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Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei

The rodent malaria parasite Plasmodium berghei is a practical model organism for experimental studies of human malaria. Plasmepsins are a class of aspartic proteinase isoforms that exert multiple pathological effects in malaria parasites. Plasmepsins residing in the food vacuole (FV) of the parasite...

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Autores principales: Liu, Peng, Robbins, Arthur H., Marzahn, Melissa R., McClung, Scott H., Yowell, Charles A., Stevens, Stanley M., Dame, John B., Dunn, Ben M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624963/
https://www.ncbi.nlm.nih.gov/pubmed/26510189
http://dx.doi.org/10.1371/journal.pone.0141758
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author Liu, Peng
Robbins, Arthur H.
Marzahn, Melissa R.
McClung, Scott H.
Yowell, Charles A.
Stevens, Stanley M.
Dame, John B.
Dunn, Ben M.
author_facet Liu, Peng
Robbins, Arthur H.
Marzahn, Melissa R.
McClung, Scott H.
Yowell, Charles A.
Stevens, Stanley M.
Dame, John B.
Dunn, Ben M.
author_sort Liu, Peng
collection PubMed
description The rodent malaria parasite Plasmodium berghei is a practical model organism for experimental studies of human malaria. Plasmepsins are a class of aspartic proteinase isoforms that exert multiple pathological effects in malaria parasites. Plasmepsins residing in the food vacuole (FV) of the parasite hydrolyze hemoglobin in red blood cells. In this study, we cloned PbPM4, the FV plasmepsin gene of P. berghei that encoded an N-terminally truncated pro-segment and the mature enzyme from genomic DNA. We over-expressed this PbPM4 zymogen as inclusion bodies (IB) in Escherichia coli, and purified the protein following in vitro IB refolding. Auto-maturation of the PbPM4 zymogen to mature enzyme was carried out at pH 4.5, 5.0, and 5.5. Interestingly, we found that the PbPM4 zymogen exhibited catalytic activity regardless of the presence of the pro-segment. We determined the optimal catalytic conditions for PbPM4 and studied enzyme kinetics on substrates and inhibitors of aspartic proteinases. Using combinatorial chemistry-based peptide libraries, we studied the active site preferences of PbPM4 at subsites S1, S2, S3, S1’, S2’ and S3’. Based on these results, we designed and synthesized a selective peptidomimetic compound and tested its inhibition of PbPM4, seven FV plasmepsins from human malaria parasites, and human cathepsin D (hcatD). We showed that this compound exhibited a >10-fold selectivity to PbPM4 and human malaria parasite plasmepsin 4 orthologs versus hcatD. Data from this study furthesr our understanding of enzymatic characteristics of the plasmepsin family and provides leads for anti-malarial drug design.
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spelling pubmed-46249632015-11-06 Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei Liu, Peng Robbins, Arthur H. Marzahn, Melissa R. McClung, Scott H. Yowell, Charles A. Stevens, Stanley M. Dame, John B. Dunn, Ben M. PLoS One Research Article The rodent malaria parasite Plasmodium berghei is a practical model organism for experimental studies of human malaria. Plasmepsins are a class of aspartic proteinase isoforms that exert multiple pathological effects in malaria parasites. Plasmepsins residing in the food vacuole (FV) of the parasite hydrolyze hemoglobin in red blood cells. In this study, we cloned PbPM4, the FV plasmepsin gene of P. berghei that encoded an N-terminally truncated pro-segment and the mature enzyme from genomic DNA. We over-expressed this PbPM4 zymogen as inclusion bodies (IB) in Escherichia coli, and purified the protein following in vitro IB refolding. Auto-maturation of the PbPM4 zymogen to mature enzyme was carried out at pH 4.5, 5.0, and 5.5. Interestingly, we found that the PbPM4 zymogen exhibited catalytic activity regardless of the presence of the pro-segment. We determined the optimal catalytic conditions for PbPM4 and studied enzyme kinetics on substrates and inhibitors of aspartic proteinases. Using combinatorial chemistry-based peptide libraries, we studied the active site preferences of PbPM4 at subsites S1, S2, S3, S1’, S2’ and S3’. Based on these results, we designed and synthesized a selective peptidomimetic compound and tested its inhibition of PbPM4, seven FV plasmepsins from human malaria parasites, and human cathepsin D (hcatD). We showed that this compound exhibited a >10-fold selectivity to PbPM4 and human malaria parasite plasmepsin 4 orthologs versus hcatD. Data from this study furthesr our understanding of enzymatic characteristics of the plasmepsin family and provides leads for anti-malarial drug design. Public Library of Science 2015-10-28 /pmc/articles/PMC4624963/ /pubmed/26510189 http://dx.doi.org/10.1371/journal.pone.0141758 Text en © 2015 Liu et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Liu, Peng
Robbins, Arthur H.
Marzahn, Melissa R.
McClung, Scott H.
Yowell, Charles A.
Stevens, Stanley M.
Dame, John B.
Dunn, Ben M.
Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei
title Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei
title_full Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei
title_fullStr Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei
title_full_unstemmed Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei
title_short Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei
title_sort enzymatic characterization of recombinant food vacuole plasmepsin 4 from the rodent malaria parasite plasmodium berghei
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624963/
https://www.ncbi.nlm.nih.gov/pubmed/26510189
http://dx.doi.org/10.1371/journal.pone.0141758
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