Characterization of SENP7, a SUMO-2/3-specific isopeptidase.

The modification of proteins by SUMO (small ubiquitin-related modifier) plays important roles in regulating the activity, stability and cellular localization of target proteins. Similar to ubiquitination, SUMO modification is a dynamic process that can be reversed by SENPs [SUMO-1/sentrin/SMT3 (suppressor of mif two 3 homologue 1)-specific peptidases]. To date, six SENPs have been discovered in humans, although knowledge of their regulation, specificity and biological functions is limited. In the present study, we report that SENP7 has a restricted substrate specificity, being unable to process SUMO precursors and displaying paralogue-specific isopeptidase activity. The C-terminal catalytic domain of SENP7 efficiently depolymerized poly-SUMO-2 chains but had undetectable activity against poly-SUMO-1 chains. SENP7 also displayed isopeptidase activity against di-SUMO-2- and SUMO-2-modified RanGAP1 (Ran GTPase-activating protein 1) but had limited activity against SUMO-1-modified RanGAP1. in vivo, full-length SENP7 was localized to the nucleoplasm and preferentially reduced the accumulation of high-molecular-mass conjugates of SUMO-2 and SUMO-3 compared with SUMO-1. Small interfering RNA-mediated ablation of SENP7 expression led to the accumulation of high-molecular-mass SUMO-2 species and to the accumulation of promyelocytic leukaemia protein in subnuclear bodies. These findings suggest that SENP7 acts as a SUMO-2/3-specific protease that is likely to regulate the metabolism of poly-SUMO-2/3 rather than SUMO-1 conjugation in vivo.

The structure of SENP1-SUMO-2 complex suggests a structural basis for discrimination between SUMO paralogues during processing.

The SUMO (small ubiquitin-like modifier)-specific protease SENP1 (sentrin-specific protease 1) can process the three forms of SUMO to their mature forms and deconjugate SUMO from modified substrates. It has been demonstrated previously that SENP1 processed SUMO-1 more efficiently than SUMO-2, but displayed little difference in its ability to deconjugate the different SUMO paralogues from modified substrates. To determine the basis for this substrate specificity, we have determined the crystal structure of SENP1 in isolation and in a transition-state complex with SUMO-2. The interface between SUMO-2 and SENP1 has a relatively poor complementarity, and most of the recognition is determined by interaction between the conserved C-terminus of SUMO-2 and the cleft in the protease. Although SENP1 is rather similar in structure to the related protease SENP2, these proteases have different SUMO-processing activities. Electrostatic analysis of SENP1 in the region where the C-terminal peptide, removed during maturation, would project indicates that it is the electrostatic complementarity between this region of SENP1 and the C-terminal peptides of the various SUMO paralogues that mediates selectivity.

[Protective immunity induced by the nucleic acid vaccine of SjC 21.7 in mice].

OBJECTIVE: To observe the protective immunity induced by the nucleic acid vaccine of 21.7 kDa membrane protein molecule of Schistosoma japonicum Chinese mainland strain (SjC 21.7) in BALB/c mice. METHODS: A pair of primers (P1 and P2) was synthesized according to the DNA sequence of the SjC21.7. The ORF sequence of SjC21.7 was amplified by PCR, and the Kozark sequence was added to the position of initiator. The gene fragment was inserted into the eukaryotic expression plasmid pcDNA3.1 to form the recombinant plasmid SjC21.7-pcDNA3.1. Forty-eight BALB/c mice were divided into three groups: control, test and boost. Each mouse was injected in quadriceps femoris with plasmid pcDNA3.1 (control) or recombinant plasmid SjC21.7-pcDNA3.1 (test, boost); for the boost group, with additional P35-pcDNA3.1 and P40-pcDNA3.1. All mice were immunized three times with an interval of 2 weeks, challenged each with 45 cercariae of S. japonicum at the 30th day after final immunization. At day 45 after challenge, all mice were sacrificed, the numbers of worms and hepatic eggs were counted. Antibody level in the sera of mice before and two weeks after immunization was determined with ELISA. The expression of the target gene in quadriceps femoris was observed with immunohistochemistry. RESULTS: The immunohistochemistry analysis showed that there were specific antigens expressed in the local tissue of the test group mice. There was specific IgG in the serum of partial mice in test and boost groups. Compared with the control group, the worm reduction rate was 29.9% and its egg reduction rate 13.8% in the test group; 31.9% and 28.0% respectively in the boost group. The egg reduction rate in the boost group was higher than that of the test group (P < 0.05). CONCLUSION: The SjC21.7 nucleic acid vaccine could induce partial protective immunity against Schistosoma japonicum in BALB/c mice.

Structural basis of NEDD8 ubiquitin discrimination by the deNEDDylating enzyme NEDP1.

NEDD8 (neural precursor cell expressed developmentally downregulated gene 8)-specific protease NEDP1 processes preNEDD8 to its mature form and deconjugates NEDD8 from substrates such as p53 and cullins. Although NEDD8 and ubiquitin are highly related in sequence and structure, their attachment to a protein leads to different biological effects. It is therefore critical that NEDP1 discriminates between NEDD8 and ubiquitin, and this requires remarkable precision in molecular recognition. To determine the basis of this specificity, we have determined the crystal structure of NEDP1 in isolation and in a transition state complex with NEDD8. This reveals that NEDP1 is a cysteine protease of the Ulp family. Binding of NEDD8 induces a dramatic conformational change in a flexible loop that swings over the C-terminus of NEDD8 locking it into an extended beta-structure optimal for catalysis. Structural, mutational and biochemical studies have identified key residues involved in molecular recognition. A single-residue difference in the C-terminus of NEDD8 and ubiquitin contributes significantly to the ability of NEDP1 to discriminate between them. In vivo analysis indicates that NEDP1 mutants perturb deNEDDylation of the tumour suppressor p53.

NEDP1, a highly conserved cysteine protease that deNEDDylates Cullins.

The ubiquitin-like protein NEDD8 is essential for activity of SCF-like ubiquitin ligase complexes. Here we identify and characterize NEDP1, a human NEDD8-specific protease. NEDP1 is highly conserved throughout evolution and equivalent proteins are present in yeast, plants, insects, and mammals. Bacterially expressed NEDP1 is capable of processing NEDD8 in vitro to expose the diglycine motif required for conjugation and can deconjugate NEDD8 from modified substrates. NEDP1 appears to be specific for NEDD8 as neither ubiquitin nor SUMO bearing COOH-terminal extensions are utilized as substrates. Inhibition studies and mutagenesis indicate that NEDP1 is a cysteine protease with sequence similarities to SUMO-specific proteases and the class of viral proteases typified by the adenovirus protease. In vivo NEDP1 deconjugates NEDD8 from a wide variety of substrates including the cullin component of SCF-like complexes. Thus NEDP1 is likely to play an important role in ubiquitin-mediated proteolysis by controlling the activity of SCF complexes.