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The reversible, covalent addition of ubiquitin to target proteins is a highly conserved and flexible regulatory mechanism. Monoubiquitination can change the activity of a ubiquinated target protein, whereas the addition of a ubiquitin chain can direct a protein to the 26S proteasome for rapid proteolytic degradation. Many proteins are modified by ubiquitin or ubiquitin-like proteins through the activity of the well-known E1-E2-E3 enzyme cascade, in which the E3 ubiquitin ligase binds the<span class="external-identifiers"> <a target="_blank" rel="external noopener noreferrer" href="https://doi.org/10.1016/j.cell.2009.07.008">doi:10.1016/j.cell.2009.07.008</a> <a target="_blank" rel="external noopener" href="https://www.ncbi.nlm.nih.gov/pubmed/19632171">pmid:19632171</a> <a target="_blank" rel="external noopener" href="https://fatcat.wiki/release/bf6rd27cz5do7ntw6qvh2zld6e">fatcat:bf6rd27cz5do7ntw6qvh2zld6e</a> </span>
more »... te and recruits the ubiquitin transferase activity of the E2 ubiquitin-conjugating enzyme. In humans, more than 500 predicted E3 ubiquitin ligases act on thousands of gene products and often recognize specifically modified forms of those proteins. Opposing this army of E3 ligases are ?95 deubiquitinating enzymes (DUBs), proteases that cleave the isopeptide bond between the C-terminal glycine of ubiquitin and a side-chain lysine of a target protein or ubiquitin itself (Reyes-Turcu et al., 2009 ). There are five distinct DUB domains, of which one is a metalloprotease (JAMM domain) and four are cysteine proteases (USP, OTU, MJD, and UCH domains). It remains generally unclear how DUBs recognize their substrates. We also only have a modest understanding of the biological roles for most DUBs (Nijman et al., 2005); some have been linked to ubiquitin processing, histone modification, cell-cycle and DNA repair, kinase signaling, and endocytosis. With so much uncharted territory, how do we connect DUBs to their biology? In this issue of Cell, Sowa and colleagues (2009) approach this problem by purifying and identifying by mass spectrometry 774 high-confidence interacting proteins of 75 epitope-tagged DUBs (Figure 1 ). The authors further propose a standardized scoring method to quantify the confidence in the interactions they identify and to track improvements in the methodology itself. The three keys to proteomic identification of interacting proteins are the selectivity of the purification, the accuracy of the mass spectrometric identification of proteins, and the bioinformatic and statistical analysis of the proteomic hits. How do Sowa and colleagues approach these steps? Using a retroviral expression vector, the authors establish stable cultured cell lines expressing hemagglutinin (HA) epitope-tagged versions of the DUBs. The tagged "bait" protein is immunoprecipitated along with interacting proteins from extracts of the cells by resin harboring anti-HA antibodies. These interacting proteins are cleaved into small fragments by trypsin, and duplicate samples from the purification are analyzed by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) ( Figure 1 ). With this standard single-epitope tag affinity purification scheme, substantial nonspecific interacting proteins are also isolated. These "background" interactions are partially corrected for by control purifications, including those with the other 74 DUBs. However, this purification approach may be improved by using two independent epitope tags and thus two sequential affinity purification steps to achieve a more selective purification. Another powerful method of stringent purification involves the specific isolation of correctly localized proteins in the cell with green fluorescent protein tags
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