Z-Arg-Leu-Arg-Gly-Gly-AMC

Several genes related to the ubiquitin (Ub)-proteasome pathway, including those coding for proteasome subunits and conjugation enzymes, are differentially expressed during the Schistosoma mansoni life cycle. Although deubiquitinating enzymes have been reported to be negative regulators of protein ubiquitination and shown to play an important role in Ub-dependent processes, little is known about their role in S. mansoni . In this study, we analysed the Ub carboxyl-terminal hydrolase (UCHs) proteins found in the database of the parasite's genome. An in silico ana- lysis (GeneDB and MEROPS) identified three different UCH family members in the genome, Sm UCH-L3, Sm UCH-L5 and Sm BAP-1 and a phylogenetic analysis confirmed the evolutionary conservation of the proteins. We performed quantitative reverse transcription-polymerase chain reaction and observed a differential expression profile for all of the investigated transcripts between the cercariae and adult worm stages. These results were corroborated by low rates of Z-Arg-Leu-Arg-Gly-Gly-AMC hydrolysis in a crude extract obtained from cercariae in parallel with high Ub conjugate levels in the same extracts. We suggest that the accumulation of ubiquitinated proteins in the cercaria and early schistosomulum stages is related to a decrease in 26S proteasome activity. Taken together, our data suggest that UCH family members contribute to regulating the activity of the Ub-proteasome system during the life cycle of this parasite.

The YUH1 gene coding for ubiquitin C-terminal hydrolase 1, a deubiquitinating enzyme, was cloned from the Saccharomyces cerevisiae genomic DNA and expressed in Escherichia coli. YUH1 was fused with the 6 histidine tag at the N-terminus (H6YUH1) or C-terminus (YUH1H6) and purified by an immobilized metal affinity chromatography with high purity. By using a fluorogenic substrate, Z-Arg-Leu-Arg-Gly-Gly-AMC, the deubiquitinating activities for H6YUH1 (1.72U/mg) and YUH1H6 (1.61U/mg) were about 18 times higher than 0.092U/mg for H6UBP1, ubiquitin specific protease 1 of S. cerevisiae containing the 6 histidine residue at the N-terminus which is normally used in protein engineering. YUH1 had the optimal temperature of 27 degrees C and acidity of pH 8.5. Analysis of thermal deactivation kinetics of H6YUH1 estimated 3.2 and 1.4h of half lives at 4 and 52 degrees C, respectively. Immobilization onto the Ni-NTA affinity resin and environmental modulation were carried out to improve the stability of YUH1. Incubation of the immobilized YUH1 in 50% glycerol solution at -20 degrees C resulted in 52% of decrease in specific activity for 7days, corresponding to a 2.7-fold increase compared with that of the free YUH1 incubated in the same solution at 4 degrees C.

We have investigated the specificity of isopeptidase T toward peptide-AMC substrates based on the C-termini of ubiquitin. The substrates investigated were Z-Gly-Gly-AMC, Z-Arg-Gly-Gly-AMC, Z-Leu-Arg-Gly-Gly-AMC, and Z-Arg-Leu-Arg-Gly-Gly-AMC and were hydrolyzed by isopeptidase T with kc/Km values of < 0.1, 1, 18, and 95 M-1 s-1, respectively. In the course of these experiments, we observed that the hydrolytic activity of isopeptidase T toward these substrates is modulated by ubiquitin in a biphasic fashion. While submicromolar concentrations of ubiquitin activate isopeptidase T, higher concentrations are inhibitory. In the activation phase, the extent of stimulation of kc/Km varies with substrate and is 8-, 50-, and 70-fold for Z-Arg-Gly-Gly-AMC, Z-Leu-Arg-Gly-Gly-AMC, and Z-Arg-Leu-Arg-Gly-Gly-AMC, respectively. Kd for ubiquitin in this phase is, of course, independent of substrate and equals 0.10 +/- 0.03 microM. At higher concentrations, ubiquitin is inhibitory and titrates kc/Km with an average Ki value of 3.0 +/- 1.3 microM for all three substrates. To explain these observations, we propose a structural model for isopeptidase T that involves two binding sites for ubiquitin. We propose that the two sites are adjacent to one another and are the extended active site that binds two ubiquitin moieties of a polyubiquitin chain for isopeptide bond hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)