RESUMO
Different ubiquitin modifications to proliferating cell nuclear antigen (PCNA) signal distinct modes of lesion bypass in the RAD6 pathway of DNA damage tolerance. The modification of PCNA with monoubiquitin signals an error-prone bypass, whereas the extension of this modification into a Lys-63-linked polyubiquitin chain promotes error-free bypass. Chain formation is catalyzed by the Mms2/Ubc13 conjugating enzyme variant/conjugating enzyme (UEV.E2) complex together with the Rad5 ubiquitin ligase. In vitro studies of this UEV.E2 complex have identified a ubiquitin binding site that is mainly localized on Mms2. However, the role of this site in DNA damage tolerance and the molecular features of the ubiquitin/Mms2 interaction are poorly understood. Here we identify two molecular determinants, the side chains of Mms2-Ile-57 and ubiquitin-Ile-44, that are required for chain assembly in vitro and error-free lesion bypass in vivo. Mutating either of these side chains to alanine elicits a severe 10-20-fold inhibition of chain synthesis that is caused by compromised binding of the acceptor ubiquitin to Mms2. These results suggest that the ubiquitin binding site of Mms2 is necessary for error-free lesion bypass in the RAD6 pathway and provide new insights into ubiquitin recognition by UEV proteins.
Assuntos
Dano ao DNA , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Sítios de Ligação/genética , Genes Fúngicos , Variação Genética , Modelos Moleculares , Complexos Multiproteicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína LigasesRESUMO
A20 is a cytoplasmic protein required for the termination of tumor necrosis factor (TNF)-induced signals. We show here that mice doubly deficient in either A20 and TNF or A20 and TNF receptor 1 developed spontaneous inflammation, indicating that A20 is also critical for the regulation of TNF-independent signals in vivo. A20 was required for the termination of Toll-like receptor-induced activity of the transcription factor NF-kappaB and proinflammatory gene expression in macrophages, and this function protected mice from endotoxic shock. A20 accomplished this biochemically by directly removing ubiquitin moieties from the signaling molecule TRAF6. The critical function of this deubiquitinating enzyme in the restriction of TLR signals emphasizes the importance of the regulation of ubiquitin conjugation in innate immune cells.
Assuntos
Glicoproteínas de Membrana/fisiologia , Proteínas/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Antígenos CD/fisiologia , Cisteína Endopeptidases , Peptídeos e Proteínas de Sinalização Intracelular , Lipopolissacarídeos/farmacologia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Proteínas Nucleares , Proteínas/metabolismo , Receptores do Fator de Necrose Tumoral/fisiologia , Receptores Tipo I de Fatores de Necrose Tumoral , Choque Séptico/prevenção & controle , Transdução de Sinais , Fator 6 Associado a Receptor de TNF , Receptores Toll-Like , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Fator de Necrose Tumoral alfa/fisiologia , Ubiquitina/metabolismoRESUMO
Ubiquitin (Ub) regulates diverse functions in eukaryotes through its attachment to other proteins. The defining step in this protein modification pathway is the attack of a substrate lysine residue on Ub bound through its C-terminus to the active site cysteine residue of a Ub-conjugating enzyme (E2) or certain Ub ligases (E3s). So far, these E2 and E3 cysteine residues are the only enzyme groups known to participate in the catalysis of conjugation. Here we show that a strictly conserved E2 asparagine residue is critical for catalysis of E2- and E2/RING E3-dependent isopeptide bond formation, but dispensable for upstream and downstream reactions of Ub thiol ester formation. In contrast, the strictly conserved histidine and proline residues immediately upstream of the asparagine are dispensable for catalysis of isopeptide bond formation. We propose that the conserved asparagine side chain stabilizes the oxyanion intermediate formed during lysine attack. The E2 asparagine is the first non-covalent catalytic group to be proposed in any Ub conjugation factor.