Heat shock induces a massive but differential inactivation of SUMO-specific proteases

Covalent conjugation of the small ubiquitin-like modifier (SUMO) to proteins is a highly dynamic and reversible process. Cells maintain a fine-tuned balance between SUMO conjugation and deconjugation. In response to stress stimuli such as heat shock, this balance is altered resulting in a dramatic increase in the levels of SUMO conjugates. Whether this reflects an activation of the conjugation cascade, a decrease in the activity of SUMO-specific proteases (SENPs), or both, remains unknown. Here, we show that from the five human SENPs detected in HeLa cells (SENP1/2/3/6/7) the activities of all but one (SENP6) were largely diminished after 30min of heat shock. The decreased activity is not due to changes in their steady-state levels. Rather, in vitro experiments suggest that these SENPs are intrinsically heat-sensitive, a property most likely emerging from their catalytic domains. Heat shock inactivation seems to be a specific property of SENPs because numerous members of the related deubiquitinase family of cysteine proteases are not affected by this stress condition. Overall, our results suggest that SENPs are particularly sensitive to heat shock, a property that may be important for the adaptation of cells to this stress condition.     

Small ubiquitin-related modifier (SUMO)-specific proteases: profiling the specificities and activities of human SENPs

SENPs are proteases that participate in the regulation of SUMOylation by generating mature small ubiquitin-related modifiers (SUMO) for protein conjugation (endopeptidase activity) and removing conjugated SUMO from targets (isopeptidase activity). Using purified recombinant catalytic domains of 6 of the 7 human SENPs, we demonstrate the specificity of their respective activities on SUMO-1, -2, and -3. The primary mode of recognition of substrates is via the SUMO domain, and the C-terminal tails direct endopeptidase specificity. Broadly speaking, SENP1 is the most efficient endopeptidase, whereas SENP2 and -5-7 have substantially higher isopeptidase than endopeptidase activities. We developed fluorogenic tetrapeptide substrates that are cleaved by SENPs, enabling us to characterize the environmental profiles of each enzyme. Using these synthetic substrates we reveal that the SUMO domain enhances catalysis of SENP1, -2, -5, -6, and -7, demonstrating substrate-induced activation of SENPs by SUMOs.     

The deSUMOylase SENP7 promotes chromatin relaxation for homologous recombination DNA repair

SUMO conjugation is known to occur in response to double‐stranded DNA breaks in mammalian cells, but whether SUMO deconjugation has a role remains unclear. Here, we show that the SUMO/Sentrin/Smt3‐specific peptidase, SENP7, interacts with the chromatin repressive KRAB‐associated protein 1 (KAP1) through heterochromatin protein 1 alpha (HP1α). SENP7 promotes the removal of SUMO2/3 from KAP1 and regulates the interaction of the chromatin remodeler CHD3 with chromatin. Consequently, in the presence of CHD3, SENP7 is required for chromatin relaxation in response to DNA damage, for homologous recombination repair and for cellular resistance to DNA‐damaging agents. Thus, deSUMOylation by SENP7 is required to promote a permissive chromatin environment for DNA repair.     

SENP3‐mediated deSUMOylation of dynamin‐related protein 1 promotes cell death following ischaemia

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.     

SENP1与前列腺癌

SUMO (small ubiquitin-related modifier)是一种小泛素相关修饰物,能共价结合许多调控基因转录的重要蛋白,包括转录因子、转录辅助因子等．SUMO化修饰对蛋白-蛋白之间的相互作用、亚细胞定位、基因转录的活性以及靶蛋白的稳定性等具有重要的调节作用． SUMO化修饰是一个动态可逆的过程,将SUMO从靶蛋白上去除,称为去SUMO化（desumoylation）,去SUMO化是SUMO特异蛋白酶（SUMO-specific proteases,SENPｓ）的主要功能．由于SUMO化是近几年才发现的一种新的蛋白质翻译后修饰系统,对其生物学功能还不十分清楚．前列腺癌是男性最常见的恶性肿瘤,最近的研究发现,SENP1在前列腺癌细胞中高表达,而且雄激素能诱导SENP1的表达,表明SENP1与前列腺癌的发生、发展密切相关．在本篇综述中,我们将就SENP1作一介绍．     

Characterization of a family of nucleolar SUMO-specific proteases with preference for SUMO-2 or SUMO-3

SUMOylation is a reversible process regulated by a family of sentrin/SUMO-specific proteases (SENPs). Of the six SENP family members, except for SENP1 and SENP2, the substrate specificities of the rest of SENPs are not well defined. Here, we have described SENP5, which has restricted substrate specificity. SENP5 showed SUMO-3 C-terminal hydrolase activity but could not process pro-SUMO-1 in vitro. Furthermore, SENP5 showed more limited isopeptidase activity in vitro. In vivo, SENP5 showed isopeptidase activity against SUMO-2 and SUMO-3 conjugates but not against SUMO-1 conjugates. Native SENP5 localized mainly to the nucleolus but was also found in the nucleus. The N terminus of SENP5 contains a stretch of amino acids responsible for the nucleolar localization of SENP5. N-terminal-truncated SENP5 co-localized with PML, a known SUMO substrate. Using PML SUMOylation mutants as model substrates, we showed that SENP5 can remove poly-SUMO-2 or poly-SUMO-3 from the Lys160 or Lys490 positions of PML. However, SENP5 could not remove SUMO-1 from the Lys160 or Lys490 positions of PML. Nonetheless, SENP5 could remove SUMO-1, -2, and -3 from the Lys65 position of PML. Thus, SENP5 also possesses limited SUMO-1 isopeptidase activity. We were also able to show that SENP3 has substrate specificity similar to that of SENP5. Thus, SENP3 and SENP5 constitute a subfamily of SENPs that regulate the formation of SUMO-2 or SUMO-3 conjugates and, to a less extent, SUMO-1 modification.     

Activity profiling of human deSUMOylating enzymes (SENPs) with synthetic substrates suggests an unexpected specificity of two newly characterized members of the family

SENPs [Sentrin/SUMO (small ubiquitin-related modifier)-specific proteases] include proteases that activate the precursors of SUMOs, or deconjugate SUMOs attached to target proteins. SENPs are usually assayed on protein substrates, and for the first time we demonstrate that synthetic substrates can be convenient tools in determining activity and specificity of these proteases. We synthesized a group of short synthetic peptide fluorogenic molecules based on the cleavage site within SUMOs. We demonstrate the activity of human SENP1, 2, 5, 6, 7 and 8 on these substrates. A parallel positional scanning approach using a fluorogenic tetrapeptide library established preferences of SENPs in the P3 and P4 positions that allowed us to design optimal peptidyl reporter substrates. We show that the specificity of SENP1, 2, 5 and 8 on the optimal peptidyl substrates matches their natural protein substrates, and that the presence of the SUMO domain enhances catalysis by 2-3 orders of magnitude. We also show that SENP6 and 7 have an unexpected specificity that distinguishes them from other members of the family, implying that, in contrast to previous predictions, their natural substrate(s) may not be SUMO conjugates.     

Redox regulation of the stability of the SUMO protease SENP3 via interactions with CHIP and Hsp90

The molecular chaperone heat shock protein 90 (Hsp90) and the co-chaperone/ubiquitin ligase carboxyl terminus of Hsc70-interacting protein (CHIP) control the turnover of client proteins. How this system decides to stabilize or degrade the client proteins under particular physiological or pathological conditions is unclear. We report here a novel client protein, the SUMO2/3 protease SENP3, that is sophisticatedly regulated by CHIP and Hsp90. SENP3 is maintained at a low basal level under non-stress condition due to Hsp90-independent CHIP-mediated ubiquitination. Upon mild oxidative stress, SENP3 undergoes thiol modification, which recruits Hsp90. Hsp90/SENP3 association protects SENP3 from CHIP-mediated ubiquitination and subsequent degradation, but this effect of Hsp90 requires the presence of CHIP. Our data demonstrate for the first time that CHIP and Hsp90 interplay with a client alternately under non-stress and stress conditions, and the choice between stabilization and degradation is made by the redox state of the client. In addition, enhanced SENP3/Hsp90 association is found in cancer. These findings provide new mechanistic insight into how cells regulate the SUMO protease in response to oxidative stress.     

Structural insights into the SENP6 Loop1 structure in complex with SUMO2

The SENP proteases regulate the SUMO conjugates in the cell by cleaving SUMO from target proteins. SENP6 and SENP7 are the most divergent members of the SENP/ULP protease family in humans by the presence of insertions in their catalytic domains. Loop1 insertion is determinant for the SUMO2/3 activity and specificity on SENP6 and SENP7. To gain structural insights into the role of Loop1, we have designed a chimeric SENP2 with the insertion of Loop1 into its sequence. The structure of SENP2‐Loop1 in complex with SUMO2 was solved at 2.15 Å resolution, and reveals the details of an interface exclusive to SENP6/7 and the formation of unique contacts between both proteins. Interestingly, functional data with SUMO substrates showed an increase of the proteolytic activity in the SENP2‐Loop1 chimera for diSUMO2 and polySUMO2 substrates.