The Study Of Catalytic Mechanism And Substrate Specificity Of Ubiquitin Specificity Enzymes USP2 And USP8

Protein ubiquitination is a post-translational modification that regulates a wide variety of cellular processes by affecting the stability, localization and/or function of the modified proteins. Conjugation of ubiquitin to target proteins is accomplished via a complex but relatively well-understood mechanism that involves the sequential action of three classes of enzymes: E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme), and E3 (ubiquitin protein ligase), which activate and transfer ubiquitin (Ub) or ubiquitin-like (Ubl) modifiers to theε-amino group of an internal lysine residue of target proteins. Attachment of Lys48-linked polyubiquitin chains promotes protein degradation by the Ub-proteasome system, while non-proteasomal functions of Ub involve the attachment of differently linked polyubiquitin chains and individual Ub molecules.Ubiquitination is a reversible process. The isopeptide bond that links the C-terminal glycine of ubiquitin to a lysine side chain on the target protein, or on another ubiquitin molecule in a polyubiquitin chain, can be cleaved by deubiquitinating enzymes (DUBs). These enzymes are also responsible for the activation of ubiquitin and ubiquitin-like modifiers by C-terminal processing of their precursors. Approximately 100 putative mammalian DUBs have been identified in the human genome, that can be grouped into five different families, four of which are cysteine proteases: the ubiquitin-C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs), ovarian-tumor (OTU) domain-containing enzymes (otubains, families) and the Machado-Joseph domain (MJD) DUBs.The ubiquitin-specific protease (USP) structural class represents the largest and most diverse family of deubiquitinating enzymes (DUBs). Many USPs assume important biological roles and emerge as potential targets for therapeutic intervention. A clear understanding of USP catalytic mechanism requires a functional evaluation of the proposed key active site residues. Crystallographic data of ubiquitin aldehyde adducts of USP catalytic cores provided structural details on the catalytic triad residues, namely the conserved Cys and His, and a variable putative third residue, and inferred indirect structural roles for two other conserved residues (Asn and Asp), in stabilizing via a bridging water molecule the oxyanion of the tetrahedral intermediate (TI). We have expressed the catalytic domain of USP2 and USP8, and probed by site-directed mutagenesis the role of these active site residues in the hydrolysis of peptide and isopeptide substrates, including a synthetic K48-linked diubiquitin substrate for which a label-free mass spectrometry assay has been developed to monitor cleavage.For USP2, hydrolysis of ubiquitin-AMC, a model substrate, was not affected by the mutations. Molecular dynamics simulations of USP2 free and complexed with the TI of a bona-fide isopeptide substrate were carried out. We found that Asn218 is structurally poised to directly stabilize the oxyanion developed in the acylation step, being structurally supported by the adjacent absolutely conserved Asp482. Mutagenesis data functionally confirmed this structural role independent of the nature (isopeptide vs peptide) of the bond being cleaved. We also found that Asn481, structurally located as the third member of the catalytic triad, does not fulfill this role functionally. A dual supporting role is inferred from double-point mutation and structural data for the absolutely conserved residue Asp482, in oxyanion hole formation, and in maintaining the correct alignment and protonation of His464 for catalytic competency.USP8 has different results: on hydrolysis of Ub-AMC the single mutant Asn781had the same results to USP2, but the other D1084A and D1085A showed low catalytic acitvity, especially the double-point mutations N781A/D1085A and D1084A/D1085A, the value of Kcat/Km reduced 2 or 3 orders of magnitude compare to WT, respectively. That means the two residues Asp1084 and Asp1085 participate in the catalysis of USP8 directly. The same results showed on hydrolysis substrate Ub-V77. In addition we found that USP8 had a very low catalytic activity on hydrolysis Di-Ub, which is 1/10 of USP8 to Ub-AMC or Ub-V77.These results suggest that USP2 and USP8 have a similly catalytic mechanism but have their own feathures. In USP2, the catalytic triad is composed by residues Cys223, His464, Asp481and Asn482 and, the residue Asn218 is structurally poised to directly stabilize the oxyanion, being structurally supported by the adjacent absolutely conserved Asp482. In USP8 the catalytic triad including Cys786, His575 and Asp1084, the residue Asn781 plays a subsidiary role and Asp1085 is more importane in oxyanion hole. Besides, USP2 can specific hydrolyze Di-Ub, but it is not a good substrate of USP8.