Distribution and paralogue specificity of mammalian deSUMOylating enzymes.

The covalent attachment of SUMO (small ubiquitin-like protein modifier) to target proteins results in modifications in their activity, binding interactions, localization or half-life. The reversal of this modification is catalysed by SENPs (SUMO-specific processing proteases). Mammals contain four SUMO paralogues and six SENP enzymes. In the present paper, we describe a systematic analysis of human SENPs, integrating estimates of relative selectivity for SUMO1 and SUMO2, and kinetic measurements of recombinant C-terminal cSENPs (SENP catalytic domains). We first characterized the reaction of each endogenous SENP and cSENPs with HA-SUMO-VS [HA (haemagglutinin)-tagged SUMO-vinyl sulfones], active-site-directed irreversible inhibitors of SENPs. We found that all cSENPs and endogenous SENP1 react with both SUMO paralogues, whereas all other endogenous SENPs in mammalian cells and tissues display high selectivity for SUMO2-VS. To obtain more quantitative data, the kinetic properties of purified cSENPs were determined using SUMO1- or SUMO2-AMC (7-amino-4-methylcoumarin) as substrate. All enzymes bind their respective substrates with high affinity. cSENP1 and cSENP2 process either SUMO substrate with similar affinity and catalytic efficiency; cSENP5 and cSENP6 show marked catalytic specificity for SUMO2 as measured by Km and kcat, whereas cSENP7 works only on SUMO2. Compared with cSENPs, recombinant full-length SENP1 and SENP2 show differences in SUMO selectivity, indicating that paralogue specificity is influenced by the presence of the variable N-terminal domain of each SENP. Our data suggest that SUMO2 metabolism is more dynamic than that of SUMO1 since most SENPs display a marked preference for SUMO2.

Identification and developmental expression of Xenopus laevis SUMO proteases.

SUMO proteins are small ubiquitin-related modifiers. All SUMOs are synthesized as propeptides that are post-translationally cleaved prior to conjugation. After processing, SUMOs become covalently conjugated to cellular targets through a pathway that is similar to ubiquitination. Ubiquitin like protein proteases/Sentrin specific proteases (Ulp/SENPs) mediate both processing and deconjugation of SUMOs. The action of Ulp/SENPs makes SUMOylation a highly dynamic post-translational modification. To investigate how Ulp/SENPs are regulated in a developmental context, we isolated and characterized all Ulp/SENPs in Xenopus laevis. Xenopus possess homologues of mammalian SENP3, 5, 6 and 7. All of these enzymes reacted with HA-tagged vinyl sulfone derivatives of SUMO-2 (HA-SU2-VS) but not SUMO-1 (HA-SU1-VS), suggesting that they act primarily on SUMO-2 and -3. In contrast, Xenopus possess a single member of the SENP1/SENP2 subfamily of Ulp/SENPs, most closely related to mammalian SENP1. Xenopus SENP1 reacted with HA-SU1-VS and HA-SU2-VS, suggesting that it acts on all SUMO paralogues. We analyzed the mRNA and protein levels for each of the Ulp/SENPs through development; we found that they show distinct patterns of expression that may involve both transcriptional and post-transcriptional regulation. Finally, we have characterized the developmental function of the most abundant Ulp/SENP found within Xenopus eggs, SENP3. Depletion of SENP3 using morpholino antisense oligonucleotides (morpholinos) caused accumulation of high molecular weight SUMO-2/3 conjugated species, defects in developing embryos and changes in the expression of some genes regulated by the transforming growth factor beta (TGF-beta) pathway. These findings collectively indicate that SUMO proteases are both highly regulated and essential for normal development.

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.

SUSP1 antagonizes formation of highly SUMO2/3-conjugated species.

Small ubiquitin-related modifier (SUMO) processing and deconjugation are mediated by sentrin-specific proteases/ubiquitin-like proteases (SENP/Ulps). We show that SUMO-specific protease 1 (SUSP1), a mammalian SENP/Ulp, localizes within the nucleoplasm. SUSP1 depletion within cell lines expressing enhanced green fluorescent protein (EGFP) fusions to individual SUMO paralogues caused redistribution of EGFP-SUMO2 and -SUMO3, particularly into promyelocytic leukemia (PML) bodies. Further analysis suggested that this change resulted primarily from a deficit of SUMO2/3-deconjugation activity. Under these circumstances, PML bodies became enlarged and increased in number. We did not observe a comparable redistribution of EGFP-SUMO1. We have investigated the specificity of SUSP1 using vinyl sulfone inhibitors and model substrates. We found that SUSP1 has a strong paralogue bias toward SUMO2/3 and that it acts preferentially on substrates containing three or more SUMO2/3 moieties. Together, our findings argue that SUSP1 may play a specialized role in dismantling highly conjugated SUMO2 and -3 species that is critical for PML body maintenance.

Derivitization of the C-terminus of ubiquitin and ubiquitin-like proteins using intein chemistry: methods and uses.

Here we describe a general method to synthesize and use a panel of reagents with selectivity for deubiquitinating enzymes exhibiting specificity for each ubiquitin-like protein. A substrate (Ubl-AMC), a reversible inhibitor (Ubl-aldehyde), and an active-site-directed irreversible inhibitor (Ubl-vinylsulfone) are described for each Ubl. Because of space constraints, we give details for only the Nedd8 derivatives, but these methods have been used in our laboratory to produce these derivatives of Ubiquitin, Nedd8, Sumo-1, Sumo-2, and Isg15. These reagents are useful in defining the specificity of DUBs, as well as in purifying and identifying these important proteins. The reagents are selective and useful in crude extracts. The reactivity of these reagents reveals differences in both the S1 and S1' sites of deubiquitinating enzymes. Only active enzymes are efficiently detected with these reagents. Published results indicate that specificity is not strictly defined by the evolutionary relationships of these DUBs.

Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family.

The ubiquitin (Ub)-proteasome system includes a large family of deubiquitinating enzymes (DUBs). Many members are assigned to this enzyme class by sequence similarity but without evidence for biological activity. A panel of novel DUB-specific probes was generated by a chemical ligation method. These probes allowed identification of DUBs and associated components by tandem mass spectrometry, as well as rapid demonstration of enzymatic activity for gene products whose functions were inferred from primary structure. We identified 23 active DUBs in EL4 cells, including the tumor suppressor CYLD1. At least two DUBs tightly interact with the proteasome 19S regulatory complex. An OTU domain-containing protein, with no sequence homology to any known DUBs, was isolated. We show that this polypeptide reacts with the C terminus of Ub, thus demonstrating DUB-like enzymatic activity for this novel superfamily of proteases.

Biophysical behavior of Scomberoides commersonianus skin collagen.

Some biophysical characteristics of the skin collagen from Scomberoides commersonianus were measured and compared to those of rat tail tendon. Stress-strain data indicate that the strain at break as well as the tensile strength of the fish skin without scales increased significantly. The maximum tension in case of rat skin is at least a factor of two higher than that observed in fish skin. The much lower hydrothermal isometric tension measurements observed in fish skin are attributable to a lesser number of heat stable crosslinks. Stress relaxation measurements in the fish skin indicate that more than one relaxation process may be involved in the stabilization of collagenous matrix. The observed differences in the biophysical behavior of fish skin may well arise from combination of changes in extent of hydroxylation of proline in collagen synthesis, hydrogen bond network and fibril orientation as compared to rat tail tendon.