br Assessing DUB activity The Ub field
Assessing DUB activity The Ub field has benefited greatly from the past efforts of biochemists and chemical biologists who have reconstituted Ub signals in vitro and generated tools for capturing or measuring DUB activities, many of which can be readily produced or are commercially available (Ekkebus, Flierman, Geurink, & Ovaa, 2014; van Tilburg, Elhebieshy, & Ovaa, 2016). Critically, the nature of the DUB in question and the type of information sought must be considered before choosing a suitable substrate, as each comes with advantages and disadvantages. In the next subsections, we highlight three WP 1130 of Ub substrates and demonstrate their utility in characterizing the activity of a bacterial DUB encoded by Chlamydia trachomatis, ChlaDUB1.
Understanding DUB structure
Summary and outlook Advances in understanding DUB activity and structure have progressed hand-in-hand with developments in synthesizing suitable substrate molecules. Ub ABPs, fluorescent substrates, and natural substrates together offer a large repertoire of tools for in vitro studies. Though each comes with advantages and disadvantages (Fig. 1), together they offer rich information into many levels of DUB specificity and structure (see Section 3.1 and Fig. 2). However, pharmacological efforts targeting DUB activity demand high-throughput, and the ideal solution to this requirement remains to be seen. Advances in fluorescent substrates are producing more and more suitable mimetics to the natural substrate (Geurink et al., 2012). On the flip side, novel approaches to monitoring hydrolysis of natural substrates, such as MALDI-TOF mass spectrometry (Ritorto et al., 2014), may allow for high-throughput screening with physiologically relevant substrates. Recent years have seen major in-roads in our understanding of DUB mechanism and structure, but with every answer come many new questions as to how the complexity of Ub signaling is controlled. As our appreciation of the in vivo Ub code expands, so must our toolset for biochemical and structural studies in vitro. We now know that Ub can be coated by additional posttranslational modifications such as UBLs, phosphorylation, or acetylation (Swatek & Komander, 2016) and that Ub modification of other proteins is not limited to their Lys residues but can also be attached through the amino-terminus or Cys and Ser residue side chains (McDowell & Philpott, 2013). DUB substrates with Ub linked to the target protein in these ways are becoming available (Huguenin-Dezot et al., 2016; Sun, Meledin, Mali, & Brik, 2018) or are on the horizon, and these will undoubtedly reveal fascinating new details of DUB specificity and function.
Introduction Reversible posttranslational modifications of proteins are a universal means for regulation of a variety of cellular processes; such transient modifications help cells to respond quickly and efficiently to changes in their environment. A well-known example of protein modification is the covalent attachment of ubiquitin (Ub), a small protein of 76 amino acids (Schlesinger, Goldstein, & Niall, 1975), to lysine residues of target substrate proteins. Ubiquitination is the result of the sequential action of a set of three enzymes in a conjugation cascade. The wide spectrum of cellular functions affected by ubiquitinated proteins includes innate and adaptive immunity, proteasomal degradation of proteins, DNA repair, vesicular trafficking, and autophagy, to name a few (Grabbe, Husnjak, & Dikic, 2011). Ub itself is also able to become ubiquitinated via any one of its seven lysine (K) residues or its N-terminal amine group, giving rise to polyubiquitin chains. The variety of different possible linkages of polyubiquitin chains allows for a variety of signaling functions, forming the basis of a complex “ubiquitin code.” In addition to Ub, Ub-like proteins such as NEDD8, ISG15, and SUMO also are covalently linked to other proteins. These modifications have important roles in host invasion response cascades to various pathogens (Li, Chai, & Liu, 2016; Radoshevich et al., 2015) and in host biology more generally.