Overexpression, purification and characterisation of the Plasmodium falciparum Hsp70-z (PfHsp70-z) protein.

Six Hsp70-like genes are represented on the genome of Plasmodium falciparum. Of these two occur i0n the cytosol: P. falciparum Hsp70-z (PfHsp70-z) and PfHsp70-1. PfHsp70-1 is a well characterised canonical Hsp70 that facilitates protein quality control and is crucial for the development of malaria parasites. There is very little known about PfHsp70-z. However, PfHsp70-z is known to be essential and is implicated in suppressing aggregation of asparagine-rich proteins of P. falciparum. In addition, its expression at the clinical stage of malaria correlates with disease prognosis. Based on structural evidence PfHsp70-z belongs to the Hsp110 family of proteins. Since Hsp110 proteins have been described as nucleotide exchange factors (NEFs) of their canonical Hsp70 counterparts, it has been speculated that PfHsp70-z may serve as a NEF of PfHsp70-1. In the current study, P. falciparum cells cultured in vitro were subjected to heat stress, triggering the enhanced expression of PfHsp70-z. Biochemical assays conducted using recombinant PfHsp70-z protein demonstrated that the protein is heat stable and possesses ATPase activity. Furthermore, we observed that PfHsp70-z is capable of self-association. The structural-functional features of PfHsp70-z provide further evidence for its role as a chaperone and possible nucleotide exchange factor of PfHsp70-1.This project was through a grant (L1/402/ 14-1) provided to AS by the Deutsche Forchungsgemeinshaft (DFG) under the theme, “German–African Cooperation Projects in Infectology”. The authors are grateful to the Department of Science and Technology/National Research Foundation (NRF) of South Africa for providing an equipment grant (grant UID, 75464) to AS and (grant UID, 78558) to EP. HWD was awarded a research grant (64788) by the NRF (South Africa). AS is a recipient of a Georg Foster researchNCS201

Overexpression, purification and characterisation of the Plasmodium falciparum Hsp70-z (PfHsp70-z) protein.

Six Hsp70-like genes are represented on the genome of Plasmodium falciparum. Of these two occur i0n the cytosol: P. falciparum Hsp70-z (PfHsp70-z) and PfHsp70-1. PfHsp70-1 is a well characterised canonical Hsp70 that facilitates protein quality control and is crucial for the development of malaria parasites. There is very little known about PfHsp70-z. However, PfHsp70-z is known to be essential and is implicated in suppressing aggregation of asparagine-rich proteins of P. falciparum. In addition, its expression at the clinical stage of malaria correlates with disease prognosis. Based on structural evidence PfHsp70-z belongs to the Hsp110 family of proteins. Since Hsp110 proteins have been described as nucleotide exchange factors (NEFs) of their canonical Hsp70 counterparts, it has been speculated that PfHsp70-z may serve as a NEF of PfHsp70-1. In the current study, P. falciparum cells cultured in vitro were subjected to heat stress, triggering the enhanced expression of PfHsp70-z. Biochemical assays conducted using recombinant PfHsp70-z protein demonstrated that the protein is heat stable and possesses ATPase activity. Furthermore, we observed that PfHsp70-z is capable of self-association. The structural-functional features of PfHsp70-z provide further evidence for its role as a chaperone and possible nucleotide exchange factor of PfHsp70-1.This project was through a grant (L1/402/ 14-1) provided to AS by the Deutsche Forchungsgemeinshaft (DFG) under the theme, “German–African Cooperation Projects in Infectology”. The authors are grateful to the Department of Science and Technology/National Research Foundation (NRF) of South Africa for providing an equipment grant (grant UID, 75464) to AS and (grant UID, 78558) to EP. HWD was awarded a research grant (64788) by the NRF (South Africa). AS is a recipient of a Georg Foster researchNCS201

Select pyrimidinones inhibit the propagation of the malarial parasite, Plasmodium falciparum

Plasmodium falciparum, the Apicomplexan parasite that is responsible for the most lethal forms of human malaria, is exposed to radically different environments and stress factors during its complex lifecycle. In any organism, Hsp70 chaperones are typically associated with tolerance to stress. We therefore reasoned that inhibition of P. falciparum Hsp70 chaperones would adversely affect parasite homeostasis. To test this hypothesis, we measured whether pyrimidinone-amides, a new class of Hsp70 modulators, could inhibit the replication of the pathogenic P. falciparum stages in human red blood cells. Nine compounds with IC50 values from 30 nM to 1.6 μM were identified. Each compound also altered the ATPase activity of purified P. falciparum Hsp70 in single-turnover assays, although higher concentrations of agents were required than was necessary to inhibit P. falciparum replication. Varying effects of these compounds on Hsp70s from other organisms were also observed. Together, our data indicate that pyrimidinone-amides constitute a novel class of anti-malarial agents. © 2009 Elsevier Ltd. All rights reserved

Proteomic analysis of Plasmodium falciparum histone deacetylase 1 complex proteins

Plasmodium falciparum histone deacetylases (PfHDACs) are an important class of epigenetic regulators that alter protein lysine acetylation, contributing to regulation of gene expression and normal parasite growth and development. PfHDACs are therefore under investigation as drug targets for malaria. Despite this, our understanding of the biological roles of these enzymes is only just beginning to emerge. In higher eukaryotes, HDACs function as part of multi-protein complexes and act on both histone and non-histone substrates. Here, we present a proteomics analysis of PfHDAC1 immunoprecipitates, identifying 26 putative P. falciparum complex proteins in trophozoite-stage asexual intraerythrocytic parasites. The co-migration of two of these (P. falciparum heat shock proteins 70-1 and 90) with PfHDAC1 was validated using Blue Native PAGE combined with Western blot. These data provide a snapshot of possible PfHDAC1 interactions and a starting point for future studies focused on elucidating the broader function of PfHDACs in Plasmodium parasites

Cotrimoxazole reduces systemic inflammation in HIV infection by altering the gut microbiome and immune activation

Wellcome TrustCanadian Institutes of Health ResearchMedical Research CouncilDepartment for International Development under MRC/DFID Concordat agreement and EDCTP2 programme supported by the European UnionMRC Clinical Trials Unit at UC

Small but crucial : the novel small heat shock protein Hsp21 mediates stress adaptation and virulence in Candida albicans

Peer reviewedPublisher PD

Expression of a malarial Hsp70 improves defects in chaperone-dependent activities in ssa1 mutant yeast

Plasmodium falciparum causes the most virulent form of malaria and encodes a large number of molecular chaperones. Because the parasite encounters radically different environments during its lifecycle, many members of this chaperone ensemble may be essential for P. falciparum survival. Therefore, Plasmodium chaperones represent novel therapeutic targets, but to establish the mechanism of action of any developed therapeutics, it is critical to ascertain the functions of these chaperones. To this end, we report the development of a yeast expression system for PfHsp70-1, a P. falciparum cytoplasmic chaperone. We found that PfHsp70-1 repairs mutant growth phenotypes in yeast strains lacking the two primary cytosolic Hsp70s, SSA1 and SSA2, and in strains harboring a temperature sensitive SSA1 allele. PfHsp70-1 also supported chaperone-dependent processes such as protein translocation and ER associated degradation, and ameliorated the toxic effects of oxidative stress. By introducing engineered forms of PfHsp70-1 into the mutant strains, we discovered that rescue requires PfHsp70-1 ATPase activity. Together, we conclude that yeast can be co-opted to rapidly uncover specific cellular activities mediated by malarial chaperones. © 2011 Bell et al

The malarial exported PFA0660w is an Hsp40 co-chaperone of PfHsp70-x

Plasmodium falciparum, the human pathogen responsible for the most dangerous malaria infection, survives and develops in mature erythrocytes through the export of proteins needed for remodelling of the host cell. Molecular chaperones of the heat shock protein (Hsp) family are prominent members of the exportome, including a number of Hsp40s and a Hsp70. PFA0660w, a type II Hsp40, has been shown to be exported and possibly form a complex with PfHsp70-x in the infected erythrocyte cytosol. However, the chaperone properties of PFA0660w and its interaction with human and parasite Hsp70s are yet to be investigated. Recombinant PFA0660w was found to exist as a monomer in solution, and was able to significantly stimulate the ATPase activity of PfHsp70-x but not that of a second plasmodial Hsp70 (PfHsp70-1) or a human Hsp70 (HSPA1A), indicating a potential specific functional partnership with PfHsp70-x. Protein binding studies in the presence and absence of ATP suggested that the interaction of PFA0660w with PfHsp70-x most likely represented a co-chaperone/chaperone interaction. Also, PFA0660w alone produced a concentrationdependent suppression of rhodanese aggregation, demonstrating its chaperone properties. Overall, we have provided the first biochemical evidence for the possible role of PFA0660w as a chaperone and as co-chaperone of PfHsp70-x. We propose that these chaperones boost the chaperone power of the infected erythrocyte, enabling successful protein trafficking and folding, and thereby making a fundamental contribution to the pathology of malaria

Global response of Plasmodium falciparum to hyperoxia: a combined transcriptomic and proteomic approach

<p>Abstract</p> <p>Background</p> <p>Over its life cycle, the <it>Plasmodium falciparum </it>parasite is exposed to different environmental conditions, particularly to variations in O₂pressure. For example, the parasite circulates in human venous blood at 5% O₂pressure and in arterial blood, particularly in the lungs, at 13% O₂pressure. Moreover, the parasite is exposed to 21% O₂levels in the salivary glands of mosquitoes.</p> <p>Methods</p> <p>To study the metabolic adaptation of <it>P. falciparum </it>to different oxygen pressures during the intraerythrocytic cycle, a combined approach using transcriptomic and proteomic techniques was undertaken.</p> <p>Results</p> <p>Even though hyperoxia lengthens the parasitic cycle, significant transcriptional changes were detected in hyperoxic conditions in the late-ring stage. Using PS 6.0™ software (Ariadne Genomics) for microarray analysis, this study demonstrate up-expression of genes involved in antioxidant systems and down-expression of genes involved in the digestive vacuole metabolism and the glycolysis in favour of mitochondrial respiration. Proteomic analysis revealed increased levels of heat shock proteins, and decreased levels of glycolytic enzymes. Some of this regulation reflected post-transcriptional modifications during the hyperoxia response.</p> <p>Conclusions</p> <p>These results seem to indicate that hyperoxia activates antioxidant defence systems in parasites to preserve the integrity of its cellular structures. Moreover, environmental constraints seem to induce an energetic metabolism adaptation of <it>P. falciparum</it>. This study provides a better understanding of the adaptive capabilities of <it>P. falciparum </it>to environmental changes and may lead to the development of novel therapeutic targets.</p