Assembly of a Polymeric Chain of SUMO1 on Human Topoisomerase I in Vitro

Abstract

Human (h) DNA topoisomerase I has been identified as a major SUMO1 target in camptothecin-treated cells. In response to TOP1-mediated DNA damage induced by camptothecin, multiple SUMO1 molecules are conjugated to the N-terminal domain of a single TOP1 molecule. To investigate the molecular mechanism of SUMO1 conjugation to TOP1, an in vitro system using purified SAE1/2, Ubc9, SUMO1, and TOP1 peptides was developed. Consistent with results from in vivo studies, multiple SUMO1 molecules were found to be conjugated to the N-terminal domain of a single TOP1 molecule. Systematic analysis has identified a single major SUMO1 conjugation site located between amino acid residues 110 and 125 that contains a single lysine residue at 117 (Lys-117). Using a short peptide spanning this region, we showed that a poly-SUMO1 chain was assembled in this peptide at Lys-117. Interestingly, a Ubc9-poly-SUMO1 intermediate had accumulated to a high level when the sumoylation assay was performed in the absence of hTOP1 substrate, suggesting a possibility that the poly-SUMO1 chain is formed on Ubc9 first and then transferred en bloc onto hTOP1. This is the first definitive demonstration of the assembly of a poly-SUMO1 chain on protein substrate. These results offer new insight into hTOP1 polysumoylation in response to TOP1-mediated DNA damage and may have general implications in protein polysumoylation.Human SUMO1 (small ubiquitin-related modifier) (also named UBL1, PIC1, GMP1, SMT3C, and sentrin in the literature) is a ubiquitin-like protein (1-3). It shares about 18% identity to ubiquitin (4). Human SUMO1 and its yeast homolog, Smt3p, also share a similar activation and conjugation pathway with ubiquitin but employ distinct sets of E1 2 and E2 enzymes (5-9). The E1 enzymes for activating human SUMO1 and yeast Smt3p are the heterodimeric proteins SAE1/SAE2 and Aos1p/Uba2p, respectively (6, 9). Ubc9 is the only E2 identified for SUMO1/Smt3p, although a dozen of E2 enzymes have been identified for ubiquitin in yeast (10 -14). Recently, proteases that specifically activate SUMO1/Smt3p precursors and cleave SUMO1/Smt3p from their protein conjugates have been identified in yeast and mammalian cells (15)(16)(17)(18). Many proteins such as RanGAP1 (19), promyelocytic leukemia protein (20), IB␣ (21), RAD51, RAD52 (22, 23), p53 (24), and centromere proteins (25, 26), which have diverse functions, have been shown to interact with Ubc9/SUMO1 or be covalently modified by SUMO1. SUMO1 appears to primarily target nuclear proteins (23). Although the function of SUMO1 is still elusive, there are a few interesting observations that may shed light on the function of SUMO1. First, Pods (promyelocytic leukemia protein oncogenic domains, also called nuclear bodies or ND10) (27) and the nuclear envelope appear to be the major site of localization of SUMO1 conjugates in the nucleus (28). Second, SUMO1 and ubiquitin appear to share the same conjugation site(s) on some target proteins (e.g. IB␣ and MDM2) (21). Third, stress-caused protein unfolding (e.g. heat s...

Keratin Hypersumoylation Alters Filament Dynamics and Is a Marker for Human Liver Disease and Keratin Mutation

“…Our in vitro sumoylation assay that showed SUMO-1 chain formation with IFs (Fig. 1B) is similar to at least one previous report (47). Additionally, the existence of mixed SUMO/ubiquitin chains has also been reported.…”

“…Although the exact functional significance of each type of modification is poorly defined, there is evidence for a structural role of polymeric SUMO-2/3 chains in kinetochore function during cell division (45,46) as well as a role for sumoylation in the ubiquitin-proteasome system (36,37). Unlike SUMO-2 and -3, which form polymeric chains in vivo via internal sumoylation sites, SUMO-1, by lacking such a sites, is not known to polymerize in vivo, but it does so in vitro (47). However, in vivo SUMO-1 can be linked to the end of SUMO-2/3 chains, resulting in a mixed SUMO chain (37,48).…”

A Novel Post-translational Modification of Nucleolin, SUMOylation at Lys-294, Mediates Arsenite-induced Cell Death by Regulating gadd45α mRNA Stability

“…Due to the lack of this motif, SUMO1 usually occurs in mono-form and leads to functional modification of the target proteins (12). * This work was supported, in whole or in part, by National Institutes of Health However, the existence of polymeric chains of SUMO1 has also been reported in the case of nuclear proteins of RanBP2 (13) and topoisomerase 1 (14), which might be formed by a noncovalent bond between the C-terminal glycine residue of SUMO1 and the ⑀-amino group of an internal lysine residue of another SUMO1 (14).…”

Characterization of The Interaction Between RIN13 (RPM1-Interacting13) and Sumo Proteins in Arabidopsis Thaliana

“…The in vitro results are well consistent with the in vivo observation ( Figure 3B), and thus eliminated the possibility of two-hybrid false positive. Combined, the results suggested by, 2006; Eckhoff & Dohmen, 2015;Park et al, 2011;Yang, Hsu, Ting, Liu, & Hwang, 2006). In addition, human SUMO2 and SUMO3 formed chains in vitro and in vivo, primarily through a conserved acceptor, Lys11 (Park et al, 2011;Tatham et al, 2001).…”