-Mediated Proteostasis","abstract":"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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":585745,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9578,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1499601,"name":"Yicheng Weng","orcid":"0000-0002-4192-0917","position":1,"is_corresponding":false},{"id":494660,"name":"Qingyun Zheng","orcid":null,"position":2,"is_corresponding":false},{"id":561027,"name":"Qian Qu","orcid":"0000-0003-4648-1869","position":3,"is_corresponding":false},{"id":266717,"name":"Satchal K. Erramilli","orcid":"0000-0001-8694-7681","position":4,"is_corresponding":false},{"id":1499602,"name":"Zhen Su","orcid":"0000-0002-5697-319X","position":5,"is_corresponding":false},{"id":1499970,"name":"Yujuan Duan","orcid":null,"position":6,"is_corresponding":false},{"id":1499971,"name":"Yunxi Han","orcid":null,"position":7,"is_corresponding":false},{"id":1499972,"name":"Xiaoguo Zhai","orcid":null,"position":8,"is_corresponding":false},{"id":342824,"name":"Jingxian Li","orcid":"0000-0003-1087-5428","position":9,"is_corresponding":false},{"id":260362,"name":"Anthony A. Kossiakoff","orcid":"0000-0003-3174-9359","position":10,"is_corresponding":false},{"id":577522,"name":"Man Pan","orcid":"0000-0001-7216-8193","position":11,"is_corresponding":false},{"id":342826,"name":"Minglei Zhao","orcid":"0000-0001-5832-6060","position":12,"is_corresponding":false},{"id":561028,"name":"Lei Liu","orcid":"0000-0001-6290-8602","position":13,"is_corresponding":false},{"id":814175,"name":"Yuanyuan Yu","orcid":"0000-0002-5379-5148","position":14,"is_corresponding":false},{"id":1499600,"name":"Chunyan Han","orcid":"0009-0004-3735-8935","position":0,"is_corresponding":true}],"reference_count":86,"raw_metadata":null,"created_at":"2026-07-19T02:59:24.273134Z","pmid":"41446272","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}