The Dynamics and Mechanism of SUMO Chain Deconjugation by SUMO-specific Proteases

Abstract

SUMOylation of proteins is a cyclic process that requires both conjugation and deconjugation of SUMO moieties. Besides modification by a single SUMO, SUMO chains have also been observed, yet the dynamics of SUMO conjugation/deconjugation remain poorly understood. Using a non-deconjugatable form of SUMO we demonstrate the underappreciated existence of SUMO chains in vivo, we highlight the importance of SUMO deconjugation, and we demonstrate the highly dynamic nature of the SUMO system. We show that SUMO-specific proteases (SENPs) play a crucial role in the dynamics of SUMO chains in vivo by constant deconjugation. Preventing deSUMOylation in Schizosaccharomyces pombe results in slow growth and a sensitivity to replication stress, highlighting the biological requirement for deSUMOylation dynamics. Furthermore, we present the mechanism of SUMO chain deconjugation by SENPs, which occurs via a stochastic mechanism, resulting in cleavage anywhere within a chain. Our results offer mechanistic insights into the workings of deSUMOylating proteases and highlight their importance in the homeostasis of (poly)SUMO-modified substrates.Reversible post-translational modification of proteins by ubiquitin-like covalent modifiers is a widely utilized mechanism to alter the fate, binding partners, function, or localization of a given target protein (1). A prime example is the covalent attachment of multiple ubiquitin moieties to a lysine side-chain of a target protein (2), leading to the formation of ubiquitin chains, which have different functions depending on the lysine utilized in the internal ubiquitin linkage. Recently, small ubiquitin-like modifier (SUMO) 2 has also been shown to form chains in vitro (3, 4) and in vivo (5, 6).The SUMO conjugation pathway consists of SUMO activation, transfer, and ligation enzyme machinery analogous to the ubiquitin pathway (7-9). Somewhere upwards of 500 cellular proteins are SUMOylated in vivo (10), yet the extent of observable SUMOylation is only a snapshot due to the presence of SUMO-specific proteases, SENPs. The SUMO cycle begins and ends with specific proteolytic events: the processing of pro-SUMO and the deconjugation of SUMO from the target protein by SENPs (11). In addition to monoSUMOylation, there is now growing evidence that SUMO, like ubiquitin, forms polymeric chains. Thus, the non-covalent interaction of the SUMO conjugating enzyme, Ubc9, with SUMO has presented a possible mechanism for SUMO chain formation (4, 12) and indeed in vitro, all SUMO molecules (Smt3, the Saccharomyces cerevisiae SUMO homologue, and human SUMO1, -2, and -3) have been observed to form SUMO chains, primarily via lysine residues located near their N termini (13). In vitro the SUMOylation system is quite permissive, allowing for multiple . The initial indication that SUMO chains exist in vivo came from transfection experiments yielding multimers of SUMO2 conjugated to HDAC4 (3) and from S. cerevisiae (15). More recently, evidence for in vivo SUMO chains comes from mass-spectrometry experiments using HeLa ...

The Human Cytomegalovirus IE1 Protein Antagonizes PML Nuclear Body-Mediated Intrinsic Immunity via the Inhibition of PML De Novo SUMOylation

“…In the present study, we report for the first time that IE1 is able to prevent the de novo SUMOylation of PML. By using a nondeconjugatable SUMO mutant, described by Békés and colleagues, we were able to overcome the rapid and reversible dynamics of PML SUMOylation (43). These dynamics are caused by the presence of both SUMO conjugating enzymes and deconjugating SUMO proteases inside PML NBs, which are considered to represent nuclear "SUMO hot spots" (19).…”

“…Unfortunately, these dynamics impede the ability to discriminate whether IE1 mediates proteolytic PML deSUMOylation or inhibition of PML de novo SUMOylation. To overcome these dynamics, we generated a nondeconjugatable mutant of SUMO2 by mutagenizing SUMO residue 90 from glutamine to proline (43). Since SUMO proteases are necessary not only for deconjugation of SUMO but also for processing and thereby conjugation, we utilized an already processed version of SUMO2, termed SUMO2 Q90P.…”

Swapping Small Ubiquitin-like Modifier (SUMO) Isoform Specificity of SUMO Proteases SENP6 and SENP7

“…Another important difference between both SUMO isoforms is that SUMO2/3 can form polySUMO2/3 chains through an N-terminal lysine residue (11)(12)(13). One function of the poly-SUMO2/3 chains might be the stabilization of PML nuclear bodies (14), which is also the signal for the ubiquitin-dependent degradation by the recruitment of the E3 ubiquitin ligase RNF4 (15,16). A recent study has analyzed the dynamics of conjugation/deconjugation of polySUMO chains, highlighting in Schizosaccharomyces pombe the role of deconjugation for the correct homeostasis of the cell (17).…”

The Human 343delT HSPB5 Chaperone Associated with Early-onset Skeletal Myopathy Causes Defects in Protein Solubility

“…MYC-WT and MYC343delT were PCR-amplified from pCMV-MYC vectors and cloned into a pCS2 vector that contained an SP6 promoter required for the TnT assay (see below). For SUMOylated constructs, a gBlock gene fragment (Integrated DNA Technologies) was purchased, containing the non-cleavable SUMO3 Q89P sequence obtained from the pcDNA3 MYC-SUMO3Q89P vector (Addgene plasmid 48963) (46), and cloned into the pCMV-MYC-WT and 343delT constructs. The resulting constructs, pCMV-MYC-SUMO3 Q89P-WT and pCMV-MYC-SUMO3 Q89P-343delT, were a fusion in the N to C-terminal direction of MYC-SUMO3Q89P-WT and 343delT HSPB5, respectively.…”

“…These plasmids contain a version of WT and 343delT HSPB5 that are N-terminally MYC-tagged followed by SUMO, preceding the HSPB5 variant. We used a non-cleavable version of SUMO3 Q89P (46) in this construct to prevent removal of SUMO by endogenous deSUMOylases. MYC tag-only versions of the constructs were used as controls.…”