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  • br Introduction The ubiquitination status of

    2020-04-09


    Introduction The ubiquitination status of a target protein is achieved via a delicate balance between two opposing forces: ubiquitin E3 ligases and DUBs. It has been postulated that the majority of proteins in a cell are regulated and modified by ubiquitin at some point (Hershko and Ciechanover, 1998); however, it has proved difficult to demonstrate the ubiquitination status of these proteins, as many of the modifications only exist transiently in vivo, owing largely to the multitude of DUBs present in cells. There are nearly 100 DUBs encoded in the human genome, which comprise approximately one-fifth of all proteases (Rawlings et al., 2010). DUBs are divided into five subfamilies based on catalytic mechanism and the fold of the active site domain (Reyes-Turcu et al., 2009). The largest family are the ubiquitin-specific proteases (USPs), followed by the ovarian tumor DUBs (OTUs), the ubiquitin C-terminal hydrolases (UCHs), the Josephin DUBs, and, last, the JAMM/MPN+ family member DUBs. Except for the JAMM/MPN+ family of DUBs, which are zinc-dependent metalloproteases, all other characterized DUBs are cysteine proteases. As the ubiquitin-modified status of a protein can fundamentally alter its properties and change its biological role, it is critical to capture the ubiquitinated state of a protein in order to investigate its biological function both in vitro and in vivo. The ubiquitin system has recently been exploited with the design of unique ubiquitin mutants that inhibit specific DUBs (Ernst et al., 2013, Zhang et al., 2013). Although the specificity of such mutants is remarkable, generating a novel mutant for each DUB has to begin de novo and is quite laborious, especially when the physiological substrates of many DUBs remain unknown. In this study, we designed and generated a DUB-resistant ubiquitin to capture and identify transiently ubiquitinated DUB substrates. Building on previous work in the SUMO conjugation and deconjugation pathway (Békés et al., 2011), we have generated a ubiquitin mutant (UbL73P) that is pleiotropically resistant to cleavage by multiple DUB families. This uncleavable ubiquitin mutant is conjugated to protein substrates in mammalian Rosiglitazone and leads to ubiquitin-conjugate stabilization. Ectopic expression of the DUB-resistant ubiquitin mutant stabilized monoubiquitinated PCNA, leading to the aberrant recruitment of translesion synthesis (TLS) polymerases in the absence of DNA damage, mimicking the effect of USP1 loss. Additional studies with DUB-resistant ubiquitin revealed a ubiquitin switch in the clearance of the DNA damage response (DDR) at shelterin-deficient chromosomal ends and captured ubiquitin-stabilized substrates by mass spectrometry. Our work provides a framework to study deubiquitination-dependent events both in vitro and in mammalian cells through the generation and use of the DUB-resistant ubiquitin tool.
    Results
    Discussion In summary, we have introduced and characterized a ubiquitin point mutant capable of conjugating to cognate substrates and incorporating into ubiquitin chains, yet remains refractory to cleavage by DUBs. This provides a unique tool to enable the generation, identification, and study of substrates with stabilized ubiquitination states. Previous work by Békés et al. (2011) on the related ubiquitin-like molecule SUMO2 showed that mutation in the P4 position of the SUMO cleavage site for deSUMOylating enzymes (SENPs) from glutamine to proline (Figure S1A, Q90P, which is also found naturally in the pseudogene, SUMO4 [Bohren et al., 2007]), results in resistance to cleavage by deSUMOylating enzymes, allowing the generation of stabilized SUMO2-conjugates in cells. We then asked whether mutation of the corresponding residue (Leu73) on ubiquitin could affect ubiquitin cleavage reactions by DUBs, and we indeed show that it does. Interestingly, in an alanine-scanning mutagenesis study of yeast ubiquitin more than a decade ago, it was shown that UbL73A allows for ubiquitin conjugation; however, the mutant is partially defective in an S. cerevisiae endocytosis assay (Sloper-Mould et al., 2001). A recent study has also identified bulky Leu73 mutations that differentially affect conjugation and deconjugation activities (Zhao et al., 2012). Furthermore, yeast provided with the UbL73A mutant as the only source of ubiquitin cannot support vegetative growth (Sloper-Mould et al., 2001). It is possible that the Leu73 mutant phenotype of ubiquitin in yeast is a composite of defects in conjugation as well as deconjugation. Leu73 contributes to part of the hydrophobic patch in ubiquitin centered around Ile36, which is utilized by some E3 ligases to synthesize polyubiquitin chains (Plechanovová et al., 2012). Therefore, in accordance with our in vitro E1/E2/E3 conjugation data, it is possible that certain substrates, whose E3 ligases solely rely on ubiquitin Ile36 hydrophobic interactions, would not be efficiently conjugated by UbL73P. This selectivity at the E3 level, together with the selectivity of the E1 enzymes, Ube1 and Uba6, in differentially charging E2 enzymes, suggests that the conjugation of UbL73P in vivo would be skewed toward conjugation pathways that can tolerate it. Nevertheless, those UbL73P conjugates would remain stable and resistant to DUBs in cells, as is the case for monoubiquitinated PCNA, the identified Ubc13, unknown factor(s) in the telomeric DDR pathway, and others.