Ubiquitination is a reversible posttranslational modificatio
Ubiquitination is a reversible posttranslational modification. The removal of Ub is carried out by enzymes known as deubiquitinases (DUBs). The antagonistic role played by these enzymes in the Ub pathway regulates the function of the ubiquitinated proteins, while maintaining the free Ub pool in eukaryotic 5-Formyl-CTP sale (Wilkinson, 1997). DUBs are a large group of enzymes, classified into multiple cysteine- or metallo-protease families, that function by cleaving the isopeptide bond between Ub and its substrate protein (Nijman et al., 2005; Ronau, Beckmann, & Hochstrasser, 2016).
Bacterial pathogens, such as Legionella pneumophila, have developed creative ways to survive, colonize, and proliferate within eukaryotic hosts, often by targeting immune response cascades and the Ub system. The pathogen achieves this by translocating over 300 effector molecules through the Dot/Icm type IV secretion system into the host cell cytoplasm (Luo, 2011; Luo & Isberg, 2004). Legionella, through these effector molecules, has cultivated quite a refined mechanism to commandeer the eukaryotic ubiquitination pathway and allow propagation of the Legionella-containing vacuole (LCV) for bacterial survival within its hosts.
The SidE family of effectors from Legionella contains four members, of which SdeA is the prototype (Luo, 2011; Luo & Isberg, 2004). Past studies have shown that these proteins are essential for intracellular bacterial replication (Bardill, Miller, & Vogel, 2005) and that they associate with the LCV at the early stages of bacterial infection. All the members of this family contain a CE-clan cysteine protease DUB domain at their N-terminal end; the DUB domain catalyzes Ub removal via a nucleophilic cysteine thiol in the canonical catalytic triad (Cys-His-Asp). Surprisingly, this module showed a preference for K63-linked polyubiquitin chains (Sheedlo et al., 2015), which may allow for efficient removal of Ub from the phagosome.
DUBs show different layers of specificity. These enzymes not only differ in their preference for Ub and Ub-like proteins, but they also show differences in the type of Ub–Ub lysine linkage they prefer to cleave. The generation of modified Ub-derived probes with reactive C-terminal ends (Borodovsky et al., 2002; Eger, Scheffner, Marx, & Rubini, 2012; Ekkebus et al., 2013; El Oualid et al., 2010), such as Ub vinyl-sulfone (Ub-VS), Ub vinyl methyl ester (Ub-VME), and Ub propargylamide (Ub-Prg), has been fundamental for gaining insights into the mechanism of cysteine-dependent DUBs. These probes have also played a crucial role in identifying new DUBs in cells. To illustrate the utility of these tools, we describe in detail the methods we use to express and purify the very unique bacterial enzyme, SdeA, and we also describe the assays we have employed to analyze its deubiquitinating activity.
Constructs used for in vitro functional analyses
Assays for characterization of the DUB activity of SdeA There are nearly 20 small Ub/Ub-like proteins in humans (van der Veen & Ploegh, 2012). They all contain the same distinctive Ub fold. A subset of these are called ubiquitin-like modifiers, which modify proteins using a mechanism that is quite similar to ubiquitination. Prominent examples include SUMO, NEDD8, and ISG15. These modifiers, especially NEDD8 and SUMO, are of similar size and shape to Ub as well, and at least SUMO also forms polymers. Despite these similarities, DUBs are able to discriminate between Ub and Ub-like modifiers. A key component of this selectivity is the ability of DUBs to recognize the C-terminal segment of Ub, which is the region proximal to the substrate attachment site. However, NEDD8 and ISG15 have 7–10 amino acid residues at the C-terminal end that are similar or identical to Ub. Described below is a gel shift assay that was used to test if SdeA was indeed a DUB. The assay employed suicide probes to test the ability of the enzyme to react with Ub and form a covalent adduct.