GSK180 br Chain specific ubiquitin probes The conventional

Chain specific ubiquitin probes The conventional way of assessing linkage specificity is by measuring turnover rates of different diubiquitin molecules as substrates. Although this method has been applied successfully in several studies [7••, 8] it is limited to isolated DUBs, whereas linkage specificities may also arise from yet unknown interactions as part of larger protein complexes [48]. ABPs on the other hand are not limited to purified GSK180 and can be used to study DUBs in a complex biological setting. Recently, diubiquitin-based ABPs bearing a warhead between the ubiquitin modules were developed to allow study of linkage specificities in a relevant context, that is, cell lysates. One of the main factors that determine DUB linkage specificity is the presence of one or multiple ubiquitin-binding pockets in DUBs (Figure 4A, top panel). These allow for selective recognition of different ubiquitin chain topologies and hence the correct positioning of a polyubiquitylated substrate. The proposed S1 and S1′ sites position a polyubiquitin chain across the active-site to allow cleavage between ubiquitin molecules in a chain. The S1′ site binds the ubiquitin moiety C-terminal to the scissile bond and the distal ubiquitin moiety in the chain will bind the S1 site. For cleavage of polyubiquitin chains from a substrate, S1 and S2 binding pockets are likely used. The most proximal ubiquitin in the chain binds to the S1 site and the more distal ubiquitin binds to the S2 site. In this manner the chain is positioned such that the bond between chain and substrate can be cleaved. The previously described monoubiquitin-based probes only bind the S1 site (Figure 4A, second panel) and therefore provide no information on linkage specificity. The recently developed diubiquitin-based probes are all designed to bind the S1 and S1′ sites (Figure 4A, third panel), after which the DUB active site thiol reacts with the internal warhead. Initial steps toward diubiquitin probes were published by Iphöfer et al. [49]. They developed the first chain-specific ubiquitin isopeptide probes in which HA-tagged ubiquitin is linked through its C-terminus to a lysine sidechain in a 13-amino acid peptide derived from ubiquitin. The C-terminal glycine residue of ubiquitin was replaced by a vinyl amide warhead (Figure 4B-2). Both the Lys48 and Lys63 diubiquitin mimics were synthesized and used to label purified DUBs. In addition, they were able to capture and identify 22 DUBs from a cell lysate and to quantify their relative binding efficiencies for the Lys48- and Lys63-derived probes. The first full-length diubiquitin probes were reported by McGouran et al. [50]. They developed probes that mimic all eight natural occurring diubiquitin linkages. The distal HA-tagged ubiquitin containing the vinyl amide warhead and a terminal alkyne moiety was linked to the proximal ubiquitin containing an azidohomoalanine residue by means of a bio-orthogonal ‘click reaction’ (Figure 4B-3). In a quantitative proteomics analysis of HEK293T cell lysate, they quantified the relative selectivity of 28 DUBs for the various ubiquitin linkages. While reported data from diubiquitin turnover assays shows overlap with the apparent linkage preferences observed, there are some major differences. These differences likely arise from the difference between isolated proteins versus DUBs in their cognate environment or simply the difference in detection methods (e.g. kinetics in a cleavage assay versus end-point in probe labeling). These differences are often observed and an important point of further investigation. Li et al. [51] reported the development of a diubiquitin probe in which the native diubiquitin linkage is more accurately resembled (Figure 4B-4). From a structural point of view this probe has the correct linker length (Figure 4B-1). The authors mutated Lys48 and Lys63 from the HA-tagged proximal ubiquitin module into a cysteine residue, which they reacted to the distal ubiquitin containing the vinyl amide warhead and a bromide. Similar to the probes described above it was shown that the probes could label isolated DUBs as well as DUBs in a cell lysate with distinct profiles.