scholarly journals Characterization of Plasmodium falciparum NEDD8 and identification of cullins as its substrates

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Manish Bhattacharjee ◽  
Navin Adhikari ◽  
Renu Sudhakar ◽  
Zeba Rizvi ◽  
Divya Das ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Drug Target ◽  
Parasite Development ◽  
Wild Type ◽  
Post Translational Modifications ◽  
Apicomplexan Parasites ◽  
Cellular Processes ◽  
Ring E3 ◽  
Physiological Substrates

AbstractA variety of post-translational modifications of Plasmodium falciparum proteins, including phosphorylation and ubiquitination, are shown to have key regulatory roles during parasite development. NEDD8 is a ubiquitin-like modifier of cullin-RING E3 ubiquitin ligases, which regulates diverse cellular processes. Although neddylation is conserved in eukaryotes, it is yet to be characterized in Plasmodium and related apicomplexan parasites. We characterized P. falciparum NEDD8 (PfNEDD8) and identified cullins as its physiological substrates. PfNEDD8 is a 76 amino acid residue protein without the C-terminal tail, indicating that it can be readily conjugated. The wild type and mutant (Gly75Ala/Gly76Ala) PfNEDD8 were expressed in P. falciparum. Western blot of wild type PfNEDD8-expressing parasites indicated multiple high molecular weight conjugates, which were absent in the parasites expressing the mutant, indicating conjugation of NEDD8 through Gly76. Immunoprecipitation followed by mass spectrometry of wild type PfNEDD8-expressing parasites identified two putative cullins. Furthermore, we expressed PfNEDD8 in mutant S. cerevisiae strains that lacked endogenous NEDD8 (rub1Δ) or NEDD8 conjugating E2 enzyme (ubc12Δ). The PfNEDD8 immunoprecipitate also contained S. cerevisiae cullin cdc53, further substantiating cullins as physiological substrates of PfNEDD8. Our findings lay ground for investigation of specific roles and drug target potential of neddylation in malaria parasites.

scholarly journals Characterization of Plasmodium falciparum NEDD8 and identification of cullins as its substrates

2020 ◽  
Author(s):  
Manish Bhattacharjee ◽  
Navin Adhikari ◽  
Renu Sudhakar ◽  
Zeba Rizvi ◽  
Divya Das ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Plasmodium Falciparum ◽  
Western Blot ◽  
Wild Type ◽  
Malaria Parasites ◽  
Post Translational Modifications ◽  
Functional Conservation ◽  
Cellular Processes ◽  
Physiological Substrates

ABSTRACTA variety of post-translational modifications of Plasmodium falciparum proteins, including phosphorylation and ubiquitination, are shown to have key regulatory roles. The neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) is a ubiquitin-like modifier of cullin-RING E3 ubiquitin ligases, which regulate diverse cellular processes, including the cell-cycle. Although neddylation pathway is conserved in eukaryotes, it is yet to be characterized in Plasmodium and related apicomplexan parasites. Towards studying the neddylation pathway in malaria parasites, we characterized P. falciparum NEDD8 (PfNEDD8) and identified cullins as its physiological substrates. PfNEDD8 is a 76 amino acid residue protein without the C-terminal tail, indicating that it can be readily conjugated. The wild type and mutant (Gly75Gly76 mutated to Ala75Ala76) PfNEDD8 were expressed in P. falciparum. Western blot of wild type PfNEDD8-expressing parasites indicated multiple high molecular weight conjugates, which were absent in the parasites expressing the mutant, indicating conjugation of NEDD8 to proteins through Gly76. Immunoprecipitation followed by mass spectrometry of wild type PfNEDD8-expressing parasites identified several proteins, including two putative cullins. Furthermore, we expressed PfNEDD8 in mutant S. cerevisiae strains that lacked endogenous NEDD8 (Δrub1) or NEDD8 conjugating E2 enzyme (ΔUbc12). The western blot of complemented strains and mass spectrometry of PfNEDD8 immunoprecipitate showed conjugation of PfNEDD8 to S. cerevisiae cullin cdc53, demonstrating functional conservation and cullins as the physiological substrates of PfNEDD8. The characterization of PfNEDD8 and identification of cullins as its substrates make ground for investigation of specific roles and drug target potential of neddylation pathway in malaria parasites.

scholarly journals Molecular Characterization of the CytochromebGene andIn VitroAtovaquone Susceptibility of Plasmodium falciparum Isolates from Kenya

2015 ◽  
Vol 59 (3) ◽  
pp. 1818-1821 ◽  
Author(s):  
Luicer A. Ingasia ◽  
Hoseah M. Akala ◽  
Mabel O. Imbuga ◽  
Benjamin H. Opot ◽  
Fredrick L. Eyase ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Genetic Polymorphism ◽  
Molecular Characterization ◽  
Mutant Allele ◽  
Inhibitory Concentration ◽  
Wild Type Allele ◽  
Wild Type ◽  
Western Kenya ◽  
Content Type

ABSTRACTThe prevalence of a genetic polymorphism(s) at codon 268 in the cytochromebgene, which is associated with failure of atovaquone-proguanil treatment, was analyzed in 227Plasmodium falciparumparasites from western Kenya. The prevalence of the wild-type allele was 63%, and that of the Y268S (denoting a Y-to-S change at position 268) mutant allele was 2%. There were no pure Y268C or Y268N mutant alleles, only mixtures of a mutant allele(s) with the wild type. There was a correlation between parasite 50% inhibitory concentration (IC50) and parasite genetic polymorphism; mutant alleles had higher IC50s than the wild type.

scholarly journals Disassembly activity of actin-depolymerizing factor (ADF) is associated with distinct cellular processes in apicomplexan parasites

2015 ◽  
Vol 26 (17) ◽  
pp. 3001-3012 ◽  
Author(s):  
Silvia Haase ◽  
Dennis Zimmermann ◽  
Maya A. Olshina ◽  
Mark Wilkinson ◽  
Fabio Fisher ◽  
...  
Keyword(s):  
Parasite Development ◽  
Actin Depolymerizing Factor ◽  
Phenotypic Effect ◽  
Basic Residue ◽  
Human Malaria Parasite ◽  
Apicomplexan Parasites ◽  
Functional Conservation ◽  
Cellular Processes ◽  
Organelle Segregation ◽  
Biochemical Analyses

Proteins of the actin-depolymerizing factor (ADF)/cofilin family have been shown to be crucial for the motility and survival of apicomplexan parasites. However, the mechanisms by which ADF proteins fulfill their function remain poorly understood. In this study, we investigate the comparative activities of ADF proteins from Toxoplasma gondii and Plasmodium falciparum, the human malaria parasite, using a conditional T. gondii ADF-knockout line complemented with ADF variants from either species. We show that P. falciparum ADF1 can fully restore native TgADF activity, demonstrating functional conservation between parasites. Strikingly, mutation of a key basic residue (Lys-72), previously implicated in disassembly in PfADF1, had no detectable phenotypic effect on parasite growth, motility, or development. In contrast, organelle segregation was severely impaired when complementing with a TgADF mutant lacking the corresponding residue (Lys-68). Biochemical analyses of each ADF protein confirmed the reduced ability of lysine mutants to mediate actin depolymerization via filament disassembly although not severing, in contrast to previous reports. These data suggest that actin filament disassembly is essential for apicomplexan parasite development but not for motility, as well as pointing to genus-specific coevolution between ADF proteins and their native actin.

scholarly journals Biochemical and Structural Characterization of Selective Allosteric Inhibitors of the Plasmodium falciparum Drug Target, Prolyl-tRNA-synthetase

2016 ◽  
Vol 3 (1) ◽  
pp. 34-44 ◽  
Author(s):  
Stephen Nakazawa Hewitt ◽  
David M. Dranow ◽  
Benjamin G. Horst ◽  
Jan A. Abendroth ◽  
Barbara Forte ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Structural Characterization ◽  
Drug Target ◽  
Trna Synthetase ◽  
Allosteric Inhibitors

scholarly journals Phosphorylation in the Charged Linker Modulates Interactions and Secretion of Hsp90β

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1701
Author(s):  
Lorenz Weidenauer ◽  
Manfredo Quadroni
Keyword(s):  
Mass Spectrometry ◽  
K562 Cells ◽  
Phosphorylation Sites ◽  
Activity Regulation ◽  
Wild Type ◽  
Post Translational Modifications ◽  
Linker Region ◽  
Cellular Processes ◽  
Tryptic Cleavage ◽  
Client Proteins

Hsp90β is a major chaperone involved in numerous cellular processes. Hundreds of client proteins depend on Hsp90β for proper folding and/or activity. Regulation of Hsp90β is critical to coordinate its tasks and is mediated by several post-translational modifications. Here, we focus on two phosphorylation sites located in the charged linker region of human Hsp90β, Ser226 and Ser255, which have been frequently reported but whose function remains unclear. Targeted measurements by mass spectrometry indicated that intracellular Hsp90β is highly phosphorylated on both sites (>90%). The level of phosphorylation was unaffected by various stresses (e.g., heat shock, inhibition with drugs) that impact Hsp90β activity. Mutating the two serines to alanines increased the amount of proteins interacting with Hsp90β globally and increased the sensitivity to tryptic cleavage in the C-terminal domain. Further investigation revealed that phosphorylation on Ser255 and to a lesser extent on Ser226 is decreased in the conditioned medium of cultured K562 cells, and that a non-phosphorylatable double alanine mutant was secreted more efficiently than the wild type. Overall, our results show that phosphorylation events in the charged linker regulate both the interactions of Hsp90β and its secretion, through changes in the conformation of the chaperone.

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

scholarly journals Glucose-6-phosphate dehydrogenase–6-phosphogluconolactonase: a unique bifunctional enzyme from Plasmodium falciparum

2011 ◽  
Vol 436 (3) ◽  
pp. 641-650 ◽  
Author(s):  
Esther Jortzik ◽  
Boniface M. Mailu ◽  
Janina Preuss ◽  
Marina Fischer ◽  
Lars Bode ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Pentose Phosphate Pathway ◽  
Drug Target ◽  
Pentose Phosphate ◽  
Bifunctional Enzyme ◽  
Functional Differences ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
First Time ◽  
Human Rbcs

The survival of malaria parasites in human RBCs (red blood cells) depends on the pentose phosphate pathway, both in Plasmodium falciparum and its human host. G6PD (glucose-6-phosphate dehydrogenase) deficiency, the most common human enzyme deficiency, leads to a lack of NADPH in erythrocytes, and protects from malaria. In P. falciparum, G6PD is combined with the second enzyme of the pentose phosphate pathway to create a unique bifunctional enzyme named GluPho (glucose-6-phosphate dehydrogenase–6-phosphogluconolactonase). In the present paper, we report for the first time the cloning, heterologous overexpression, purification and kinetic characterization of both enzymatic activities of full-length PfGluPho (P. falciparum GluPho), and demonstrate striking structural and functional differences with the human enzymes. Detailed kinetic analyses indicate that PfGluPho functions on the basis of a rapid equilibrium random Bi Bi mechanism, where the binding of the second substrate depends on the first substrate. We furthermore show that PfGluPho is inhibited by S-glutathionylation. The availability of recombinant PfGluPho and the major differences to hG6PD (human G6PD) facilitate studies on PfGluPho as an excellent drug target candidate in the search for new antimalarial drugs.

scholarly journals Actin from the apicomplexan Neospora caninum (NcACT) has different isoforms in 2D electrophoresis

Parasitology ◽  
2018 ◽  
Vol 146 (1) ◽  
pp. 33-41
Author(s):  
Luciana Baroni ◽  
Letícia Pollo-Oliveira ◽  
Albert JR Heck ◽  
AF Maarten Altelaar ◽  
Ana Patrícia Yatsuda
Keyword(s):  
Neospora Caninum ◽  
Mass Spectrometry Data ◽  
2D Electrophoresis ◽  
Post Translational Modifications ◽  
Actin Isoforms ◽  
One Dimensional ◽  
Apicomplexan Parasites ◽  
Dense Granules ◽  
Cellular Processes ◽  
Cellular Extract

AbstractApicomplexan parasites have unconventional actins that play a central role in important cellular processes such as apicoplast replication, motility of dense granules, endocytic trafficking and force generation for motility and host cell invasion. In this study, we investigated the actin of the apicomplexan Neospora caninum – a parasite associated with infectious abortion and neonatal mortality in livestock. Neospora caninum actin was detected and identified in two bands by one-dimensional (1D) western blot and in nine spots by the 2D technique. The mass spectrometry data indicated that N. caninum has at least nine different actin isoforms, possibly caused by post-translational modifications. In addition, the C4 pan-actin antibody detected specifically actin in N. caninum cellular extract. Extracellular N. caninum tachyzoites were treated with toxins that act on actin, jasplakinolide and cytochalasin D. Both substances altered the peripheric cytoplasmic localization of actin on tachyzoites. Our findings add complexity to the study of the apicomplexan actin in cellular processes, since the multiple functions of this important protein might be regulated by mechanisms involving post-translational modifications.

scholarly journals Protein O- and C-Glycosylation pathways in Toxoplasma gondii and Plasmodium falciparum

Parasitology ◽  
2019 ◽  
Vol 146 (14) ◽  
pp. 1755-1766 ◽  
Author(s):  
Giulia Bandini ◽  
Andreia Albuquerque-Wendt ◽  
Jan Hegermann ◽  
John Samuelson ◽  
Françoise H. Routier
Keyword(s):  
Plasmodium Falciparum ◽  
Toxoplasma Gondii ◽  
Protein Glycosylation ◽  
Specific Gene ◽  
Post Translational Modifications ◽  
Apicomplexan Parasites ◽  
Causative Agents ◽  
Thrombospondin Type 1 Repeats ◽  
Great Public Health

AbstractApicomplexan parasites are amongst the most prevalent and morbidity-causing pathogens worldwide. They are responsible for severe diseases in humans and livestock and are thus of great public health and economic importance. Until the sequencing of apicomplexan genomes at the beginning of this century, the occurrence of N- and O-glycoproteins in these parasites was much debated. The synthesis of rudimentary and divergent N-glycans due to lineage-specific gene loss is now well established and has been recently reviewed. Here, we will focus on recent studies that clarified classical O-glycosylation pathways and described new nucleocytosolic glycosylations in Toxoplasma gondii, the causative agents of toxoplasmosis. We will also review the glycosylation of proteins containing thrombospondin type 1 repeats by O-fucosylation and C-mannosylation, newly discovered in Toxoplasma and the malaria parasite Plasmodium falciparum. The functional significance of these post-translational modifications has only started to emerge, but the evidence points towards roles for these protein glycosylation pathways in tissue cyst wall rigidity and persistence in the host, oxygen sensing, and stability of proteins involved in host invasion.

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