More unusual modifications also regulate DUBs Recent reports
More unusual modifications also regulate DUBs. Recent reports have illustrated how reactive oxygen species (ROS) can regulate DUB activity 87, 88, 89, 90. ROS can serve as potent signaling molecules by reacting with active site cysteines of tyrosine phosphatases and some cysteine peptidases to form reversible sulfenic TAK-875 adducts or irreversible sulfinic or sulfonic acid adducts . Oxidation of active site cysteines to sulfenic acid appears to be widespread in the OTU, USP, and UCH classes of DUBs, where it reduces DUB activity. Functionally, the modification may have important roles in cells, as exemplified by the oxidative inactivation of the PCNA deubiquitinase USP1 . This results in the accumulation of monoubiquitinated PCNA, a mark of cellular stress responses 88, 89.
Multiple layers of regulation In the previous sections, we discussed separately for illustrative purposes how certain types of regulation impinge on DUB activity. In practice, however, many regulatory mechanisms coexist. This situation sometimes even occurs within a single domain. The RPN13 DEUBAD domain, for example, is responsible for both the activation of UCH-L5 and recruiting the enzyme to the proteasome 62, 63, 64. We expect that more factors exist that have multiple regulatory roles that occur simultaneously. The concept of multiple regulatory layers can best be exemplified by considering USP7 (Figure 2 and Table 1). The USP7 CD can exist in a catalytically incompetent state that can be activated by ubiquitin binding and USP7 HUBL-45 27, 28. This active state can be further reinforced allosterically by the external factor GMPS, which binds to HUBL-123 28, 47 and target recruitment is promoted by its N-terminal TRAF domain 23, 24. Another example of multiple layers of regulation impinging on a single protein is the tumor suppressor BAP1. This enzyme is activated by ASXL1 to deubiquitylate H2A and can be targeted to certain genomic loci by its association with transcriptional regulators 20, 61, 92. BAP1 can furthermore be spatially separated from its targets by ubiquitylation of its C-terminal nuclear localization signal (NLS) by UBE2O, causing mislocalization to the cytoplasm . This type of multilayered regulation is likely a feature of many DUBs and multiprotein DUB complexes, and contributes to the tight control of deubiquitylation.
Concluding remarks Research on the mechanisms of DUB regulation has advanced significantly over the past few years. The regulation takes place at different layers and a notable feature is the diversity of the known mechanisms. This variety is also present at the biochemical level: the regulatory mechanisms range from solely impinging on catalytic activity (kcat) to primarily substrate interaction (KM), to combinations of both. The accumulated regulatory effects of the layers determine DUB activity and, ultimately, the fate of ubiquitylated substrates. Even though we understand some aspects of DUB mechanism, there are important outstanding questions (Box 5), such as how DUB activity is regulated within the large macromolecular complexes. For example, recently determined UCH-L5/RPN13 structures give insights into the basic activation mechanisms of UCH-L5 65, 66, but do not explain the polyubiquitin hydrolysis activity of UCH-L5 as part of the proteasome. Similar challenges exist for other DUBs and can only be addressed by studying large holo-enzyme complexes over different activation states.
Introduction The ubiquitin modification is required in the regulation of both proteolytic and non-proteolytic events, targeting proteins for proteasomal or lysosomal degradation, protein interactions, protein activity, protein localization to signaling transduction (Swatek & Komander, 2016). Like the balance of phosphorylation events by kinases and phosphatases, ubiquitin modification could be simply divided into two parts: the covalent attachment of ubiquitin mediated by ubiquitination enzymes including E1 activating enzyme, E2 conjugating enzyme and E3 ligase and the reversible removal of ubiquitin catalyzed by deubiquitinating enzymes (DUBs) (Hershko and Ciechanover, 1998, Komander and Rape, 2012).