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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, Jr, Stanley M ; Dame, John B ; Dunn, Ben M Permyakov, Eugene A.

PloS one, 2015-10, Vol.10 (10), p.e0141758-e0141758 [Periódico revisado por pares]

United States: Public Library of Science

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Título:
Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei
Autor: Liu, Peng ; Robbins, Arthur H ; Marzahn, Melissa R ; McClung, Scott H ; Yowell, Charles A ; Stevens, Jr, Stanley M ; Dame, John B ; Dunn, Ben M
Permyakov, Eugene A.
Assuntos: Amino Acid Substitution ; Amino acids ; Analysis ; Animals ; Antimicrobial agents ; Aspartic Acid Endopeptidases - antagonists & inhibitors ; Aspartic Acid Endopeptidases - chemistry ; Aspartic Acid Endopeptidases - genetics ; Aspartic Acid Endopeptidases - metabolism ; Aspartic proteinase ; Biochemistry ; Biotechnology ; Blood cells ; Care and treatment ; Catalysis ; Catalytic activity ; Catalytic Domain ; Cathepsin D ; Chemical properties ; Chemotherapy ; Cloning ; Combinatorial analysis ; Combinatorial chemistry ; Complications and side effects ; Deoxyribonucleic acid ; Design ; DNA ; Drug development ; E coli ; Enzyme Activation ; Enzymes ; Erythrocytes ; Food ; Food vacuoles ; Gene Expression ; Health aspects ; Hemoglobin ; Inclusion bodies ; Infectious diseases ; Interdisciplinary aspects ; Isoforms ; Kinetics ; Malaria ; Molecular biology ; Parasites ; Pathological effects ; Peptide libraries ; Pharmaceutical sciences ; Physiological aspects ; Plasmodium ; Plasmodium (Protozoa) ; Plasmodium berghei ; Plasmodium berghei - genetics ; Plasmodium berghei - metabolism ; Plasmodium falciparum ; Proenzymes ; Protein Refolding ; Proteinase ; Proteins ; Protozoan Proteins - antagonists & inhibitors ; Protozoan Proteins - chemistry ; Protozoan Proteins - genetics ; Protozoan Proteins - metabolism ; Reaction kinetics ; Recombinant Proteins - genetics ; Recombinant Proteins - isolation & purification ; Recombinant Proteins - metabolism ; Substrate inhibition ; Vaccines ; Vector-borne diseases ; Veterinary colleges ; Veterinary medicine
É parte de: PloS one, 2015-10, Vol.10 (10), p.e0141758-e0141758
Notas: ObjectType-Article-1
SourceType-Scholarly Journals-1
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content type line 23
Competing Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: PL BMD. Performed the experiments: PL MRM SHM SMS. Analyzed the data: PL AHR SMS BMD. Contributed reagents/materials/analysis tools: CAY SMS JBD. Wrote the paper: PL BMD.
Current address: Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, Florida, United States of America
Current address: Department of Neurology and N. Bud Grossman Center for Memory Research and Care, University of Minnesota, Minneapolis, Minnesota, United States of America
Current address: Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, United States of America
Descrição: 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.
Editor: United States: Public Library of Science
Idioma: Inglês

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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, Jr, Stanley M ; Dame, John B ; Dunn, Ben M Permyakov, Eugene A.

United States: Public Library of Science

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