RESUMO
Sixteen ovarian tumor (OTU) family deubiquitinases (DUBs) exist in humans, and most members regulate cell-signaling cascades. Several OTU DUBs were reported to be ubiquitin (Ub) chain linkage specific, but comprehensive analyses are missing, and the underlying mechanisms of linkage specificity are unclear. Using Ub chains of all eight linkage types, we reveal that most human OTU enzymes are linkage specific, preferring one, two, or a defined subset of linkage types, including unstudied atypical Ub chains. Biochemical analysis and five crystal structures of OTU DUBs with or without Ub substrates reveal four mechanisms of linkage specificity. Additional Ub-binding domains, the ubiquitinated sequence in the substrate, and defined S1' and S2 Ub-binding sites on the OTU domain enable OTU DUBs to distinguish linkage types. We introduce Ub chain restriction analysis, in which OTU DUBs are used as restriction enzymes to reveal linkage type and the relative abundance of Ub chains on substrates.
Assuntos
Endopeptidases/química , Endopeptidases/metabolismo , Neoplasias Ovarianas/enzimologia , Ubiquitinação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Endopeptidases/genética , Feminino , Humanos , Modelos Moleculares , Neoplasias Ovarianas/metabolismo , Estrutura Terciária de Proteína , Tioléster Hidrolases/química , Tioléster Hidrolases/metabolismo , Ubiquitinas/metabolismoRESUMO
Protein ubiquitination is a highly versatile post-translational modification that regulates as diverse processes as protein degradation and kinase activation. Deubiquitinases hydrolyse ubiquitin modifications from proteins and are hence key regulators of the ubiquitin system. Ovarian tumour deubiquitinases comprise a family of fourteen human enzymes, many of which regulate cellular signalling pathways. Ovarian tumour deubiquitinases are cysteine proteases that cleave polyubiquitin chains in vitro and in cells, but little is currently known about their regulation. Here we show that ovarian tumour deubiquitinases are susceptible to reversible oxidation of the catalytic cysteine residue. High-resolution crystal structures of the catalytic domain of A20 in four different oxidation states reveal that the reversible form of A20 oxidation is a cysteine sulphenic acid intermediate, which is stabilised by the architecture of the catalytic centre. Using chemical tools to detect sulphenic acid intermediates, we show that many ovarian tumour deubiquitinases undergo reversible oxidation upon treatment with H2O2, revealing a new mechanism to regulate deubiquitinase activity.