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.     

In Vitro Studies Reveal a Sequential Mode of Chain Processing by the Yeast SUMO (Small Ubiquitin-related Modifier)-specific Protease Ulp2

Sumoylation is a post-translational modification essential in most eukaryotes that regulates stability, localization, activity, or interaction of a multitude of proteins. It is a reversible process wherein counteracting ligases and proteases, respectively, mediate the conjugation and deconjugation of SUMO molecules to/from target proteins. Apart from attachment of single SUMO moieties to targets, formation of poly-SUMO chains occurs by the attachment of additional SUMO molecules to lysine residues in the N-terminal extensions of SUMO. In Saccharomyces cerevisiae there are apparently only two SUMO(Smt3)-specific proteases: Ulp1 and Ulp2. Ulp2 has been shown to be important for the control of poly-SUMO conjugates in cells and to dismantle SUMO chains in vitro, but the mechanism by which it acts remains to be elucidated. Applying an in vitro approach, we found that Ulp2 acts sequentially rather than stochastically, processing substrate-linked poly-SUMO chains from their distal ends down to two linked SUMO moieties. Furthermore, three linked SUMO units turned out to be the minimum length of a substrate-linked chain required for efficient binding to and processing by Ulp2. Our data suggest that Ulp2 disassembles SUMO chains by removing one SUMO moiety at a time from their ends (exo mechanism). Apparently, Ulp2 recognizes surfaces at or near the N terminus of the distal SUMO moiety, as attachments to this end significantly reduce cleavage efficiency. Our studies suggest that Ulp2 controls the dynamic range of SUMO chain lengths by trimming them from the distal ends.     

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.     

Nucleolar protein B23/nucleophosmin regulates the vertebrate SUMO pathway through SENP3 and SENP5 proteases

Ubiquitin-like protein/sentrin-specific proteases (Ulp/SENPs) mediate both processing and deconjugation of small ubiquitin-like modifier proteins (SUMOs). Here, we show that Ulp/SENP family members SENP3 and SENP5 localize within the granular component of the nucleolus, a subnucleolar compartment that contains B23/nucleophosmin. B23/nucleophosmin is an abundant shuttling phosphoprotein, which plays important roles in ribosome biogenesis and which has been strongly implicated in hematopoietic malignancies. Moreover, we found that B23/nucleophosmin binds SENP3 and SENP5 in Xenopus laevis egg extracts and that it is essential for stable accumulation of SENP3 and SENP5 in mammalian tissue culture cells. After either codepletion of SENP3 and SENP5 or depletion of B23/nucleophosmin, we observed accumulation of SUMO proteins within nucleoli. Finally, depletion of these Ulp/SENPs causes defects in ribosome biogenesis reminiscent of phenotypes observed in the absence of B23/nucleophosmin. Together, these results suggest that regulation of SUMO deconjugation may be a major facet of B23/nucleophosmin function in vivo.     

SUMOylation alters CRMP2 regulation of calcium influx in sensory neurons

The axon/dendrite specification collapsin response mediator protein 2 (CRMP2) bidirectionally modulates N-type voltage-gated Ca²⁺ channels (CaV2.2). Here we demonstrate that small ubiquitin-like modifier (SUMO) protein modifies CRMP2 via the SUMO E2-conjugating enzyme Ubc9 in vivo. Removal of a SUMO conjugation site KMD in CRMP2 (K374A/M375A/D376A; CRMP2_AAA) resulted in loss of SUMOylated CRMP2 without compromising neurite branching, a canonical hallmark of CRMP2 function. Increasing SUMOylation levels correlated inversely with calcium influx in sensory neurons. CRMP2 deSUMOylation by SUMO proteases SENP1 and SENP2 normalized calcium influx to those in the CRMP2_AAA mutant. Thus, our results identify a novel role for SUMO modification in CRMP2/CaV2.2 signaling pathway.     

Modification in reverse: the SUMO proteases

SUMOs (small ubiquitin-like modifiers) are ubiquitin-related proteins that become covalently conjugated to cellular target proteins that are involved in a variety of processes. Frequently, this modification has a key role in regulating the activities of those targets and, thus, their cellular functions. SUMO conjugation is a highly dynamic process that can be rapidly reversed by the action of members of the Ubl (ubiquitin-like protein)-specific protease (Ulp) family. The same family of enzymes is also responsible for maturation of newly synthesized SUMOs prior to their initial conjugation. Recent advances in structural, biochemical and cell biological analysis of Ulp/SENPs reveal their high degree of specificity towards SUMO paralogs, in addition to discrimination between processing, deconjugation and chain-editing reactions. The dissimilar sub-nuclear localization patterns of Ulp/SENPs and phenotypes of Ulp/SENP mutants further indicate that different Ulp/SENPs have distinct and non-redundant roles.     

Nucleocytoplasmic shuttling modulates activity and ubiquitination-dependent turnover of SUMO-specific protease 2

Small ubiquitin-related modifier (SUMO) proteins are conjugated to numerous polypeptides in cells, and attachment of SUMO plays important roles in regulating the activity, stability, and subcellular localization of modified proteins. SUMO modification of proteins is a dynamic and reversible process. A family of SUMO-specific proteases catalyzes the deconjugation of SUMO-modified proteins. Members of the Sentrin (also known as SUMO)-specific protease (SENP) family have been characterized with unique subcellular localizations. However, little is known about the functional significance of or the regulatory mechanism derived from the specific localizations of the SENPs. Here we identify a bipartite nuclear localization signal (NLS) and a CRM1-dependent nuclear export signal (NES) in the SUMO protease SENP2. Both the NLS and the NES are located in the nonhomologous domains of SENP2 and are not conserved among other members of the SENP family. Using a series of SENP2 mutants and a heterokaryon assay, we demonstrate that SENP2 shuttles between the nucleus and the cytoplasm and that the shuttling is blocked by mutations in the NES or by treating cells with leptomycin B. We show that SENP2 can be polyubiquitinated in vivo and degraded through proteolysis. Restricting SENP2 in the nucleus by mutations in the NES impairs its polyubiquitination, whereas a cytoplasm-localized SENP2 made by introducing mutations in the NLS can be efficiently polyubiquitinated, suggesting that SENP2 is ubiquitinated in the cytoplasm. Finally, treating cells with MG132 leads to accumulation of polyubiquitinated SENP2, indicating that SENP2 is degraded through the 26S proteolysis pathway. Thus, the function of SENP2 is regulated by both nucleocytoplasmic shuttling and polyubiquitin-mediated degradation.     

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.     

DeSUMOylating enzymes--SENPs

Modification of proteins by ubiquitin and SUMO (small ubiquitin-like modifiers) is a dynamic and reversible process. Similar to the ubiquitin pathway, where the action of deubiquitinating enzymes removes ubiquitin from ubiquitin-adducts, SUMO is also removed intact from its substrates by proteases belonging to the sentrin-specific proteases (SENPs) family. In addition to their isopeptidase activity, SENPs also execute another essential function as endopeptidases by removing the short C-terminal extension from immature SUMOs. The defining characteristics of SENPs are their predicted conserved molecular scaffold-defined as members of peptidase Clan CE, conserved catalytic mechanism, and their reported activity on SUMO or Nedd8 conjugated proteins (or the respective precursors). We discuss recent progress on the human SENPs and their substrates.     

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.     

Structure of the human SENP7 catalytic domain and poly-SUMO deconjugation activities for SENP6 and SENP7

Small ubiquitin-like modifier (SUMO) proteases regulate the abundance and lifetime of SUMO-conjugated substrates by antagonizing reactions catalyzed by SUMO-conjugating enzymes. Six SUMO proteases constitute the human SENP/ULP protease family (SENP1-3 and SENP5-7). SENP6 and SENP7 include the most divergent class of SUMO proteases, which also includes the yeast enzyme ULP2. We present the crystal structure of the SENP7 catalytic domain at a resolution of 2.4 angstroms. Comparison with structures of human SENP1 and SENP2 reveals unique elements that differ from previously characterized structures of SUMO-deconjugating enzymes. Biochemical assays show that SENP6 and SENP7 prefer SUMO2 or SUMO3 in deconjugation reactions with rates comparable with those catalyzed by SENP2, particularly during cleavage of di-SUMO2, di-SUMO3, and poly-SUMO chains composed of SUMO2 or SUMO3. In contrast, SENP6 and SENP7 exhibit lower rates for processing pre-SUMO1, pre-SUMO2, or pre-SUMO3 in comparison with SENP2. Structure-guided mutational analysis reveals elements unique to the SENP6 and SENP7 subclass of SENP/ULP proteases that contribute to protease function during deconjugation of poly-SUMO chains.     

Distinct roles for Arabidopsis SUMO protease ESD4 and its closest homolog ELS1

SUMO conjugation affects a broad range of processes in Arabidopsis thaliana, including flower initiation, pathogen defense, and responses to cold, drought and salt stress. We investigated two sequence-related SUMO-specific proteases that are both widely expressed and show that they differ significantly in their properties. The closest homolog of SUMO protease ESD4, ESD4-LIKE SUMO PROTEASE 1 (ELS1, alternatively called AtULP1a) has SUMO-specific proteolytic activity, but is functionally distinct from ESD4, as shown by intracellular localization, mutant phenotype and heterologous expression in yeast mutants. Furthermore, we show that the growth defects caused by loss of ESD4 function are not due to increased synthesis of the stress signal salicylic acid, as was previously shown for a SUMO ligase, indicating that impairment of the SUMO system affects plant growth in different ways. Our results demonstrate that two A. thaliana SUMO proteases showing close sequence similarity have distinct in vivo functions.     

The SUMO isopeptidase Ulp2 prevents accumulation of SUMO chains in yeast

The ubiquitin-related protein SUMO functions by becoming covalently attached to lysine residues in other proteins. Unlike ubiquitin, which is often linked to its substrates as a polyubiquitin chain, only one SUMO moiety is attached per modified site in most substrates. However, SUMO has recently been shown to form chains in vitro and in mammalian cells, with a lysine in the non-ubiquitin-like N-terminal extension serving as the major SUMO-SUMO branch site. To investigate the physiological function of SUMO chains, we generated Saccharomyces cerevisiae strains that expressed mutant SUMOs lacking various lysine residues. Otherwise wild-type strains lacking any of the nine lysines in SUMO were viable, had no obvious growth defects or stress sensitivities, and had SUMO conjugate patterns that did not differ dramatically from wild type. However, mutants lacking the SUMO-specific isopeptidase Ulp2 accumulated high molecular weight SUMO-containing species, which formed only when the N-terminal lysines of SUMO were present, suggesting that they contained SUMO chains. Furthermore SUMO branch-site mutants suppressed several of the phenotypes of ulp2delta, consistent with the possibility that some ulp2delta phenotypes are caused by accumulation of SUMO chains. We also found that a mutant SUMO whose non-ubiquitin-like N-terminal domain had been entirely deleted still carried out all the essential functions of SUMO. Thus, the ubiquitin-like domain of SUMO is sufficient for conjugation and all downstream functions required for yeast viability. Our data suggest that SUMO can form chains in vivo in yeast but demonstrate conclusively that chain formation is not required for the essential functions of SUMO in S. cerevisiae.     

OTS1‐dependent deSUMOylation increases tolerance to high copper levels in Arabidopsis

The conjugation of SUMO(small ubiquitin-like modifier) to protein substrates is a reversible process(SUMOylation/deSUMOylation) that regulates plant development and stress responses. The essential metal copper(Cu) is required for normal plant growth, but excess amounts are toxic. The SUMO E_3 ligase, SIZ_1, and SIZ_1-mediated SUMOylation function in plant tolerance to excess Cu. It is unknown whether deSUMOylation also contributes to Cu tolerance in plants. Here, we report that OTS_1, a protease that cleaves SUMO from its substrate proteins,participates in Cu tolerance in Arabidopsis thaliana(Arabidopsis). OTS_1 loss-of-function mutants(ots_(1-2) and ots_(1-3))displayed increased sensitivity to excess Cu. Redox homeostasis and the balance between SUMOylation and deSUMOylation were disrupted in the ots_(1-3) mutant under excess Cu conditions. The ots_(1-3) mutant accumulated higher levels of Cu in both shoots and roots compared to wild type.Specific Cu-related metal transporter genes were upregulated due to the loss-of-function of OTS_1, which might explain the high Cu levels in ots_(1-3). These results suggest that the SUMOylation/deSUMOylation machinery is activated in response to excess Cu, and modulates Cu homeostasis and tolerance by regulating both Cu uptake and detoxification. Together, our findings provide insight into the biological function and regulatory role of SUMOylation/deSUMOylation in plant tolerance to Cu.     

Crystal structure of DeSI-1, a novel deSUMOylase belonging to a putative isopeptidase superfamily

Post-translational modification by small ubiquitin-like modifier (SUMO) can be reversed by sentrin/SUMO-specific proteases (SENPs), the first known class of deSUMOylase. Recently, we identified a new deSUMOylating enzyme DeSI-1, which is distinct from SENPs and belongs to the putative deubiquitinating isopeptidase PPPDE superfamily. Herein, we report the crystal structure of DeSI-1, revealing that this enzyme forms a homodimer and that the groove between the two subunits is the active site harboring two absolutely conserved cysteine and histidine residues that form a catalytic dyad. We also show that DeSI-1 exhibits an extremely low endopeptidase activity toward precursor forms of SUMO-1 and SUMO-2, unlike SENPs.     

Drosophila Ulp1, a nuclear pore-associated SUMO protease, prevents accumulation of cytoplasmic SUMO conjugates

SUMO is a small ubiquitin-like protein that becomes covalently conjugated to a variety of target proteins, the large majority of which are found in the nucleus. Ulp1 is a member of a family of proteases that control SUMO function positively, by catalyzing the proteolytic processing of SUMO to its mature form, and negatively, by catalyzing SUMO deconjugation. In Drosophila S2 cells, depletion of Ulp1 by RNA interference results in a dramatic change in the overall spectrum of SUMO conjugates, indicating that SUMO deconjugation is substrate-specific and plays a critical role in determining the steady state targets of SUMO conjugation. Ulp1 normally serves to prevent the accumulation of SUMO-conjugated forms of a number of proteins, including the aminoacyl-tRNA synthetase EPRS. In the presence of Ulp1, most SUMO conjugates reside in the nucleus. However, in its absence, SUMO-conjugated EPRS accumulates in the cytoplasm, contributing to an overall shift of SUMO from the nucleus to the cytoplasm. The ability of Ulp1 to restrict SUMO conjugates to the nucleus is independent of its role as a SUMO-processing enzyme because Ulp1-dependent nuclear localization of SUMO is even observed when SUMO is expressed in a preprocessed form. Studies of a Ulp1-GFP fusion protein suggest that Ulp1 localizes to the nucleoplasmic face of the nuclear pore complex. We hypothesize that, as a component of the nuclear pore complex, Ulp1 may prevent proteins from leaving the nucleus with SUMO still attached.