Since both the Ub and ISG systems are
Since both the Ub and ISG15 systems are crucial for the innate immune response, many prokaryotic and viral pathogens have evolved ways to hijack them in order to create a “window-of-opportunity” for efficient replication. Several viral and bacterial proteins have been found to directly target these systems via their deubiquitinating or deISGylating activity (Li, Chai, & Liu, 2016). For example, proteases derived from severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and Crimean-Congo hemorrhagic fever virus (CCHFV) have been shown to deconjugate both Ub and ISG15 in order to suppress innate immune responses (Barretto et al., 2005; Bekes et al., 2016; Frias-Staheli et al., 2007; Lindner et al., 2005; Mielech, Kilianski, Baez-Santos, Mesecar, & Baker, 2014). In mechlorethamine to eukaryotic DUBs, proteases encoded by pathogens (bacteria and viruses) often deconjugate more than one type of Ubl. Another example is the CE clan bacterial effector proteases from, for instance, Rickettsia bellii and Chlamydia trachomatis, which were shown to display both deubiquitinating and deNeddylating activities (Lin & Machner, 2017; Pruneda et al., 2016). For many human DUBs and Ubl-specific proteases, it has been shown that their malfunction contributes to human disease, including cancer and neurodegenerative disorders (Harrigan, Jacq, Martin, & Jackson, 2018); therefore, tools to study them in detail and on a molecular level are of great interest. Often, proteases are translated as inactive proenzymes, requiring posttranslational activation by their natural regulators. In addition, their activity may be controlled by posttranslational modifications, such as acetylation, phosphorylation, ubiquitination, or methylation. In order to study the role of DUBs in biological processes, it is therefore insufficient to simply monitor the enzyme\'s abundance by antibody staining, proteomics, or mRNA quantification because this is not necessarily related to a protein\'s activity (Hewings, Flygare, Bogyo, & Wertz, 2017). A powerful method to visualize enzyme activities in a complex biological setting is the use of Activity-Based Probes (ABPs) (Ovaa, 2007; Verdoes & Verhelst, 2016). ABPs come in many flavors and their design is predominantly determined by their respective protein target(s) and the particular application of the ABP. Generally, ABPs comprise a recognition element, that directs the ABP toward its target, attached to a reactive group (or “warhead”) that reacts with the enzyme\'s active site to form a covalent adduct, either reversible or irreversible, depending on the type of enzyme and reactive group installed (see Fig. 1). The recognition element is designed to resemble structural and functional motifs of the natural substrate of the target, in the form of a short peptide, carbohydrate, nucleoside, or even a small protein. A variety of ABPs has been developed to study the activities of DUBs and these all share a common recognition element derived from full-length Ub. Typically, but not necessarily, an ABP is also equipped with a reporter group, such as a fluorophore, radioactive label or affinity tag, which is used for visualization, purification or identification of the ABP-bound target(s). These chemical tools are designed in such a way that they only bind to active enzymes covalently but do not react with their inactive counterparts. The application of ABPs is widespread. For example, these tools are commonly used in combination with mass spectrometry, to capture, isolate, and identify active enzymes from cells or cell extracts (Cravatt, Wright, & Kozarich, 2008). In addition, ABPs can be applied to determine the active fraction of a recombinantly expressed and purified enzyme or to study the effect of specific enzyme modifications or mutations with respect to the enzyme\'s activity and its substrate specificities (Mevissen et al., 2013, Mevissen et al., 2016). ABPs are also very useful tools for gaining insight into the structural characteristics of an enzyme, where an enzyme–ABP complex mimics a certain state of the reaction between the enzyme and its substrate (Basters et al., 2017; van Tilburg, Elhebieshy, & Ovaa, 2016). Also, by designing and testing different structural variants of an ABP (Flierman et al., 2016; Mulder, El Oualid, ter Beek, & Ovaa, 2014), one can identify preferences of a given enzyme for certain structural features (Bekes et al., 2015, Bekes et al., 2016; Mevissen et al., 2016). Finally, since only the active fraction of an enzyme is labeled by the ABP, it is possible to check the inhibitory potential of an inhibitor toward one or multiple enzymes in a cell or cell lysate, e.g., by means of an ABP competition assay (Altun et al., 2011; de Jong et al., 2012).