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Biochemical Properties of a Novel Cysteine Protease of <i>Plasmodium vivax</i> , Vivapain-4
Na, Byoung-Kuk,Bae, Young-An,Zo, Young-Gun,Choe, Youngchool,Kim, Seon-Hee,Desai, Prashant V.,Avery, Mitchell A.,Craik, Charles S.,Kim, Tong-Soo,Rosenthal, Philip J.,Kong, Yoon Public Library of Science 2010 PLoS neglected tropical diseases Vol.4 No.10
<▼1><P><B>Background</B></P><P>Multiple cysteine proteases of malaria parasites are required for maintenance of parasite metabolic homeostasis and egress from the host erythrocyte. In <I>Plasmodium falciparum</I> these proteases appear to mediate the processing of hemoglobin and aspartic proteases (plasmepsins) in the acidic food vacuole and the hydrolysis of erythrocyte structural proteins at neutral pH. Two cysteine proteases, vivapain (VX)-2 and VX-3 have been characterized in <I>P. vivax</I>, but comprehensive studies of <I>P. vivax</I> cysteine proteases remain elusive.</P><P><B>Findings</B></P><P>We characterized a novel cysteine protease of <I>P. vivax</I>, VX-4, of which orthologs appears to have evolved differentially in primate plasmodia with strong cladistic affinity toward those of rodent <I>Plasmodium</I>. Recombinant VX-4 demonstrated dual substrate specificity depending on the surrounding micro-environmental pH. Its hydrolyzing activity against benzyloxycarbonyl-Leu-Arg-4-methyl-coumaryl-7-amide (Z-Leu-Arg-MCA) and Z-Phe-Arg-MCA was highest at acidic pH (5.5), whereas that against Z-Arg-Arg-MCA was maximal at neutral pH (6.5–7.5). VX-4 preferred positively charged amino acids and Gln at the P1 position, with less strict specificity at P3 and P4. P2 preferences depended on pH (Leu at pH 5.5 and Arg at pH 7.5). Three amino acids that delineate the S2 pocket were substituted in VX-4 compared to VX-2 and VX-3 (Ala90, Gly157 and Glu180). Replacement of Glu180 abolished activity against Z-Arg-Arg-MCA at neutral pH, indicating the importance of this amino acid in the pH-dependent substrate preference. VX-4 was localized in the food vacuoles and cytoplasm of the erythrocytic stage of <I>P. vivax</I>. VX-4 showed maximal activity against actin at neutral pH, and that against <I>P. vivax</I> plasmepsin 4 and hemoglobin was detected at neutral/acidic and acidic pH, respectively.</P><P><B>Conclusion</B></P><P>VX-4 demonstrates pH-dependent substrate switching, which might offer an efficient mechanism for the specific cleavage of different substrates in different intracellular environments. VX-4 might function as a hemoglobinase in the acidic parasite food vacuole, a maturase of <I>P. vivax</I> plasmepsin 4 at neutral or acidic pH, and a cytoskeleton-degrading protease in the neutral erythrocyte cytosol.</P></▼1><▼2><P><B>Author Summary</B></P><P><I>Plasmodium vivax</I> affects hundreds of millions each year and results in severe morbidity and mortality. Plasmodial cysteine proteases (CPs) play crucial roles during the progression of malaria since inhibition of these molecules impairs parasite growth. These CPs might be targeted for new antimalarial drugs. We characterized a novel <I>P. vivax</I> CP, vivapain-4 (VX-4), which appeared to evolve differentially among primate <I>Plasmodium</I> species. VX-4 showed highly unique substrate preference depending on surrounding micro-environmental pH. It effectively hydrolyzed benzyloxycarbonyl-Leu-Arg-4-methyl-coumaryl-7-amide (Z-Leu-Arg-MCA) and Z-Phe-Arg-MCA at acidic pH and Z-Arg-Arg-MCA at neutral pH. Three amino acids (Ala90, Gly157 and Glu180) that delineate the S2 pocket were found to be substituted in VX-4. Alteration of Glu180 abolished hydrolytic activity against Z-Arg-Arg-MCA at neutral pH, indicating Glu180 is intimately involved in the pH-dependent substrate preference. VX-4 hydrolyzed actin at neutral pH and hemoglobin at acidic pH, and participated in plasmepsin 4 activation at neutral/acidic pH. VX-4 was localized in the food vacuoles and cytoplasm of the erythrocytic stage of <I>P. vivax</I>. The differential substrate preferences depending on pH suggested a highly efficient mechanism to enlarge biological implications of VX-4, including hemoglobin degradation, maturation of plasmepsin, and remodeling of the parasite architecture during growth and development of <I>P. vivax</I>.</P></▼2>
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Melé,ndez-Ló,pez, Samuel G.,Herdman, Scott,Hirata, Ken,Choi, Min-Ho,Choe, Youngchool,Craik, Charles,Caffrey, Conor R.,Hansell, Elisabeth,Chá,vez-Munguí,a, Bibiana,Chen, Yen Tin American Society for Microbiology 2007 EUKARYOTIC CELL Vol.6 No.7
<B>ABSTRACT</B><P>Cysteine proteinases are key virulence factors of the protozoan parasite <I>Entamoeba histolytica</I>. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of the <I>E. histolytica</I> genome project has revealed more than 40 genes encoding cysteine proteinases. We have focused on <I>E. histolytica</I> cysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasive <I>E. histolytica</I> and is highly expressed and released. Recombinant EhCP1 was expressed in <I>Escherichia coli</I> and refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis.</P>
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