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Functional and biochemical characterization of the baculovirus caspase inhibitor MaviP35.

ABSTRACT: Many viruses express proteins which prevent the host cell death that their infection would otherwise provoke. Some insect viruses suppress host apoptosis through the expression of caspase inhibitors belonging to the P35 superfamily. Although a number of P35 relatives have been identified, Autographa californica (Ac) P35 and Spodoptera littoralis (Spli) P49 have been the most extensively characterized. AcP35 was found to inhibit caspases via a suicide substrate mechanism: the caspase cleaves AcP35 within its 'reactive site loop' then becomes trapped, irreversibly bound to the cleaved inhibitor. The Maruca vitrata multiple nucleopolyhedrovirus encodes a P35 family member (MaviP35) that exhibits 81% identity to AcP35. We found that this relative shared with AcP35 the ability to inhibit mammalian and insect cell death. Caspase-mediated cleavage within the MaviP35 reactive site loop occurred at a sequence distinct from that in AcP35, and the inhibitory profiles of the two P35 relatives differed. MaviP35 potently inhibited human caspases 2 and 3, DCP-1, DRICE and CED-3 in vitro, but (in contrast to AcP35) only weakly suppressed the proteolytic activity of the initiator human caspases 8, 9 and 10. Although MaviP35 inhibited the AcP35-resistant caspase DRONC in yeast, and was sensitive to cleavage by DRONC in vitro, MaviP35 failed to inhibit the proteolytic activity of bacterially produced DRONC in vitro.

SUBMITTER: Brand IL

PROVIDER: S-EPMC3252740 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Functional and biochemical characterization of the baculovirus caspase inhibitor MaviP35.

Brand I L IL Green M M MM Civciristov S S Pantaki-Eimany D D George C C Gort T R TR Huang N N Clem R J RJ Hawkins C J CJ

Cell death & disease 20111215

Many viruses express proteins which prevent the host cell death that their infection would otherwise provoke. Some insect viruses suppress host apoptosis through the expression of caspase inhibitors belonging to the P35 superfamily. Although a number of P35 relatives have been identified, Autographa californica (Ac) P35 and Spodoptera littoralis (Spli) P49 have been the most extensively characterized. AcP35 was found to inhibit caspases via a suicide substrate mechanism: the caspase cleaves AcP3 ...[more]

PMID: 22170098

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Project description:Emergence of drug-resistant Plasmodium falciparum has created an urgent need for new drug targets. DNA polymerase ? is an essential enzyme required for chromosomal DNA replication and repair, and therefore may be a potential target for anti-malarial drug development. However, little is known of the characteristics and function of this P. falciparum enzyme.The coding sequences of DNA polymerase ? catalytic subunit (PfPol?-cat), DNA polymerase ? small subunit (PfPol?S) and proliferating cell nuclear antigen (PfPCNA) from chloroquine- and pyrimethamine-resistant P. falciparum strain K1 were amplified, cloned into an expression vector and expressed in Escherichia coli. The recombinant proteins were analysed by SDS-PAGE and identified by LC-MS/MS. PfPol?-cat was biochemically characterized. The roles of PfPol?S and PfPCNA in PfPol?-cat function were investigated. In addition, inhibitory effects of 11 compounds were tested on PfPol?-cat activity and on in vitro parasite growth using SYBR Green I assay.The purified recombinant protein PfPol?-cat, PfPol?S and PfPCNA showed on SDS-PAGE the expected size of 143, 57 and 34 kDa, respectively. Predicted amino acid sequence of the PfPol?-cat and PfPol?S had 59.2 and 24.7 % similarity respectively to that of the human counterpart. The PfPol?-cat possessed both DNA polymerase and 3'-5' exonuclease activities. It used both Mg(2+) and Mn(2+) as cofactors and was inhibited by high KCl salt (>200 mM). PfPol?S stimulated PfPol?-cat activity threefolds and up to fourfolds when PfPCNA was included in the assay. Only two compounds were potent inhibitors of PfPol?-cat, namely, butylphenyl-dGTP (BuPdGTP; IC50 of 38 µM) and 7-acetoxypentyl-(3, 4 dichlorobenzyl) guanine (7-acetoxypentyl-DCBG; IC50 of 55 µM). The latter compound showed higher inhibition on parasite growth (IC50 of 4.1 µM).Recombinant PfPol?-cat, PfPol?S and PfPCNA were successfully expressed and purified. PfPolS and PfPCNA increased DNA polymerase activity of PfPol?-cat. The high sensitivity of PfPol? to BuPdGTP can be used to differentiate parasite enzyme from mammalian and human counterparts. Interestingly, 7-acetoxypentyl-DCBG showed inhibitory effects on both enzyme activity and parasite growth. Thus, 7-acetoxypentyl-DCBG is a potential candidate for future development of a new class of anti-malarial agents targeting parasite replicative DNA polymerase.

Dataset Information

Functional and biochemical characterization of the baculovirus caspase inhibitor MaviP35.

Publications

Functional and biochemical characterization of the baculovirus caspase inhibitor MaviP35.

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