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

Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2011; 286(12):10238-47. DOI: 10.1074/jbc.M110.205153
Source: PubMed


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.

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Available from: Tharan Srikumar, Sep 25, 2015
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    • "The Authors P4 position of the DUB cleavage site in the C terminus of ubiquitin (Figure 1A); the analogous mutation in SUMO2 (Figure S1A) results in a conjugatable but deconjugation-resistant SUMO (Bé ké s et al., 2011). To test the ''uncleavability'' of UbL73P in the context of a linear peptide bond, we expressed recombinant linear diubiquitin (M1 linked) containing the L73P mutation in both ubiquitin moieties with an N-terminal Smt3-tag (Figure 1B) and tested it as a substrate for USP2 CD (Figures 1C and S1B). "
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    ABSTRACT: The ubiquitin-modification status of proteins in cells is highly dynamic and maintained by specific ligation machineries (E3 ligases) that tag proteins with ubiquitin or by deubiquitinating enzymes (DUBs) that remove the ubiquitin tag. The development of tools that offset this balance is critical in characterizing signaling pathways that utilize such ubiquitination switches. Herein, we generated a DUB-resistant ubiquitin mutant that is recalcitrant to cleavage by various families of DUBs both in vitro and in mammalian cells. As a proof-of-principle experiment, ectopic expression of the uncleavable ubiquitin stabilized monoubiquitinated PCNA in the absence of DNA damage and also revealed a defect in the clearance of the DNA damage response at unprotected telomeres. Importantly, a proteomic survey using the uncleavable ubiquitin identified ubiquitinated substrates, validating the DUB-resistant ubiquitin expression system as a valuable tool for interrogating cell signaling pathways.
    Full-text · Article · Nov 2013 · Cell Reports
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    • "In a complimentary approach, we enhanced SUMO-2/3 conjugation by overexpression of YFP-SUMO-2 Q90P and YFP-SUMO-2 DGG in HEK293 cells. SUMO-2 Q90P is resistant to SENP3-mediated deSUMOylation (Bekes et al, 2011) and should provide maximal SUMO-2 conjugation whereas SUMO-2 DGG is unable to conjugate (Li et al, 2006). As predicted, YFP-SUMO-2 Q90P reduced LDH release compared to SUMO-2 DGG in cells subjected to OGD plus reoxygenation (Supplementary Figure 7B) further highlighting the crucial role of SUMO-2/3 conjugation in cell survival after OGD plus reoxygenation. "
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    ABSTRACT: Global increases in small ubiquitin-like modifier (SUMO)-2/3 conjugation are a neuroprotective response to severe stress but the mechanisms and specific target proteins that determine cell survival have not been identified. Here, we demonstrate that the SUMO-2/3-specific protease SENP3 is degraded during oxygen/glucose deprivation (OGD), an in vitro model of ischaemia, via a pathway involving the unfolded protein response (UPR) kinase PERK and the lysosomal enzyme cathepsin B. A key target for SENP3-mediated deSUMOylation is the GTPase Drp1, which plays a major role in regulating mitochondrial fission. We show that depletion of SENP3 prolongs Drp1 SUMOylation, which suppresses Drp1-mediated cytochrome c release and caspase-mediated cell death. SENP3 levels recover following reoxygenation after OGD allowing deSUMOylation of Drp1, which facilitates Drp1 localization at mitochondria and promotes fragmentation and cytochrome c release. RNAi knockdown of SENP3 protects cells from reoxygenation-induced cell death via a mechanism that requires Drp1 SUMOylation. Thus, we identify a novel adaptive pathway to extreme cell stress in which dynamic changes in SENP3 stability and regulation of Drp1 SUMOylation are crucial determinants of cell fate.
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    • "Removal of SUMO from targets is a dynamic process, and SUMO deconjugation is important for cellular health (Bekes et al., 2011). Three proteases remove SUMO from its targets in yeast. "
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    ABSTRACT: Microtubules and microtubule-associated proteins are fundamental for multiple cellular processes including mitosis and intracellular motility, but the factors that control MAPs are poorly understood. In this report, two MAPs, the CLIP-170 homolog Bik1p and the Lis1 homolog Pac1p, interact with several proteins in the sumoylation pathway. Bik1p and Pac1p interact with Smt3p the yeast SUMO, Ubc9p an E2, and Nfi1p an E3. Bik1p interacts directly with SUMO in vitro and overexpression of Smt3p and Bik1p results in its in vivo sumoylation. Modified Pac1p is observed when the SUMO protease Ulp1p is inactivated. Both ubiquitin and Smt3p copurify with Pac1p. In contrast to ubiquitination, sumoylation does not directly tag the substrate for degradation. However, SUMO-Targeted Ubiquitin Ligases (STUbLs) can recognize a sumoylated substrate and promote its degradation via ubiquitination and the proteasome. Both Pac1p and Bik1p interact with the STUbL Nis1p-Ris1p and the protease Wss1p. Strains deleted for RIS1 or WSS1 accumulate Pac1p conjugates. This suggests a novel model in which the abundance of these MAPs may be regulated via STUbLs. Pac1p modification is also altered by Kar9p and the dynein-regulator, She1p. This work has implications for the regulation of dynein's interaction with various cargoes, including its off-loading to the cortex.
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