1. Enzymatic analysis of recombinant Japanese encephalitis virus NS2B(H)-NS3pro protease with fluorogenic model peptide substrates
Muhammad Junaid, Chakard Chalayut, Anna Sehgelmeble Torrejon, Chanan Angsuthanasombat, Iryna Shutava, Maris Lapins, Jarl E S Wikberg, Gerd Katzenmeier PLoS One. 2012;7(5):e36872. doi: 10.1371/journal.pone.0036872. Epub 2012 May 15.
Background: Japanese encephalitis virus (JEV), a member of the Flaviviridae family, causes around 68,000 encephalitis cases annually, of which 20-30% are fatal, while 30-50% of the recovered cases develop severe neurological sequelae. Specific antivirals for JEV would be of great importance, particularly in those cases where the infection has become persistent. Being indispensable for flaviviral replication, the NS2B-NS3 protease is a promising target for design of anti-flaviviral inhibitors. Contrary to related flaviviral proteases, the JEV NS2B-NS3 protease is structurally and mechanistically much less characterized. Here we aimed at establishing a straightforward procedure for cloning, expression, purification and biochemical characterization of JEV NS2B(H)-NS3pro protease. Methodology/principal findings: The full-length sequence of JEV NS2B-NS3 genotype III strain JaOArS 982 was obtained as a synthetic gene. The sequence of NS2B(H)-NS3pro was generated by splicing by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro, expressed in E. coli as soluble protein, was purified to >95% purity by a single-step immobilized metal affinity chromatography. SDS-PAGE and immunoblotting of the purified enzyme demonstrated NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 36, 21, and 10 kDa bands, respectively. Kinetic parameters, K(m) and k(cat), for fluorogenic protease model substrates, Boc-GRR-amc, Boc-LRR-amc, Ac-nKRR-amc, Bz-nKRR-amc, Pyr-RTKR-amc and Abz-(R)(4)SAG-nY-amide, were obtained using inner filter effect correction. The highest catalytic efficiency k(cat)/K(m) was found for Pyr-RTKR-amc (k(cat)/K(m): 1962.96 ± 85.0 M(-1) s(-1)) and the lowest for Boc-LRR-amc (k(cat)/K(m): 3.74±0.3 M(-1) s(-1)). JEV NS3pro is inhibited by aprotinin but to a lesser extent than DEN and WNV NS3pro. Conclusions/significance: A simplified procedure for the cloning, overexpression and purification of the NS2B(H)-NS3pro was established which is generally applicable to other flaviviral proteases. Kinetic parameters obtained for a number of model substrates and inhibitors, are useful for the characterization of substrate specificity and eventually for the design of high-throughput assays aimed at antiviral inhibitor discovery.
2. Ozone and aging up-regulate type II metacaspase gene expression and global metacaspase activity in the leaves of field-grown maize (Zea mays L.) plants
Rafiq Ahmad, Yasmine Zuily-Fodil, Chantal Passaquet, Olivier Bethenod, Romain Roche, Anne Repellin Chemosphere. 2012 May;87(7):789-95. doi: 10.1016/j.chemosphere.2011.12.081. Epub 2012 Jan 25.
Maize plants (Zea mays L. cv. NK Perform) were exposed to O(3)-enriched air, using a new field fumigation system. Transcriptional changes for three type II-metacaspase genes were studied in the leaves (ranks 10 and 12), using quantitative real-time PCR. Global metacaspase activity was measured using metacaspase-specific synthetic tripeptide Boc-GRR-AMC. Aging had little effect on mRNA accumulation whereas four to six-fold increases were observed for the most O(3)-responsive type II metacaspase genes, in the older leaves 10. Global metacaspase activity increased by 257% and 333% in leaves 12 and 10, respectively, in response to the highest cumulated concentration. In non-fumigated plants, metacaspase activity progressively increased over the course of the experiment and always was higher in the older leaves 10. Together, these results suggest that metacaspase-mediated proteolysis is a crucial step in leaf responses to both O(3) and age-mediated senescence.
