{"doi":"10.1093/hmg/ddz098","title":"Mechanism and chain specificity of RNF216/TRIAD3, the ubiquitin ligase mutated in Gordon Holmes syndrome","abstract":"<jats:title>Abstract</jats:title><jats:p>Gordon Holmes syndrome (GDHS) is an adult-onset neurodegenerative disorder characterized by ataxia and hypogonadotropic hypogonadism. GDHS is caused by mutations in the gene encoding the RING-between-RING (RBR)-type ubiquitin ligase RNF216, also known as TRIAD3. The molecular pathology of GDHS is not understood, although RNF216 has been reported to modify several substrates with K48-linked ubiquitin chains, thereby targeting them for proteasomal degradation. We identified RNF216 in a bioinformatical screen for putative SUMO-targeted ubiquitin ligases and confirmed that a cluster of predicted SUMO-interaction motifs (SIMs) indeed recognizes SUMO2 chains without targeting them for ubiquitination. Surprisingly, purified RNF216 turned out to be a highly active ubiquitin ligase that exclusively forms K63-linked ubiquitin chains, suggesting that the previously reported increase of K48-linked chains after RNF216 overexpression is an indirect effect. The linkage-determining region of RNF216 was mapped to a narrow window encompassing the last two Zn-fingers of the RBR triad, including a short C-terminal extension. Neither the SIMs nor a newly discovered ubiquitin-binding domain in the central portion of RNF216 contributes to chain specificity. Both missense mutations reported in GDHS patients completely abrogate the ubiquitin ligase activity. For the R660C mutation, ligase activity could be restored by using a chemical ubiquitin loading protocol that circumvents the requirement for ubiquitin-conjugating (E2) enzymes. This result suggests Arg-660 to be required for the ubiquitin transfer from the E2 to the catalytic cysteine. Our findings necessitate a re-evaluation of the previously assumed degradative role of RNF216 and rather argue for a non-degradative K63 ubiquitination, potentially acting on SUMOylated substrates.</jats:p>","journal":"Human Molecular Genetics","year":2019,"id":608258,"datarank":0.4887144807032224,"base_score":3.258096538021482,"endowment":3.258096538021482,"self_citation_contribution":0.4887144807032224,"citation_network_contribution":0.0,"self_endowment_contribution":0.4887144807032224,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":25,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":76961,"name":"Thomas Hermanns","orcid":"0000-0001-5350-7479","position":1,"is_corresponding":false},{"id":1562097,"name":"Tamara Blyszcz","orcid":null,"position":2,"is_corresponding":false},{"id":90540,"name":"Michael Lammers","orcid":"0000-0003-4168-4640","position":3,"is_corresponding":false},{"id":1562099,"name":"Gerrit J K Praefcke","orcid":null,"position":4,"is_corresponding":false},{"id":90539,"name":"Kay Hofmann","orcid":"0000-0002-2289-9083","position":5,"is_corresponding":false},{"id":1562096,"name":"Ramkumar Seenivasan","orcid":null,"position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Mechanism and chain specificity of RNF216/TRIAD3, the ubiquitin ligase mutated in Gordon Holmes syndrome","abstract":"<jats:title>Abstract</jats:title><jats:p>Gordon Holmes syndrome (GDHS) is an adult-onset neurodegenerative disorder characterized by ataxia and hypogonadotropic hypogonadism. GDHS is caused by mutations in the gene encoding the RING-between-RING (RBR)-type ubiquitin ligase RNF216, also known as TRIAD3. The molecular pathology of GDHS is not understood, although RNF216 has been reported to modify several substrates with K48-linked ubiquitin chains, thereby targeting them for proteasomal degradation. We identified RNF216 in a bioinformatical screen for putative SUMO-targeted ubiquitin ligases and confirmed that a cluster of predicted SUMO-interaction motifs (SIMs) indeed recognizes SUMO2 chains without targeting them for ubiquitination. Surprisingly, purified RNF216 turned out to be a highly active ubiquitin ligase that exclusively forms K63-linked ubiquitin chains, suggesting that the previously reported increase of K48-linked chains after RNF216 overexpression is an indirect effect. The linkage-determining region of RNF216 was mapped to a narrow window encompassing the last two Zn-fingers of the RBR triad, including a short C-terminal extension. Neither the SIMs nor a newly discovered ubiquitin-binding domain in the central portion of RNF216 contributes to chain specificity. Both missense mutations reported in GDHS patients completely abrogate the ubiquitin ligase activity. For the R660C mutation, ligase activity could be restored by using a chemical ubiquitin loading protocol that circumvents the requirement for ubiquitin-conjugating (E2) enzymes. This result suggests Arg-660 to be required for the ubiquitin transfer from the E2 to the catalytic cysteine. Our findings necessitate a re-evaluation of the previously assumed degradative role of RNF216 and rather argue for a non-degradative K63 ubiquitination, potentially acting on SUMOylated substrates.</jats:p>","is_dataset_classified":null,"base_score":3.258096538021482,"endowment":3.258096538021482,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"31087003","pmcid":null,"openalex_id":"https://openalex.org/W2945927883","authors":[],"funders":[{"funder_name":"Deutsche Forschungsgemeinschaft","grant_id":"SFB670","title":null},{"funder_name":"Deutsche Forschungsgemeinschaft","grant_id":"389564084","title":null},{"funder_name":"Deutsche Forschungsgemeinschaft","grant_id":"unidentified","title":"unidentified"}],"total_grants":3,"fwci":1.3257,"citation_percentile":0.80399768,"influential_citations":0,"citation_trend":[{"year":2020,"count":3},{"year":2021,"count":7},{"year":2022,"count":6},{"year":2023,"count":5},{"year":2024,"count":1},{"year":2025,"count":2},{"year":2026,"count":1}],"oa_status":"closed","license":"OUP Standard Publication Reuse","oa_locations":[{"url":"http://academic.oup.com/hmg/advance-article-pdf/doi/10.1093/hmg/ddz098/28861708/ddz098.pdf","host_type":"publisher"},{"url":"http://academic.oup.com/hmg/article-pdf/28/17/2862/29207225/ddz098.pdf","host_type":"publisher"},{"url":"https://doi.org/10.1093/hmg/ddz098","host_type":"journal"},{"url":"https://pubmed.ncbi.nlm.nih.gov/31087003","host_type":"repository"},{"url":"https://dx.doi.org/10.1093/hmg/ddz098","host_type":""}],"fields_of_study":["Ubiquitin and proteasome pathways","interferon and immune responses","Toxoplasma gondii Research Studies","0301 basic medicine","0303 health sciences","03 medical and health sciences","Amino Acid Sequence","Carrier Proteins","Cerebellar Ataxia","Enzyme Activation","Genetic Predisposition to Disease","Gonadotropin-Releasing Hormone","Humans","Hypogonadism","Mutation","Phosphorylation","Protein Binding","Protein Domains","Protein Interaction Domains and Motifs","Small Ubiquitin-Related Modifier Proteins","Sumoylation","Ubiquitin","Ubiquitin-Protein Ligases","Ubiquitination","Cerebellar Ataxia and Hypogonadotropic Hypogonadism"],"mesh_terms":["Protein Domains","Amino Acid Sequence","Carrier Proteins","Cerebellar Ataxia","Enzyme Activation","Humans","Hypogonadism","Gonadotropin-Releasing Hormone","Mutation","Phosphorylation","Protein Binding","Genetic Predisposition to Disease","Ubiquitin","Small Ubiquitin-Related Modifier Proteins","Ubiquitin-Protein Ligases","Protein Interaction Domains and Motifs","Ubiquitination","Sumoylation"],"keywords":["Biology","Ubiquitin ligase","Ubiquitin","Mechanism (biology)","DNA ligase","Genetics","Ubiquitin-Protein Ligases","Mutation","Molecular biology","Cell biology","Gene","Cerebellar Ataxia","Hypogonadism","Ubiquitination","Sumoylation","Enzyme Activation","Gonadotropin-Releasing Hormone","Protein Domains","Small Ubiquitin-Related Modifier Proteins","Humans","Genetic Predisposition to Disease","Protein Interaction Domains and Motifs","Amino Acid Sequence","Phosphorylation","Carrier Proteins","Protein Binding"],"sdg_mappings":[],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[],"source":"live","citation_network_status":"fetched"},"created_at":"2026-07-30T07:50:57.645420Z","pmid":null,"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":[]}