ap1 In humans several different small ubiquitin related modi
In humans, several different small ubiquitin-related modifier (SUMO) paralogs can be conjugated to cellular proteins. The human genome codes for five SUMO paralogs (SUMO1–5); of these, SUMO1 and the almost identical SUMO2 and SUMO3 are ubiquitously expressed. Sumoylation is executed by an enzymatic triad that covalently attaches SUMO to selected substrates in a hierarchical process. First, the unique heterodimeric SUMO E1 enzyme Aos1/Uba2 activates SUMO by ATP-driven adenylation of the SUMO C-terminus followed by formation of an energy-rich thioester bond between a Uba2 cysteine and the SUMO C-terminus. Next, SUMO is transferred to the unique E2 enzyme Ubc9 again resulting in a thioester linkage (~). Eventually, the SUMO C-terminus is conjugated to a substrate lysine residue, forming an isopeptide bond (*). The final conjugation step usually involves E3 ligases, which stabilize the interaction of the SUMO~charged E2 enzyme with the substrate. However, exceptional cases allow efficient modification also in the absence of E3 ligases (Pichler, Fatouros, Lee, & Eisenhardt, 2017). Generally, substrates can be modified at single or multiple lysines either with a single SUMO moiety or with poly SUMO chains. The most abundant sumoylation sites are acceptor lysines embedded in a SUMO consensus motif (SCM) ψKxE (ψ: hydrophobic amino ap1 with preference for V and I), but non-SCM lysines can also be modified especially upon stress conditions (Hendriks et al., 2017, Hendriks et al., 2018; Hendriks & Vertegaal, 2016). The number of known SUMO-ligating enzymes is very limited, with far fewer than the number of corresponding enzymes used for ubiquitin-protein ligation. In mammals, these SUMO pathway enzymes comprise the single E1 and E2 enzymes and a handful E3 ligases that belong to three different classes: 1. the SP-RING family consisting of PIAS1, PIAS2, PIAS3, PIAS4, and MMS21; 2. RanBP2; and 3. the ZNF451-family presented by ZNF451–1, ZNF451–2, ZNF451–3, and the primate-specific KIAA1586 protein (Cappadocia, Pichler, & Lima, 2015; Eisenhardt et al., 2015; Kahyo, Nishida, & Yasuda, 2001; Pichler, Gast, Seeler, Dejean, & Melchior, 2002; Sachdev et al., 2001). This small enzyme number is especially surprising in the light of thousands of SUMO substrates which have been identified in cells (Hendriks et al., 2017, Hendriks et al., 2018; Hendriks & Vertegaal, 2016). Detailed biochemical and structural analyses of the three known classes of bona fide SUMO E3 ligases revealed that specific donor-SUMO (SUMOD) positioning is a hallmark of E3-dependent catalysis along with the ability to bind E2 (Cappadocia et al., 2015; Eisenhardt et al., 2015; Reverter & Lima, 2005; Streich & Lima, 2016; Yunus & Lima, 2009). SUMOD is the SUMO that forms the thioester linkage with the E2 enzyme and its positioning leads to an optimal orientation, the so-called closed conformation, for nucleophilic attack of the incoming substrate lysine ɛ-amino group, resulting in efficient isopeptide formation. Thus, SUMOD positioning and rapid discharge of the SUMOD~E2 are diagnostic features of E3 ligases that can be monitored in in vitro sumoylation reactions. The interface between the E3 ligase and the E2 enzyme can vary, and ZNF451 and SP-RING ligases stabilize this interaction via noncovalent binding to a scaffold SUMO (SUMOB) on the backside of the E2 (Cappadocia et al., 2015; Eisenhardt et al., 2015; Streich & Lima, 2016). By contrast, RanBP2 does not involve such a scaffold SUMO and interacts directly with the backside of the E2 (Pichler, Knipscheer, Saitoh, Sixma, & Melchior, 2004; Reverter & Lima, 2005). Hence, E2 backside interaction is also important for efficient substrate modification of most but not all E3 ligases and thus can also be addressed by using the in vitro sumoylation assays (see below). Substrate sumoylation can be visualized by SDS polyacrylamide gel electrophoresis (PAGE) due to the stability of the isopeptide bond in SDS and DTT. SUMO attachment results in a ~15–20kDa size shift in substrate mobility as measured by SDS-PAGE (the predicted ~10kDa SUMO migrates more slowly in gels because of its flexible N-terminus). Thus, SUMO–protein conjugates can be distinguished from noncovalent SUMO interactions such as those that occur through a SUMO interaction motif (SIM) or with the backside of the E2 (Pichler et al., 2017). Also thioester bonds, such as the linkage in SUMOD~E2, can be visualized by SDS-PAGE, but in contrast to isopeptide bonds, this linkage is sensitive to reducing agents and should not exceed a final concentration of 0.1mM DTT.