11 research outputs found
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
Structure-function study of a Plasmodium falciparum Hsp70 using three dimensional modelling and in Vitro analyses
The spatial orientation of domains of the heat shock protein 70 from Plasmodium falciparum (PfHsp70) were mapped based on a three-dimensional model of the protein. Purified PfHsp70 displayed chaperone activity in vitro. Amino acid substitutions introduced in the chaperone's substrate binding cavity compromised the protein's chaperone function
Biophysical analysis of Plasmodium falciparum Hsp70-Hsp90 organising protein (PfHop) reveals a monomer that is characterised by folded segments connected by flexible linkers
Abstract
Plasmodium falciparum causes the most lethal form of malaria. The cooperation of heat shock protein (Hsp) 70 and 90 is thought to facilitate folding of select group of cellular proteins that are crucial for cyto-protection and development of the parasites. Hsp70 and Hsp90 are brought into a functional complex that allows substrate exchange by stress inducible protein 1 (STI1), also known as Hsp70-Hsp90 organising protein (Hop). P. falciparum Hop (PfHop) co-localises and occurs in complex with the parasite cytosolic chaperones, PfHsp70â1 and PfHsp90. Here, we characterised the structure of recombinant PfHop using synchrotron radiation circular dichroism (SRCD) and small-angle X-ray scattering. Structurally, PfHop is a monomeric, elongated but folded protein, in agreement with its predicted TPR domain structure. Using SRCD, we established that PfHop is unstable at temperatures higher than 40°C. This suggests that PfHop is less stable at elevated temperatures compared to its functional partner, PfHsp70â1, that is reportedly stable at temperatures as high as 80°C. These findings contribute towards our understanding of the role of the Hop-mediated functional partnership between Hsp70 and Hsp90
Characterisation of the Plasmodium falciparum Hsp70-Hsp90 organising protein (PfHop)
Malaria is caused by Plasmodium species, whose
transmission to vertebrate hosts is facilitated by mosquito
vectors. The transition from the cold blooded mosquito
vector to the host represents physiological stress to the
parasite, and additionally malaria blood stage infection is
characterised by intense fever periods. In recent years, it
has become clear that heat shock proteins play an essential
role during the parasite's life cycle. Plasmodium falciparum
expresses two prominent heat shock proteins: heat shock
protein 70 (PfHsp70) and heat shock protein 90 (PfHsp90).
Both of these proteins have been implicated in the
development and pathogenesis of malaria. In eukaryotes,
Hsp70 and Hsp90 proteins are functionally linked by an
essential adaptor protein known as the Hsp70âHsp90
organising protein (Hop). In this study, recombinant P.
falciparum Hop (PfHop) was heterologously produced in E.
coli and purified by nickel affinity chromatography. Using
specific anti-PfHop antisera, the expression and localisation
of PfHop in P. falciparum was investigated. PfHop was
shown to co-localise with PfHsp70 and PfHsp90 in parasites
at the trophozoite stage. Gel filtration and coimmunoprecipitation
experiments suggested that PfHop
was present in a complex together with PfHsp70 and
PfHsp90. The association of PfHop with both PfHsp70 and
PfHsp90 suggests that this protein may mediate the
functional interaction between the two chaperones
Use of a Chimeric Hsp70 to Enhance the Quality of Recombinant Plasmodium falciparum S-Adenosylmethionine Decarboxylase Protein Produced in Escherichia coli
S1 Fig. KPf and PfHsp70 do not co-purify with PfAdoMetDC. Western blot representing the
purification of PfAdoMetDC expressed in E. coli BL21 (DE3) Star cells rehosted with various chaperone
combinations. Lanes: UâPfAdoMetDC expressed in the absence of supplemented chaperones;
KâPfAdoMetDC co-expressed with supplemented DnaK; KPfâPfAdoMetDC expressed in
cells supplemented with KPf; Pf70 âPfAdoMetDC expressed in cells supplemented with PfHsp70;
K-ELâPfAdoMetDC expressed in cells supplemented with DnaK and GroEL-GroES; KP-ELâPfAdoMetDC
expressed in cells supplemented with KPf and GroEL-GroES; Pf70-ELâPfAdoMetDC
expressed in cells supplemented with PfHsp70 and GroEL-GroES; +Câpositive consisting of purified
PfHsp70 protein.Western blot analysis of PfHsp70 (70 kDa) detected using α-PfHsp70 antibody.
Numbers to the left represent protein markers (Fermentas) in kDa.S2 Fig. Sequence alignment of PfHsp70 and E. coli DnaK. Sequence alignment of E. coli
DnaK (accession number: BAA01595.1) and PfHsp70 (accession number: PF08_0054) were
conducted using ClustalW and Boxshade. The following structural features are highlighted: the
highly conserved linker segment (black horizontal line) which separates the ATPase domain
from the peptide binding domain. Residues Y145, N147, D148, N170 and T173 in the ATPase
domain that interact with DnaJ as reviewed by Shonhai et al (8) are shown with black arrows.
Residues G400, D526 and G539 in the peptide binding domain of DnaK that are important for
interaction with DnaJ, and the aligned residues in PfHsp70 are shown as black arrows. Identical
residues are presented in white against a black background and similar residues are shown in
black against a grey background).S1 Table. E. coli strains and plasmids used in this study.S2 Table. Description of primers used towards generation of destination plasmids.S-adenosylmethionine decarboxylase (PfAdoMetDC) from Plasmodium falciparum is a prospective
antimalarial drug target. The production of recombinant PfAdoMetDC for biochemical
validation as a drug target is important. The production of PfAdoMetDC in Escherichia
coli has been reported to result in unsatisfactory yields and poor quality product. The coexpression
of recombinant proteins with molecular chaperones has been proposed as one
way to improve the production of the former in E. coli. E. coli heat shock proteins DnaK,
GroEL-GroES and DnaJ have previously been used to enhance production of some recombinant
proteins. However, the outcomes were inconsistent. An Hsp70 chimeric protein, KPf,
which is made up of the ATPase domain of E. coli DnaK and the substrate binding domain
of P. falciparum Hsp70 (PfHsp70) has been previously shown to exhibit chaperone function
when it was expressed in E. coli cells whose resident Hsp70 (DnaK) function was impaired.
We proposed that because of its domain constitution, KPf would most likely be recognised
by E. coli Hsp70 co-chaperones. Furthermore, because it possesses a substrate binding
domain of plasmodial origin, KPf would be primed to recognise recombinant PfAdoMetDC
expressed in E. coli. First, using site-directed mutagenesis, followed by complementation
assays, we established that KPf with a mutation in the hydrophobic residue located in its
substrate binding cavity was functionally compromised. We further co-expressed PfAdo-
MetDC with KPf, PfHsp70 and DnaK in E. coli cells either in the absence or presence of over-expressed GroEL-GroES chaperonin. The folded and functional status of the produced
PfAdoMetDC was assessed using limited proteolysis and enzyme assays. PfAdo-
MetDC co-expressed with KPf and PfHsp70 exhibited improved activity compared to
protein co-expressed with over-expressed DnaK. Our findings suggest that chimeric KPf may be an ideal Hsp70 co-expression partner for the production of recombinant plasmodial
proteins in E. coli.The
National Research Foundation for an equipment
grant (UID, 75464) awarded to AS. AS is a recipient
of a Georg Foster research fellowship awarded by the
Alexander von Humboldt Foundation of Germany.
XHM is a recipient of a National Research
Foundation (South Africa) scarce skills scholarship
and also received a grant from the University of Zululand Research Committee. AB is a recipient of a
postdoctoral fellowship awarded by the NRF.http://www.plosone.orgam2016BiochemistryUP Centre for Sustainable Malaria Control (UP CSMC
Plasmodium falciparum encodes a single cytosolic type I Hsp40 that functionally interacts with Hsp70 and is upregulated by heat shock
Heat shock protein 70 (Hsp70) and heat shock protein 40 (Hsp40) function as molecular chaperones during the folding and trafficking of proteins within most cell types. However, the Hsp70âHsp40 chaperone partnerships within the malaria parasite, Plasmodium falciparum, have not been elucidated. Only one of the 43 P. falciparum Hsp40s is predicted to be a cytosolic, canonical Hsp40 (termed PfHsp40) capable of interacting with the major cytosolic P. falciparum-encoded Hsp70, PfHsp70. Consistent with this hypothesis, we found that PfHsp40 is upregulated under heat shock conditions in a similar pattern to PfHsp70. In addition, PfHsp70 and PfHsp40 reside mainly in the parasite cytosol, as assessed using indirect immunofluorescence microscopy. Recombinant PfHsp40 stimulated the ATP hydrolytic rates of both PfHsp70 and human Hsp70 similar to other canonical Hsp40s of yeast (Ydj1) and human (Hdj2) origin. In contrast, the Hsp40-stimulated plasmodial and human Hsp70 ATPase activities were differentially inhibited in the presence of pyrimidinone-based small molecule modulators. To further probe the chaperone properties of PfHsp40, protein aggregation suppression assays were conducted. PfHsp40 alone suppressed protein aggregation, and cooperated with PfHsp70 to suppress aggregation. Together, these data represent the first cellular and biochemical evidence for a PfHsp70âPfHsp40 partnership in the malaria parasite, and furthermore that the plasmodial and human Hsp70âHsp40 chaperones possess unique attributes that are differentially modulated by small molecules