Abstract
Posttranslational modifications by small ubiquitin-like modifiers (SUMOs) regulate many cellular processes, including genome integrity, gene expression, and ribosome biogenesis. The E2-conjugating enzyme Ubc9 catalyzes the conjugation of SUMOs to ϵ-amino groups of lysine residues in target proteins. Attachment of SUMO moieties to internal lysines in Ubc9 itself can further lead to the formation of polymeric SUMO chains. Mono- and poly-SUMOylations of target proteins provide docking sites for distinct adapter and effector proteins important for regulating discrete SUMO-regulated pathways. However, molecular tools to dissect pathways depending on either mono- or poly-SUMOylation are largely missing. Using a protein-engineering approach, we generated high-affinity SUMO2 variants by phage display that bind the back side binding site of Ubc9 and function as SUMO-based Ubc9 inhibitors (SUBINs). Importantly, we found that distinct SUBINs primarily inhibit poly-SUMO chain formation, whereas mono-SUMOylation was not impaired. Proof-of-principle experiments demonstrated that in a cellular context, SUBINs largely prevent heat shock-triggered poly-SUMOylation. Moreover, SUBINs abrogated arsenic-induced degradation of promyelocytic leukemia protein. We propose that the availability of the new chain-selective SUMO inhibitors reported here will enable a thorough investigation of poly-SUMO-mediated cellular processes, such as DNA damage responses and cell cycle progression.
Highlights
Posttranslational modifications by small ubiquitin-like modifiers (SUMOs) regulate many cellular processes, including genome integrity, gene expression, and ribosome biogenesis
S2 variants (S2vs) derived from such a library that lack the C-terminal di-Gly motif (⌬GG) cannot be activated by the E1 complex or become a part of SUMO2/3 chains when expressed in cells
Interface residues of SUMO2 buried in complexes with Ubc9 or de-conjugating enzymes are located on the 1 and 2, 3, and the C-terminal 4 strand extending into the C-terminal di-Gly motif
Summary
Site-specific inhibition of the small ubiquitin-like modifier (SUMO)-conjugating enzyme Ubc selectively impairs SUMO chain formation. One critical target in this process is mediator of DNA damage checkpoint 1 (MDC1), which is ubiquitinated by RNF4 in a poly-SUMO-dependent manner [11, 12]. Back side binding is critical for poly-SUMO chain formation catalyzed by tandem SIM-containing SUMO-E3 ligases [18, 19] Based on these insights, we hypothesized that high affinity inhibitors that disrupt the binding of SUMO to the back side of Ubc could be used to block SUMOylation processes. SUBINs inhibit poly-SUMOylation in vitro, limit cellular SUMO chain formation in response to heat stress, and prevent arsenic-induced degradation of PML
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