The Experts below are selected from a list of 2001 Experts worldwide ranked by ideXlab platform
Daniel E. Goldberg - One of the best experts on this subject based on the ideXlab platform.
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Plasmodium falciparum falcilysin: an unprocessed Food Vacuole enzyme
2015Co-Authors: Short Communication, Christina E. Murata, Daniel E. GoldbergAbstract:Five hundred million infections and nearly two million deaths each year are attributed to the protozoan Plasmodium falciparum, a causative agent of human malaria. With the increasing prevalence of drug resistant strains, there is an urgent need to identify new drug targets. Examination of the parasite’s unique metabolic pathways, such as hemoglobin degradation, provides candidates for chemotherapeutic de-velopment. Intraerythrocytic development of the parasite is depen-dent upon degradation of red blood cell hemoglobin. A semi-ordered pathway of proteases mediates this catabolism, which occurs in the acidic organelle called the Food Vacuole. The aspartic proteases, plasmepsins I and II, are proposed to be responsible for initial cleavage of hemoglobin in a con-served hinge region of the alpha chain [1,2]. Plasmepsins, and a family of cysteine proteases, falcipain-2 and 3, then carry out further degradation of the denatured globin [2–5]. The resulting small globin peptides serve as substrates for falcilysin (FLN) [6]. FLN was first identified in 1999 when analysis of de-graded globin fragments from the Food Vacuole revealed several peptides with cleavage sites that could not be at-tributed to the known proteases [7]. Specifically, a pro-tease preferring to cleave substrates at polar or charged residues was implicated; this stands in contrast to the other hemoglobin-degrading proteases, which favor cleavage at hydrophobic residues. Purification of the native enzyme from Food Vacuoles revealed that it is a monomeric en-zyme with a molecular weight of 130,000, by far the largest hemoglobin catabolic protease. FLN is a member of the M16 family of metalloproteases, enzymes character
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an fkbp destabilization domain modulates protein levels in plasmodium falciparum
Nature Methods, 2007Co-Authors: Christopher M Armstrong, Daniel E. GoldbergAbstract:To enhance the repertoire of molecular tools for studying malaria parasite biology, we adapted a ligand-regulatable FKBP protein destabilization domain (ddFKBP) for use in P. falciparum. We destabilized the reporter yellow fluorescent protein (YFP) and the P. falciparum protease falcipain-2 in a ligand-reversible manner by tagging with ddFKBP. The swollen Food Vacuole phenotype of falcipain-2 knockout parasites could be rescued in a Shld1 ligand-dependent fashion by falcipain-2-ddFKBP expression.
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a plasmodium falciparum dipeptidyl aminopeptidase i participates in vacuolar hemoglobin degradation
Journal of Biological Chemistry, 2004Co-Authors: Michael Klemba, Ilya Y Gluzman, Daniel E. GoldbergAbstract:Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum requires the catabolism of large amounts of host cell hemoglobin. Endoproteolytic digestion of hemoglobin to short oligopeptides occurs in an acidic organelle called the Food Vacuole. How amino acids are generated from these peptides is not well understood. To gain insight into this process, we have studied a plasmodial ortholog of the lysosomal exopeptidase cathepsin C. The plasmodial enzyme dipeptidyl aminopeptidase 1 (DPAP1) was enriched from parasite extract by two different approaches and was shown to possess hydrolytic activity against fluorogenic dipeptide substrates. To localize DPAP1 we created a transgenic parasite line expressing a chromosomally encoded DPAP1-green fluorescent protein fusion. Green fluorescent protein fluorescence was observed in the Food Vacuole of live transgenic parasites, and anti-DPAP1 antibody labeled the Food Vacuole in parasite cryosections. Together these data implicate DPAP1 in the generation of dipeptides from hemoglobin-derived oligopeptides. To assess the significance of DPAP1, we attempted to ablate DPAP1 activity from blood stage parasites by truncating the chromosomal DPAP1-coding sequence. The inability to disrupt the coding sequence indicates that DPAP1 is important for asexual proliferation. The proenzyme form of DPAP1 was found to accumulate in the parasitophorous Vacuole of mature parasites. This observation suggests a trafficking route for DPAP1 through the parasitophorous Vacuole to the Food Vacuole.
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plasmodium falciparum cysteine protease falcipain 1 is not essential in erythrocytic stage malaria parasites
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Puran Singh Sijwali, Michael Klemba, Daniel E. Goldberg, Kentaro Kato, Karl B Seydel, Jiri Gut, Julie Lehman, Louis H Miller, Philip J. RosenthalAbstract:Among potential new targets for antimalarial chemotherapy are Plasmodium falciparum cysteine proteases, known as falcipains. Falcipain-2 and falcipain-3 are Food Vacuole hemoglobinases that may have additional functions. The function of falcipain-1 remains uncertain. To better characterize the role of falcipain-1 in erythrocytic parasites, we disrupted the falcipain-1 gene and characterized recombinant parasites. Disruption of the falcipain-1 gene was confirmed with Southern blots, and loss of expression of falcipain-1 was confirmed with immunoblots and by loss of labeling with a specific protease inhibitor. Compared with wild-type parasites, falcipain-1 knockout parasites developed normally, with the same morphology, multiplication rate, and invasion efficiency, and without significant differences in sensitivity to cysteine protease inhibitors. In wild-type and knockout parasites, cysteine protease inhibitors blocked hemoglobin hydrolysis in trophozoites, with a subsequent block in rupture of erythrocytes by mature schizonts, but they did not inhibit erythrocyte invasion by merozoites. Our results indicate that although falcipain-1 is expressed by erythrocytic parasites, it is not essential for normal development during this stage or for erythrocyte invasion.
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trafficking of plasmepsin ii to the Food Vacuole of the malaria parasite plasmodium falciparum
Journal of Cell Biology, 2004Co-Authors: Michael Klemba, Ilya Y Gluzman, Wandy L. Beatty, Daniel E. GoldbergAbstract:fA amily of aspartic proteases, the plasmepsins (PMs), plays a key role in the degradation of hemoglobin in the Plasmodium falciparum Food Vacuole. To study the trafficking of proPM II, we have modified the chromosomal PM II gene in P. falciparum to encode a proPM II–GFP chimera. By taking advantage of green fluorescent protein fluorescence in live parasites, the ultrastructural resolution of immunoelectron microscopy, and inhibitors of trafficking and PM maturation, we have investigated the biosynthetic path leading to mature PM II in the Food Vacuole. Our data support a model whereby proPM II is transported through the secretory system to cytostomal Vacuoles and then is carried along with its substrate hemoglobin to the Food Vacuole where it is proteolytically processed to mature PM II.
Michael Klemba - One of the best experts on this subject based on the ideXlab platform.
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distribution and biochemical properties of an m1 family aminopeptidase in plasmodium falciparum indicate a role in vacuolar hemoglobin catabolism
Journal of Biological Chemistry, 2011Co-Authors: Daniel R T Ragheb, Kristin M Bompiani, Seema Dalal, Keith W Ray, Michael KlembaAbstract:Aminopeptidases catalyze N-terminal peptide bond hydrolysis and occupy many diverse roles across all domains of life. Here we present evidence that an M1-family aminopeptidase, PfA-M1, has been recruited to specialized roles in the human malaria parasite Plasmodium falciparum. PfA-M1 is abundant in two subcellular compartments in asexual intraerythrocytic parasites; that is, the Food Vacuole, where the catabolism of host hemoglobin takes place, and the nucleus. A unique N-terminal extension contributes to the observed dual targeting by providing a signal peptide and putative alternate translation initiation sites. PfA-M1 exists as two major isoforms, a nuclear 120-kDa species and a processed species consisting of a complex of 68- and 35-kDa fragments. PfA-M1 is both stable and active at the acidic pH of the Food Vacuole lumen. Determination of steady-state kinetic parameters for both aminoacyl-β-naphthylamide and unmodified dipeptide substrates over the pH range 5.0-8.5 reveals that k(cat) is relatively insensitive to pH, whereas K(m) increases at pH values below 6.5. At the pH of the Food Vacuole lumen (5.0-5.5), the catalytic efficiency of PfA-M1 remains high. Consistent with the kinetic data, the affinity of peptidic competitive inhibitors is diminished at acidic pH. Together, these results support a catalytic role for PfA-M1 in the Food Vacuole and indicate the importance of evaluating the potency of peptidic inhibitors at physiologically relevant pH values. They also suggest a second, distinct function for this enzyme in the parasite nucleus.
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evidence for catalytic roles for plasmodium falciparum aminopeptidase p in the Food Vacuole and cytosol
Journal of Biological Chemistry, 2009Co-Authors: Daniel Ragheb, Kristin M Bompiani, Seema Dalal, Michael KlembaAbstract:The metalloenzyme aminopeptidase P catalyzes the hydrolysis of amino acids from the amino termini of peptides with a prolyl residue in the second position. The human malaria parasite Plasmodium falciparum expresses a homolog of aminopeptidase P during its asexual intraerythrocytic cycle. P. falciparum aminopeptidase P (PfAPP) shares with mammalian cytosolic aminopeptidase P a three-domain, homodimeric organization and is most active with Mn(II) as the cofactor. A distinguishing feature of PfAPP is a 120-amino acid amino-terminal extension that appears to be removed from the mature protein. PfAPP is present in the Food Vacuole and cytosol of the parasite, a distribution that suggests roles in vacuolar hemoglobin catabolism and cytosolic peptide turnover. To evaluate the plausibility of these putative functions, the stability and kinetic properties of recombinant PfAPP were evaluated at the acidic pH of the Food Vacuole and at the near-neutral pH of the cytosol. PfAPP exhibited high stability at 37 °C in the pH range 5.0–7.5. In contrast, recombinant human cytosolic APP1 was unstable and formed a high molecular weight aggregate at acidic pH. At both acidic and slightly basic pH values, PfAPP efficiently hydrolyzed the amino-terminal X-Pro bond of the nonapeptide bradykinin and of two globin pentapeptides that are potential in vivo substrates. These results provide support for roles for PfAPP in peptide catabolism in both the Food Vacuole and the cytosol and suggest that PfAPP has evolved a dual distribution in response to the metabolic needs of the intraerythrocytic parasite.
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roles for two aminopeptidases in vacuolar hemoglobin catabolism in plasmodium falciparum
Journal of Biological Chemistry, 2007Co-Authors: Seema Dalal, Michael KlembaAbstract:During the erythrocytic stage of its life cycle, the human malaria parasite Plasmodium falciparum catabolizes large quantities of host-cell hemoglobin in an acidic organelle, the Food Vacuole. A current model for the catabolism of globin-derived oligopeptides invokes peptide transport out of the Food Vacuole followed by hydrolysis to amino acids by cytosolic aminopeptidases. To test this model, we have examined the roles of four parasite aminopeptidases during the erythrocytic cycle. Localization of tagged aminopeptidases, coupled with biochemical analysis of enriched Food Vacuoles, revealed the presence of amino acid-generating pathways in the Food Vacuole as well as the cytosol. Based on the localization data and in vitro assays, we propose a specific role for one of the plasmodial enzymes, aminopeptidase P, in the catabolism of proline-containing peptides in both the Vacuole and the cytosol. We establish an apparent requirement for three of the four aminopeptidases (including the two Food Vacuole enzymes) for efficient parasite proliferation. To gain insight into the impact of aminopeptidase inhibition on parasite development, we examined the effect of the presence of amino acids in the culture medium of the parasite on the toxicity of the aminopeptidase inhibitor bestatin. The ability of bestatin to block parasite replication was only slightly affected when 19 of 20 amino acids were withdrawn from the medium, indicating that exogenous amino acids cannot compensate for the loss of aminopeptidase activity. Together, these results support the development of aminopeptidase inhibitors as novel chemotherapeutics directed against malaria.
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a plasmodium falciparum dipeptidyl aminopeptidase i participates in vacuolar hemoglobin degradation
Journal of Biological Chemistry, 2004Co-Authors: Michael Klemba, Ilya Y Gluzman, Daniel E. GoldbergAbstract:Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum requires the catabolism of large amounts of host cell hemoglobin. Endoproteolytic digestion of hemoglobin to short oligopeptides occurs in an acidic organelle called the Food Vacuole. How amino acids are generated from these peptides is not well understood. To gain insight into this process, we have studied a plasmodial ortholog of the lysosomal exopeptidase cathepsin C. The plasmodial enzyme dipeptidyl aminopeptidase 1 (DPAP1) was enriched from parasite extract by two different approaches and was shown to possess hydrolytic activity against fluorogenic dipeptide substrates. To localize DPAP1 we created a transgenic parasite line expressing a chromosomally encoded DPAP1-green fluorescent protein fusion. Green fluorescent protein fluorescence was observed in the Food Vacuole of live transgenic parasites, and anti-DPAP1 antibody labeled the Food Vacuole in parasite cryosections. Together these data implicate DPAP1 in the generation of dipeptides from hemoglobin-derived oligopeptides. To assess the significance of DPAP1, we attempted to ablate DPAP1 activity from blood stage parasites by truncating the chromosomal DPAP1-coding sequence. The inability to disrupt the coding sequence indicates that DPAP1 is important for asexual proliferation. The proenzyme form of DPAP1 was found to accumulate in the parasitophorous Vacuole of mature parasites. This observation suggests a trafficking route for DPAP1 through the parasitophorous Vacuole to the Food Vacuole.
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plasmodium falciparum cysteine protease falcipain 1 is not essential in erythrocytic stage malaria parasites
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Puran Singh Sijwali, Michael Klemba, Daniel E. Goldberg, Kentaro Kato, Karl B Seydel, Jiri Gut, Julie Lehman, Louis H Miller, Philip J. RosenthalAbstract:Among potential new targets for antimalarial chemotherapy are Plasmodium falciparum cysteine proteases, known as falcipains. Falcipain-2 and falcipain-3 are Food Vacuole hemoglobinases that may have additional functions. The function of falcipain-1 remains uncertain. To better characterize the role of falcipain-1 in erythrocytic parasites, we disrupted the falcipain-1 gene and characterized recombinant parasites. Disruption of the falcipain-1 gene was confirmed with Southern blots, and loss of expression of falcipain-1 was confirmed with immunoblots and by loss of labeling with a specific protease inhibitor. Compared with wild-type parasites, falcipain-1 knockout parasites developed normally, with the same morphology, multiplication rate, and invasion efficiency, and without significant differences in sensitivity to cysteine protease inhibitors. In wild-type and knockout parasites, cysteine protease inhibitors blocked hemoglobin hydrolysis in trophozoites, with a subsequent block in rupture of erythrocytes by mature schizonts, but they did not inhibit erythrocyte invasion by merozoites. Our results indicate that although falcipain-1 is expressed by erythrocytic parasites, it is not essential for normal development during this stage or for erythrocyte invasion.
Philip J. Rosenthal - One of the best experts on this subject based on the ideXlab platform.
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artemisinin dipeptidyl vinyl sulfone hybrid molecules design synthesis and preliminary sar for antiplasmodial activity and falcipain 2 inhibition
Bioorganic & Medicinal Chemistry Letters, 2009Co-Authors: Rita Capela, Philip J. Rosenthal, Jiri Gut, Rudi Oliveira, Lidia M Goncalves, Ana Domingos, Francisca Lopes, Rui MoreiraAbstract:A series of artemisinin-vinyl sulfone hybrid molecules with the potential to act in the parasite Food Vacuole via endoperoxide activation and falcipain inhibition was synthesized and screened for antiplasmodial activity and falcipain-2 inhibition. All conjugates were active against the Plasmodium falciparum W2 strain in the low nanomolar range and those containing the Leu-hPhe core inhibited falcipain-2 in low micromolar range.
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plasmodium falciparum cysteine protease falcipain 1 is not essential in erythrocytic stage malaria parasites
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Puran Singh Sijwali, Michael Klemba, Daniel E. Goldberg, Kentaro Kato, Karl B Seydel, Jiri Gut, Julie Lehman, Louis H Miller, Philip J. RosenthalAbstract:Among potential new targets for antimalarial chemotherapy are Plasmodium falciparum cysteine proteases, known as falcipains. Falcipain-2 and falcipain-3 are Food Vacuole hemoglobinases that may have additional functions. The function of falcipain-1 remains uncertain. To better characterize the role of falcipain-1 in erythrocytic parasites, we disrupted the falcipain-1 gene and characterized recombinant parasites. Disruption of the falcipain-1 gene was confirmed with Southern blots, and loss of expression of falcipain-1 was confirmed with immunoblots and by loss of labeling with a specific protease inhibitor. Compared with wild-type parasites, falcipain-1 knockout parasites developed normally, with the same morphology, multiplication rate, and invasion efficiency, and without significant differences in sensitivity to cysteine protease inhibitors. In wild-type and knockout parasites, cysteine protease inhibitors blocked hemoglobin hydrolysis in trophozoites, with a subsequent block in rupture of erythrocytes by mature schizonts, but they did not inhibit erythrocyte invasion by merozoites. Our results indicate that although falcipain-1 is expressed by erythrocytic parasites, it is not essential for normal development during this stage or for erythrocyte invasion.
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gene disruption confirms a critical role for the cysteine protease falcipain 2 in hemoglobin hydrolysis by plasmodium falciparum
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Puran Singh Sijwali, Philip J. RosenthalAbstract:Erythrocytic malaria parasites degrade hemoglobin in an acidic Food Vacuole to acquire free amino acids and maintain parasite homeostasis. Hemoglobin hydrolysis appears to be a cooperative process requiring cysteine proteases (falcipains) and aspartic proteases (plasmepsins), but the specific roles of different enzymes in this process are unknown. We previously showed that falcipain-2 is a major trophozoite Food Vacuole cysteine protease. To characterize the specific role of falcipain-2, we disrupted the falcipain-2 gene and assessed the effect of this alteration. Falcipain-2-knockout trophozoites had markedly diminished cysteine protease activity and swollen, dark staining Food Vacuoles, consistent with a block in hemoglobin hydrolysis, as caused by cysteine protease inhibitors. However, more mature stages of knockout parasites were indistinguishable from wild-type parasites and developed normally. The knockout parasites had decreased and delayed expression of falcipain-2, which appeared to be directed by increased transcription of a second copy of the gene (falcipain-2′). Expression of other falcipains and plasmepsins was similar in wild-type and knockout parasites. Compared with wild-type, knockout parasites were about 3 times more sensitive to the cysteine protease inhibitors E-64 and leupeptin, and over 50-fold more sensitive to the aspartic protease inhibitor pepstatin. Our results assign a specific function for falcipain-2, the hydrolysis of hemoglobin in trophozoites. In addition, they highlight the cooperative action of cysteine and aspartic proteases in hemoglobin degradation by malaria parasites.
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characterization of native and recombinant falcipain 2 a principal trophozoite cysteine protease and essential hemoglobinase of plasmodium falciparum
Journal of Biological Chemistry, 2000Co-Authors: Bhaskar R. Shenai, Ajay Singh, Puran Singh Sijwali, Philip J. RosenthalAbstract:Abstract Trophozoites of the malaria parasitePlasmodium falciparum hydrolyze erythrocyte hemoglobin in an acidic Food Vacuole to provide amino acids for parasite protein synthesis. Cysteine protease inhibitors block hemoglobin degradation, indicating that a cysteine protease plays a key role in this process. A principal trophozoite cysteine protease was purified by affinity chromatography. Sequence analysis indicated that the protease is encoded by a previously unidentified gene, falcipain-2. Falcipain-2 was predominantly expressed in trophozoites, was concentrated in Food Vacuoles, and was responsible for at least 93% of trophozoite soluble cysteine protease activity. A construct encoding mature falcipain-2 and a small portion of the prodomain was expressed in Escherichia coli and refolded to active enzyme. Specificity for the hydrolysis of peptide substrates by native and recombinant falcipain-2 was very similar, and optimal at acid pH in a reducing environment. Under physiological conditions (pH 5.5, 1 mm glutathione), falcipain-2 hydrolyzed both native hemoglobin and denatured globin. Our results suggest that falcipain-2 can initiate cleavage of native hemoglobin in the P. falciparum Food Vacuole, that, following initial cleavages, the protease plays a key role in rapidly hydrolyzing globin fragments, and that a drug discovery effort targeted at this protease is appropriate.
Puran Singh Sijwali - One of the best experts on this subject based on the ideXlab platform.
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plasmodium falciparum atg18 localizes to the Food Vacuole via interaction with the multi drug resistance protein 1 and phosphatidylinositol 3 phosphate
bioRxiv, 2021Co-Authors: Renu Sudhakar, Divya Das, Subramanian Thanumalayan, Somesh Gorde, Puran Singh SijwaliAbstract:Autophagy is a lysosome-dependent degradative process involving over 35 Atg proteins. The autophagy repertoire in malaria parasites is limited and does not appear to be a major degradative process. To better understand the autophagy process, we investigated Plasmodium falciparum Atg18 (PfAtg18), a PROPPIN family protein, whose members like S. cerevisiae Atg18 (ScAtg18) and human WIPI2 are essential for autophagy. Wild type and mutant PfAtg18 were expressed in P. falciparum and assessed for localization, the effect of various inhibitors and antimalarials on PfAtg18 localization, and identification of PfAtg18-interacting proteins. PfAtg18 is expressed in asexual erythrocytic stages and localized to the Food Vacuole, which was also observed with other Plasmodium Atg18 proteins, indicating that Food Vacuole localization is a conserved feature. Interaction of PfAtg18 with the Food Vacuole-associated PI3P is essential for localization, as PfAtg18 mutants of PI3P-binding motifs neither bound PI3P nor localized to the Food Vacuole. Interestingly, ScAtg18 showed complete cytoplasmic localization despite binding with PI3P, indicating additional requirement for PfAtg18 localization. The Food Vacuole multi-drug resistance protein 1 (MDR1) was consistently identified in the PfAtg18 immunoprecipitate, and also interacted with PfAtg18. In contrast to PfAtg18, ScAtg18 did not interact with the MDR1, which, in addition to PI3P, could play a critical role in localization of PfAtg18. Chloroquine and amodiaquine greatly affected PfAtg18 localization, suggesting that these quinolines target PfAtg18 or the proteins that might be involved in its localization. Thus, PI3P and MDR1are critical mediators of PfAtg18 localization, and PfAtg18 may modulate MDR1 activity.
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plasmodium falciparum cysteine protease falcipain 1 is not essential in erythrocytic stage malaria parasites
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Puran Singh Sijwali, Michael Klemba, Daniel E. Goldberg, Kentaro Kato, Karl B Seydel, Jiri Gut, Julie Lehman, Louis H Miller, Philip J. RosenthalAbstract:Among potential new targets for antimalarial chemotherapy are Plasmodium falciparum cysteine proteases, known as falcipains. Falcipain-2 and falcipain-3 are Food Vacuole hemoglobinases that may have additional functions. The function of falcipain-1 remains uncertain. To better characterize the role of falcipain-1 in erythrocytic parasites, we disrupted the falcipain-1 gene and characterized recombinant parasites. Disruption of the falcipain-1 gene was confirmed with Southern blots, and loss of expression of falcipain-1 was confirmed with immunoblots and by loss of labeling with a specific protease inhibitor. Compared with wild-type parasites, falcipain-1 knockout parasites developed normally, with the same morphology, multiplication rate, and invasion efficiency, and without significant differences in sensitivity to cysteine protease inhibitors. In wild-type and knockout parasites, cysteine protease inhibitors blocked hemoglobin hydrolysis in trophozoites, with a subsequent block in rupture of erythrocytes by mature schizonts, but they did not inhibit erythrocyte invasion by merozoites. Our results indicate that although falcipain-1 is expressed by erythrocytic parasites, it is not essential for normal development during this stage or for erythrocyte invasion.
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gene disruption confirms a critical role for the cysteine protease falcipain 2 in hemoglobin hydrolysis by plasmodium falciparum
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Puran Singh Sijwali, Philip J. RosenthalAbstract:Erythrocytic malaria parasites degrade hemoglobin in an acidic Food Vacuole to acquire free amino acids and maintain parasite homeostasis. Hemoglobin hydrolysis appears to be a cooperative process requiring cysteine proteases (falcipains) and aspartic proteases (plasmepsins), but the specific roles of different enzymes in this process are unknown. We previously showed that falcipain-2 is a major trophozoite Food Vacuole cysteine protease. To characterize the specific role of falcipain-2, we disrupted the falcipain-2 gene and assessed the effect of this alteration. Falcipain-2-knockout trophozoites had markedly diminished cysteine protease activity and swollen, dark staining Food Vacuoles, consistent with a block in hemoglobin hydrolysis, as caused by cysteine protease inhibitors. However, more mature stages of knockout parasites were indistinguishable from wild-type parasites and developed normally. The knockout parasites had decreased and delayed expression of falcipain-2, which appeared to be directed by increased transcription of a second copy of the gene (falcipain-2′). Expression of other falcipains and plasmepsins was similar in wild-type and knockout parasites. Compared with wild-type, knockout parasites were about 3 times more sensitive to the cysteine protease inhibitors E-64 and leupeptin, and over 50-fold more sensitive to the aspartic protease inhibitor pepstatin. Our results assign a specific function for falcipain-2, the hydrolysis of hemoglobin in trophozoites. In addition, they highlight the cooperative action of cysteine and aspartic proteases in hemoglobin degradation by malaria parasites.
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characterization of native and recombinant falcipain 2 a principal trophozoite cysteine protease and essential hemoglobinase of plasmodium falciparum
Journal of Biological Chemistry, 2000Co-Authors: Bhaskar R. Shenai, Ajay Singh, Puran Singh Sijwali, Philip J. RosenthalAbstract:Abstract Trophozoites of the malaria parasitePlasmodium falciparum hydrolyze erythrocyte hemoglobin in an acidic Food Vacuole to provide amino acids for parasite protein synthesis. Cysteine protease inhibitors block hemoglobin degradation, indicating that a cysteine protease plays a key role in this process. A principal trophozoite cysteine protease was purified by affinity chromatography. Sequence analysis indicated that the protease is encoded by a previously unidentified gene, falcipain-2. Falcipain-2 was predominantly expressed in trophozoites, was concentrated in Food Vacuoles, and was responsible for at least 93% of trophozoite soluble cysteine protease activity. A construct encoding mature falcipain-2 and a small portion of the prodomain was expressed in Escherichia coli and refolded to active enzyme. Specificity for the hydrolysis of peptide substrates by native and recombinant falcipain-2 was very similar, and optimal at acid pH in a reducing environment. Under physiological conditions (pH 5.5, 1 mm glutathione), falcipain-2 hydrolyzed both native hemoglobin and denatured globin. Our results suggest that falcipain-2 can initiate cleavage of native hemoglobin in the P. falciparum Food Vacuole, that, following initial cleavages, the protease plays a key role in rapidly hydrolyzing globin fragments, and that a drug discovery effort targeted at this protease is appropriate.
Stefano Benedicenti - One of the best experts on this subject based on the ideXlab platform.
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effect of 808 nm diode laser on swimming behavior Food Vacuole formation and endogenous atp production of paramecium primaurelia protozoa
Photochemistry and Photobiology, 2015Co-Authors: Andrea Amaroli, Silvia Ravera, Steven Parker, Isabella Panfoli, Alberico Benedicenti, Stefano BenedicentiAbstract:Photobiomodulation (PBM) has been used in clinical practice for more than 40 years. To clarify the mechanisms of action of PBM at cellular and organism levels, we investigated its effect on Paramecium primaurelia (Protozoa) irradiated by an 808 nm infrared diode laser with a flat-top handpiece (1 W in CW). Our results led to the conclusion that: (1) the 808 nm laser stimulates the P. primaurelia without a thermal effect, (2) the laser effect is demonstrated by an increase in swimming speed and in Food Vacuole formation, (3) the laser treatment affects endogenous adenosine triphosphate (ATP) production in a positive way, (4) the effects of irradiation dose suggest an optimum exposure time of 50 s (64 J cm � 2 of fluence) to stimulate the Paramecium cells; irradiation of 25 s shows no effect or only mild effects and irradiation up to 100 s does not increase the effect observed with 50 s of treatment, (5) the increment of endogenous ATP concentration highlights the positive photobiomodulating effect of the 808 nm laser and the optimal irradiation conditions by the flat-top handpiece.