During the activation and transferring process, E1 and E2 form a thioester-linkage with SUMOs. By using an in vitro assay, it is demonstrated that SENP1 is able to cleave the thioester-linkage between SUMO-1/SUMO-3 and E1/E2. This finding suggests that SUMO proteases regulate the sumoylation pathway, not only during maturation and deconjugation, but also in the E1 activation and E2 conjugation processes. / Recently, reactive oxygen species have been demonstrated to influence the equilibrium of sumoylation-desumoylation. Here, by in vitro assay, it is shown that H2O2 induces formation of inter-molecular disulfide linkage of human SUMO protease SENP1, via the active-site Cys 603 and a unique residue Cys 613. Such reversible modification confers higher enzyme activity recovery which is also observed in yeast Ulp1, but not in human SENP2, suggesting its protective role against irreversible sulfhydryl oxidation. The physiological relevance of the disulfide-linked dimer of SENP1 is also detected in cultured cells upon oxidative stress. The modifications are further verified by the crystal structures of Ulp1 with catalytic cysteine oxidized to sulfenic, sulfinic and sulfonic acids. The current findings suggest that, in addition to SUMO conjugating enzymes, SUMO proteases may act as redox sensors and effectors, which modulate the desumoylation pathway and allow immediate specific cellular responses to oxidative stress. / SUMO (small ubiquitin-related modifier) is a member of the ubiquitin-like protein family that is highly conserved in all eukaryotic organisms and regulates cellular function of a variety of target proteins. SUMO proteins are expressed in their precursor forms and precursor processing involves cleavage of the residues after the conserved 'GG' region by the hydrolytic activity of SUMO-specific protease. The exposed second glycine then forms a covalent bond with the epsilon-amino group of a substrate lysine residue at the psiKxE motif by a cascade of SUMO El, E2 and E3 ligases. As a reversible modification, SUMO proteases can cleave SUMOs from their substrates during de-conjugation process. / To date, four SUMO family members, SUMO-1, -2, -3 and -4 and six SUMO proteases, SENP1--3 and 5-7 (where SENP stands for sentrin-specific protease) have been identified in human. By characterizing the maturation reactions of SUMO-1, -2 and -3 catalyzed by SENP1, it is demonstrated that SENP1 contains the highest maturation efficiency for SUMO-1, followed by SUMO-2 and SUMO-3. By mutagenesis study, it is further identified that the two amino acids immediately after GG motif could influence the maturation efficiency of SENP1. By comparison with another investigation which showed the preference of the maturation reaction of SUMO-2 by SENP2, the results suggest that SUMO proteases with specific tissue distribution control the availability of different mature SUMOs in human. / To gain a deeper insight into the molecular basis of maturation and de-conjugation processes catalyzed by SENP1, it has been determined, at 2.8 A resolution, the X-ray structure of a complex between the catalytic domain of SENP1C(C603S) and matured SUMO-1. The structure shows that the substituted serine residue does not undergo any local structural rearrangements at the active site as observed in the previously solved SENP2/SUMO-1 complex structure. This finding suggests that SUMO proteases require a self-conformational change prior to the cleavage reaction, and further disclose the cleavage mechanism of the hydrolytic reactions catalyzed by SUMO proteases. Moreover, analysis of the interface of SENP1 and SUMO1 has identified four amino acids that are unique in SENP1 sequence and facilitate the interaction of SENP1 and SUMO-1. / Xu, Zheng. / "July 2007." / Advisers: Shannon Au Wing Ngor; Tzi-Bun Ng. / Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0125. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 181-194). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Infectious bursal disease virus (IBDV) causes an acute and highly contagious disease affecting young chickens, which is responsible for significant losses in the poultry industry world-wide. The virus specifically infects and destroys B-cell precursors within the bursa of Fabricius, an avian lymphoid organ, leading to immunosuppression. IBDV has a bi-segmented, double-stranded RNA genome. The larger segment encodes a 110-kDa precursor polyprotein, designated NH₂-VPX-VP4-VP3-COOH, in a single open reading frame. The autocatalytic processing of this precursor into mature proteins is a critical step in viral replication and VP4 is the putative protease responsible for this cleavage. This study concerns the development of a strategy to clone and express recombinant VP4 and describes the use of VP4 as a marker for rapid and effective detection of IBDV. VP4 cDNA was produced and amplified by optimisation of a reverse transcription coupled to the polymerase chain reaction (RT-PCR), providing a clear and sensitive assay. Anti-peptide antibodies were raised against a selected peptide from VP4 and were used to probe homogenates of infected bursae for the native protein to assess their potential for immunological detection. These antibody-related results are promising though inconclusive, due to the complex nature of the assayed sample. Amplified VP4 cDNA from KwaZulu-Natal strains of IBDV isolated from 1989 to 1997 was also examined by restriction fragment length polymorphism (RFLP) analysis to determine the relatedness of local IBDV to global strains. All KwaZulu-Natal samples produced identical patterns, which were most similar to one of ten international strains examined, namely, the British strain UK661. Samples infected with IBDV were also probed for VP4 activity. Double basic amino acid cleavage sites have been proposed for the putative protease and infected samples were assayed for activity against the fluorogenic peptide Cbz-Arg-Arg-AMC. Demonstrably higher activity was found in infected versus uninfected samples, although the origin of this activity is unclear. The findings in this study suggest that VP4 warrants further attention, both as a marker for infectious bursal disease, and as a novel viral protease. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.