{"doi":"10.1101/2023.05.01.538963","title":"Lysine-independent ubiquitination and degradation of REV-ERBα involves a bi-functional degradation control sequence at its N-terminus","abstract":"REV-ERBα and REV-ERBβ proteins play crucial roles in linking the circadian system to overt daily rhythms in mammalian physiology and behavior. In most tissues, REV-ERBα protein robustly cycles such that it is detected only within a tight interval of 4-6 hours each day, suggesting both its synthesis and degradation are tightly controlled. Several ubiquitin ligases are known to drive REV-ERBα degradation, but how they interact with REV-ERBα and which lysine residues they ubiquitinate to promote degradation are unknown. In this study, we attempted to identify both ubiquitin-ligase-binding and ubiquitination sites within REV-ERBα required for its degradation. Surprisingly, mutating all lysine residues, the common sites for ubiquitin conjugation, in REV-ERBα to arginines (K20R), did very little to impair its degradation in cells. K20R were degraded much faster by co-expression of two E3 ligases, SIAH2 or SPSB4, suggesting possible N-terminal ubiquitination. To explore this, we examined if small deletions at the N-terminus of REV-ERBα would alter its degradation. Interestingly, deletion of amino acid (AA) residues 2 to 9 (delAA2-9) clearly resulted in a less stable REV-ERBα. We found that it was the length (i.e. 8 AA), and not the specific sequence, that confers stability in this region. Simultaneously, we also mapped the interaction site of the E3 ligase SPSB4 to this same region, specifically requiring AA4-9 of REV-ERBα. Thus, the first 9 AA of REV-ERBα has two opposing roles in regulating REV-ERBα turnover. Further, deleting eight additional AAs (delAA2-17) from the N-terminus strongly prevents REV-ERBα degradation. Combined, these results suggest that complex interactions within the first 25AAs potentially act as an endogenous 'switch' that allows REV-ERBα to exist in a stabilized conformation in order to accumulate at one time of day, but then rapidly shifts to a destabilized form, to enhance its removal at the end of its daily cycle.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":406860,"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":0.9538,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":534743,"name":"Jason P. DeBruyne","orcid":"0000-0001-9803-9568","position":1,"is_corresponding":false},{"id":584670,"name":"Ting-Chung Suen","orcid":"0000-0002-4708-8519","position":0,"is_corresponding":true}],"reference_count":52,"raw_metadata":null,"created_at":"2026-07-19T01:21:07.213041Z","pmid":"37205588","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":[]}