scholarly journals Plasmodium falciparum Niemann-Pick Type C1-Related Protein is a Druggable Target Required for Parasite Membrane Homeostasis

2018 ◽  
Author(s):  
Eva S. Istvan ◽  
Sudipta Das ◽  
Suyash Bhatnagar ◽  
Josh R. Beck ◽  
Edward Owen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasmodium Falciparum ◽  
Plasma Membrane ◽  
Small Molecule ◽  
Antimalarial Drug ◽  
Drug Target ◽  
Related Protein ◽  
Immuno Electron Microscopy ◽  
Increased Sensitivity ◽  
Niemann Pick

AbstractPlasmodium parasites possess a protein with homology to Niemann-Pick Type C1 proteins (Plasmodium falciparum Niemann-Pick Type C1-Related protein, PfNCR1). We isolated parasites with resistance-conferring mutations in PfNCR1 during selections with three diverse small-molecule antimalarial compounds and show that the mutations are causative for compound resistance. PfNCR1 protein knockdown results in severely attenuated growth and confers hypersensitivity to the compounds. Compound treatment or protein knockdown leads to increased sensitivity of the parasite plasma membrane (PPM) to the amphipathic glycoside saponin and engenders digestive vacuoles (DVs) that are small and malformed. Immuno-electron microscopy and split-GFP experiments localize PfNCR1 to the PPM. Our experiments show that PfNCR1 activity is critically important for the composition of the PPM and is required for DV biogenesis, suggesting PfNCR1 as a novel antimalarial drug target.

scholarly journals Plasmodium Niemann-Pick type C1-related protein is a druggable target required for parasite membrane homeostasis

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Eva S Istvan ◽  
Sudipta Das ◽  
Suyash Bhatnagar ◽  
Josh R Beck ◽  
Edward Owen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasmodium Falciparum ◽  
Plasma Membrane ◽  
Drug Target ◽  
Editorial Note ◽  
Editorial Process ◽  
Related Protein ◽  
Immuno Electron Microscopy ◽  
Increased Sensitivity ◽  
Niemann Pick

Plasmodium parasites possess a protein with homology to Niemann-Pick Type C1 proteins (Niemann-Pick Type C1-Related protein, NCR1). We isolated parasites with resistance-conferring mutations in Plasmodium falciparum NCR1 (PfNCR1) during selections with three diverse small-molecule antimalarial compounds and show that the mutations are causative for compound resistance. PfNCR1 protein knockdown results in severely attenuated growth and confers hypersensitivity to the compounds. Compound treatment or protein knockdown leads to increased sensitivity of the parasite plasma membrane (PPM) to the amphipathic glycoside saponin and engenders digestive vacuoles (DVs) that are small and malformed. Immuno-electron microscopy and split-GFP experiments localize PfNCR1 to the PPM. Our experiments show that PfNCR1 activity is critically important for the composition of the PPM and is required for DV biogenesis, suggesting PfNCR1 as a novel antimalarial drug target.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (<xref ref-type="decision-letter" rid="SA1">see decision letter</xref>).

scholarly journals A specific non-bisphosphonate inhibitor of the bifunctional farnesyl/geranylgeranyl diphosphate synthase in malaria parasites

2017 ◽  
Author(s):  
Jolyn E. Gisselberg ◽  
Zachary Herrera ◽  
Lindsey Orchard ◽  
Manuel Llinás ◽  
Ellen Yeh
Keyword(s):  
Plasmodium Falciparum ◽  
Physicochemical Properties ◽  
Small Molecule ◽  
Antimalarial Drug ◽  
Drug Target ◽  
Drug Targets ◽  
Geranylgeranyl Diphosphate Synthase ◽  
Geranylgeranyl Diphosphate ◽  
Malaria Parasites ◽  
Starting Point

SummaryIsoprenoid biosynthesis is essential for Plasmodium falciparum (malaria) parasites and contains multiple validated antimalarial drug targets, including a bifunctional farnesyl and geranylgeranyl diphosphate synthase (FPPS/GGPPS). We identified MMV019313 as an inhibitor of PfFPPS/GGPPS. Though PfFPPS/GGPPS is also inhibited by a class of bisphosphonate drugs, MMV019313 has significant advantages for antimalarial drug development. MMV019313 has superior physicochemical properties compared to charged bisphosphonates that have poor bioavailability and strong bone affinity. We also show that it is highly selective for PfFPPS/GGPPS and showed no activity against human FPPS or GGPPS. Inhibition of PfFPPS/GGPPS by MMV019313, but not bisphosphonates, was disrupted in an S228T variant, demonstrating that MMV019313 and bisphosphonates have distinct modes-of-inhibition against PfFPPS/GGPPS. Altogether MMV019313 is the first specific, non-bisphosphonate inhibitor of PfFPPS/GGPPS. Our findings uncover a new small molecule binding site in this important antimalarial drug target and provide a promising starting point for development of Plasmodium-specific FPPS/GGPPS inhibitors.

scholarly journals alpha-2 Macroglobulin receptor/Ldl receptor-related protein(Lrp)-dependent internalization of the urokinase receptor.

1995 ◽  
Vol 131 (6) ◽  
pp. 1609-1622 ◽  
Author(s):  
M Conese ◽  
A Nykjaer ◽  
C M Petersen ◽  
O Cremona ◽  
R Pardi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Confocal Microscopy ◽  
Cell Surface ◽  
Cell Line ◽  
Plasminogen Activator ◽  
Protein Synthesis Inhibitor ◽  
Related Protein ◽  
Urokinase Plasminogen Activator Receptor ◽  
Immuno Electron Microscopy ◽  
Pai 1

The GPI-anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA). On the contrary, uPAR-bound complexes of uPA with its serpin inhibitors PAI-1 (plasminogen activator inhibitor type-1) or PN-1 (protease nexin-1) are readily internalized in several cell types. Here we address the question whether uPAR is internalized as well upon binding of uPA-serpin complexes. Both LB6 clone 19 cells, a mouse cell line transfected with the human uPAR cDNA, and the human U937 monocytic cell line, express in addition to uPAR also the endocytic alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP/alpha 2-MR) which is required to internalize uPAR-bound uPA-PAI-1 and uPA-PN-1 complexes. Downregulation of cell surface uPAR molecules in U937 cells was detected by cytofluorimetric analysis after uPA-PAI-1 and uPA-PN-1 incubation for 30 min at 37 degrees C; this effect was blocked by preincubation with the ligand of LRP/alpha 2-MR, RAP (LRP/alpha 2-MR-associated protein), known to block the binding of the uPA complexes to LRP/alpha 2-. MR. Downregulation correlated in time with the intracellular appearance of uPAR as assessed by confocal microscopy and immuno-electron microscopy. After 30 min incubation with uPA-PAI-1 or uPA-PN-1 (but not with free uPA), confocal microscopy showed that uPAR staining in permeabilized LB6 clone 19 cells moved from a mostly surface associated to a largely perinuclear position. This effect was inhibited by the LRP/alpha 2-MR RAP. Perinuclear uPAR did not represent newly synthesized nor a preexisting intracellular pool of uPAR, since this fluorescence pattern was not modified by treatment with the protein synthesis inhibitor cycloheximide, and since in LB6 clone 19 cells all of uPAR was expressed on the cell surface. Immuno-electron microscopy confirmed the plasma membrane to intracellular translocation of uPAR, and its dependence on LRP/alpha 2-MR in LB6 clone 19 cells only after binding to the uPA-PAI-1 complex. After 30 min incubation at 37 degrees C with uPA-PAI-1, 93% of the specific immunogold particles were present in cytoplasmic vacuoles vs 17.6% in the case of DFP-uPA. We conclude therefore that in the process of uPA-serpin internalization, uPAR itself is internalized, and that internalization requires the LRP/alpha 2-MR.

scholarly journals Caveolin-1 is incorporated into mature respiratory syncytial virus particles during virus assembly on the surface of virus-infected cells

2002 ◽  
Vol 83 (3) ◽  
pp. 611-621 ◽  
Author(s):  
Gaie Brown ◽  
James Aitken ◽  
Helen W. McL. Rixon ◽  
Richard J. Sugrue
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Respiratory Syncytial Virus ◽  
Confocal Microscopy ◽  
Cell Surface ◽  
Filamentous Form ◽  
Caveolin 1 ◽  
Immuno Electron Microscopy ◽  
Infected Cells ◽  
Syncytial Virus

We have employed immunofluorescence microscopy and transmission electron microscopy to examine the assembly and maturation of respiratory syncytial virus (RSV) in the Vero cell line C1008. RSV matures at the apical cell surface in a filamentous form that extends from the plasma membrane. We observed that inclusion bodies containing viral ribonucleoprotein (RNP) cores predominantly appeared immediately below the plasma membrane, from where RSV filaments form during maturation at the cell surface. A comparison of mock-infected and RSV-infected cells by confocal microscopy revealed a significant change in the pattern of caveolin-1 (cav-1) fluorescence staining. Analysis by immuno-electron microscopy showed that RSV filaments formed in close proximity to cav-1 clusters at the cell surface membrane. In addition, immuno-electron microscopy showed that cav-1 was closely associated with early budding RSV. Further analysis by confocal microscopy showed that cav-1 was subsequently incorporated into the envelope of RSV filaments maturing on the host cell membrane, but was not associated with other virus structures such as the viral RNPs. Although cav-1 was incorporated into the mature virus, it was localized in clusters rather than being uniformly distributed along the length of the viral filaments. Furthermore, when RSV particles in the tissue culture medium from infected cells were examined by immuno-negative staining, the presence of cav-1 on the viral envelope was clearly demonstrated. Collectively, these findings show that cav-1 is incorporated into the envelope of mature RSV particles during egress.

scholarly journals Mitochondrial type II NADH dehydrogenase of Plasmodium falciparum is dispensable and not the functional target of putative NDH2 quinolone inhibitors

2018 ◽  
Author(s):  
Hangjun Ke ◽  
Suresh M. Ganesan ◽  
Swati Dass ◽  
Joanne M. Morrisey ◽  
Sovitj Pou ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Antimalarial Drug ◽  
Drug Target ◽  
Nadh Dehydrogenase ◽  
Antimalarial Activity ◽  
Reductase Activity ◽  
Proton Pumping ◽  
Type Ii ◽  
Long Time ◽  
Specific Inhibitors

AbstractThe battle against malaria has been substantially impeded by the recurrence of drug resistance in Plasmodium falciparum, the deadliest human malaria parasite. To counter the problem, novel antimalarial drugs are urgently needed, especially those that target unique pathways of the parasite, since they are less likely to have side effects. The mitochondrial type II NADH dehydrogenase of P. falciparum, PfNDH2 (PF3D7_0915000), has been considered a good prospective antimalarial drug target for over a decade, since malaria parasites lack the conventional multi-subunit NADH dehydrogenase, or Complex I, present in the mammalian mitochondrial electron transport chain (mtETC). Instead, Plasmodium parasites contain a single subunit NDH2, which lacks proton pumping activity and is absent in humans. A significant amount of effort has been expended to develop PfNDH2 specific inhibitors, yet the essentiality of PfNDH2 has not been convincingly verified. Herein, we knocked out PfNDH2 in P. falciparum via a CRISPR/Cas9 mediated approach. Deletion of PfNDH2 does not alter the parasite’s susceptibility to multiple mtETC inhibitors, including atovaquone and ELQ-300. We also show that the antimalarial activity of the fungal NDH2 inhibitor HDQ and its new derivative CK-2-68 is due to inhibition of the parasite cytochrome bc1 complex rather than PfNDH2. These compounds directly inhibit the ubiquinol-cytochrome c reductase activity of the malarial bc1 complex. Our results call into question the validity of PfNDH2 as an antimalarial drug target.ImportanceFor a long time, PfNDH2 has been considered an attractive antimalarial drug target. However, the conclusion that PfNDH2 is essential was based on preliminary and incomplete data. Here we generate a PfNDH2 KO (knockout) parasite in the blood stages of Plasmodium falciparum, showing that the gene is not essential. We also show that previously reported PfNDH2-specific inhibitors kill the parasites primarily via targeting the cytochrome bc1 complex, not PfNDH2. Overall, we provide genetic and biochemical data that help to resolve a long-debated issue in the field regarding the potential of PfNDH2 as an antimalarial drug target.

scholarly journals Comparison of Efficacies of Cysteine Protease Inhibitors against Five Strains of Plasmodium falciparum

2001 ◽  
Vol 45 (3) ◽  
pp. 949-951 ◽  
Author(s):  
Ajay Singh ◽  
Philip J. Rosenthal
Keyword(s):  
Plasmodium Falciparum ◽  
Protease Inhibitors ◽  
Cysteine Protease ◽  
Antimalarial Drug ◽  
Drug Target ◽  
Antimalarial Drugs ◽  
Cross Resistance ◽  
Cysteine Protease Inhibitors ◽  
Antimalarial Agents ◽  
Parasite Strains

ABSTRACT Falcipain-2, a cysteine protease and essential hemoglobinase ofPlasmodium falciparum, is a potential antimalarial drug target. We compared the falcipain-2 sequences and sensitivities to cysteine protease inhibitors of five parasite strains that differ markedly in sensitivity to established antimalarial drugs. The sequence of falcipain-2 was highly conserved, and the sensitivities of all of the strains to falcipain-2 inhibitors were very similar. Thus, cross-resistance between cysteine protease inhibitors and other antimalarial agents is not expected in parasites that are now circulating and falcipain-2 remains a promising chemotherapeutic target.

scholarly journals Visualization of Receptor-mediated Endocytosis in Yeast

1999 ◽  
Vol 10 (3) ◽  
pp. 799-817 ◽  
Author(s):  
Jon Mulholland ◽  
James Konopka ◽  
Birgit Singer-Kruger ◽  
Marino Zerial ◽  
David Botstein
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Receptor Internalization ◽  
Time Dependent ◽  
Carboxypeptidase Y ◽  
Cortical Actin ◽  
Double Labeling ◽  
Receptor Mediated Endocytosis ◽  
Immuno Electron Microscopy ◽  
Actin Patch

We studied the ligand-induced endocytosis of the yeast α-factor receptor Ste2p by immuno-electron microscopy. We observed and quantitated time-dependent loss of Ste2p from the plasma membrane of cells exposed to α-factor. This ligand-induced internalization of Ste2p was blocked in the well-characterized endocytosis-deficient mutant sac6Δ. We provide evidence that implicates furrow-like invaginations of the plasma membrane as the site of receptor internalization. These invaginations are distinct from the finger-like plasma membrane invaginations within actin cortical patches. Consistent with this, we show that Ste2p is not located within the cortical actin patch before and during receptor-mediated endocytosis. In wild-type cells exposed to α-factor we also observed and quantitated a time-dependent accumulation of Ste2p in intracellular, membrane-bound compartments. These compartments have a characteristic electron density but variable shape and size and are often located adjacent to the vacuole. In immuno-electron microscopy experiments these compartments labeled with antibodies directed against the rab5 homologue Ypt51p (Vps21p), the resident vacuolar protease carboxypeptidase Y, and the vacuolar H+-ATPase Vph1p. Using a new double-labeling technique we have colocalized antibodies against Ste2p and carboxypeptidase Y to this compartment, thereby identifying these compartments as prevacuolar late endosomes.

Characterization of Plasmodium falciparum 6-Phosphogluconate Dehydrogenase as an Antimalarial Drug Target

2018 ◽  
Vol 430 (21) ◽  
pp. 4049-4067 ◽  
Author(s):  
Kristina Haeussler ◽  
Karin Fritz-Wolf ◽  
Max Reichmann ◽  
Stefan Rahlfs ◽  
Katja Becker
Keyword(s):  
Plasmodium Falciparum ◽  
Antimalarial Drug ◽  
Drug Target ◽  
Phosphogluconate Dehydrogenase
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