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KMID : 1140120060110030162
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Cancer Prevention Research
2006 Volume.11 No. 3 p.162 ~ p.170
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Activation of Malarial Protease, Plasmepsin and Calpain Involved in Eryptosis by Paclitaxel, an Anti-cancer Drug
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Choi Yun-Young
Jhee Ok-Hwa
Kang Ju-Seop
Yun Young-Gab
Park Hyun
Lee Yun-Sik
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Abstract
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In Plasmodium falciparum, ten genes are known to encode plasmepsins or related enzymes. Of these, plasmepsin I, II, IV, and histo-aspartic protease (HAP) are known as food vacuole proteases having the capacity to digest hemoglobin. The importance of correct targeting of plasmepsins to the food vacuole for their processing is evidenced by the observation that the processing of plasmepsins is blocked by the treatment of parasites with brefeldin A, an inhibitor of anterograde protein traffic from the endoplasmic reticulum. As for the trafficking route of plasmepsins to food vacuole, it has been suggested that plasmepsin II is targeted to the food vacuole through to the secretory system of the cytostomal vacuole in a brefeldin A-sensitive manner, rather than a direct trafficking route from the ER to the food vacuole. A calpains, intracellular cystein proteases with numerous isoforms in organisms ranging from mammals to bacteria, and the calpain activity is related to physiological function, apoptosis and several biological activities, like cell migration and disassembly of the focal adhesion. To study the mechanism of calpain, and plasmepsin II and IV in malaria, we used their wild type and mutant proteins in which the catalytic Asp residue in two active-site motifs was mutated and active catalytic domain of calpain, as well as protease inhibitors. In this study, we summarized a proposed pathway for hemoglobin degradation and a role of calpain in the processing of each plasmepsin, and a genetic map and catalytic domain region of calpain in Plasmodium falciparum. The expressions and activities of cloned calpain catalytic domains and the effects of proteases inhibitor for plasmepsin II and IV were tested. Side effects of cytostatic treatment include development of anemia resulting from either decreased generation or accelerated clearance of circulating erythrocytes. Recent experiments revealed a novel kind of stress- induced erythrocyte death, i.e. eryptosis, which is characterized by enhanced cytosolic Ca2 levels, increased ceramide formation and exposure of phosphatidylserine at the cell surface. The present study explored whether cytostatic treatment with paclitaxel (Taxol ) triggers eryptosis. To know a role and localization of calpain, and activation of calpain in erythrocyte by treatment of paclitaxel, we designed four potential specific sites for antibodies against for the catalytic domain of calpain, and two specific antibodies, Cal4 and Cal5, which are recognized the catalytic domain of calpain were obtained. For NMR spectroscopy, we designed the expression vector for the calpain of the active catalytic domain and moves to the purification procedure. (Cancer Prev Res 11, 162-170, 2006)
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KEYWORD
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Parasites, Malarial protease, Plasmepsins, Calpains, Catalytic domain
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