Abstract
Understanding function and specificity of de-ubiquitylating enzymes (DUBs) is a major goal of current research, since DUBs are key regulators of ubiquitylation events and have been shown to be mutated in human diseases. Most DUBs are cysteine proteases, relying on a catalytic triad of cysteine, histidine and aspartate to cleave the isopeptide bond between two ubiquitin units in a poly-ubiquitin chain. We have discovered that the two Drosophila melanogaster homologues of human OTUD4, CG3251 and Otu, contain a serine instead of a cysteine in the catalytic OTU (ovarian tumor) domain. DUBs that are serine proteases instead of cysteine- or metallo-proteases have not been described. In line with this, neither CG3251 nor Otu protein were active to cleave ubiquitin chains. Re-introduction of a cysteine in the catalytic center did not render the enzymes active, indicating that further critical features for ubiquitin binding or cleavage have been lost in these proteins. Sequence analysis of OTUD4 homologues from various other species showed that within this OTU subfamily, loss of the catalytic cysteine has occurred frequently in presumably independent events, as well as gene duplications or triplications, suggesting DUB-independent functions of OTUD4 proteins. Using an in vivo RNAi approach, we show that CG3251 might function in the regulation of Inhibitor of Apoptosis (IAP)-antagonist-induced apoptosis, presumably in a DUB-independent manner.
Highlights
Posttranslational modification of proteins with mono- or poly-ubiquitin is a highly versatile tool to control for example protein stability, protein localization or signaling processes
Analysis of the potential catalytic triad of CG3251 revealed a lack of the catalytic cysteine, which is replaced by serine (S40, Fig 1A and 1B), while D37 and H143 represent the additional two amino acids of the triad
Alignment of the Ovarian Tumor- (OTU) domains of CG3251 (UniProt accession number Q9VR20), Otu and OTUD4 is shown in Fig 1B and the position of amino acids of the catalytic triad is highlighted in yellow
Summary
Posttranslational modification of proteins with mono- or poly-ubiquitin is a highly versatile tool to control for example protein stability, protein localization or signaling processes. Ubiquitin gets covalently linked to lysine residues of the target protein or to lysines within another ubiquitin molecule, which leads to the formation of poly-ubiquitin chains. These chains can be linked through seven different internal lysines. Different chain types are recognized by proteins with specialized ubiquitin-binding domains (UBD), which mediate downstream events such as proteasomal degradation or formation of protein complexes [1]. An important regulatory mechanism is the removal of ubiquitin chains by de-ubiquitylating enzymes (DUBs). Five different classes of DUBs have been described, based on their type of protease domain: Ubiquitin-specific proteases (USPs), Ubiquitin-COOH-terminal hydrolases
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