73 research outputs found
Molecular characterisation of the chaperone properties of Plasmodium falciparum heat shock protein 70
Heat shock protein 70 (called DnaK in prokaryotes) is one of the most prominent groups of chaperones whose role is to prevent and reverse protein misfolding. PfHsp70 is a heatinducible cytoplasm/nuclear localised Plasmodium falciparum Hsp70. PfHsp70 is thought to confer chaperone cytoprotection to P. falciparum during the development of malaria fever. The objective of this study was to examine the chaperone properties of PfHsp70 using a bioinformatics approach, coupled to in vivo and in vitro analysis. Structural motifs that qualify PfHsp70 as a typical Hsp70 chaperone were identified. Although PfHsp70 has a higher similarity to human Hsc70 than E. coli DnaK, in vivocomplementation assays showed that PfHsp70 was able to reverse the thermosensitivity of E. coli dnaK756 (a temperature sensitive strain whose DnaK is functionally compromised). Two residues (V401 and Q402) in the linker region of PfHsp70 that are critical for its in vivo function were identified. Constructs were generated that encoded the ATPase domain of PfHsp70 and the peptide binding domain of E. coli DnaK (to generate PfK chimera); and the ATPase domain of E. coli DnaK fused to the peptide binding domain of PfHsp70 (KPf). The two chimeras were tested for their ability to reverse the thermosensitivity of E. coli dnaK756 cells. Whilst KPf was able to reverse the thermosensitivity of the E. coli dnaK756 cells, PfK could not. Previously, PfHsp70 purification involved urea denaturation. Using a detergent, polyethylenimine (PEI), PfHsp70 was natively purified. Natively purified PfHsp70 had a basal ATPase activity approximately two times higher than the previously reported activity for the protein purified through urea denaturation. PfJ4, a type II Hsp40, could not stimulate the ATPase activity of PfHsp70 in vitro. Arch and hydrophobic pocket substitutions (A419Y, Y444A and V451F) were introduced in the PfHsp70 peptide binding domain. Similar substitutions were also introduced in the KPf chimera. PfHsp70-V451F (hydrophobic pocket mutant) had marginally compromised in vivo function. However, a similar mutation (V436F), introduced in KPf abrogated the in vivo function of this chimera. The arch and hydrophobic pocket derivatives of PfHsp70 exhibited marginally compromised in vivo function, whilst equivalent mutations in KPf did not affect its in vivo function. The ability of PfHsp70 and its arch/hydrophobic pocket mutants to suppress the heatinduced aggregation of malate dehydrogenase (MDH) in vitro was investigated. Whilst PfHsp70 arch mutants displayed marginal functional loss in vivo, data from in vitro studies revealed that their functional deficiencies were more severe. This is the first study in which an Hsp70 from a parasitic eukaryote was able to suppress the thermosensitivity of an E. coli DnaK mutant strain. Findings from the in vivo and in vitro assays conducted on PfHsp70 suggest that this protein plays a key role in the life-cycle of P. falciparum. Furthermore, this study raised insights that are pertinent to the current dogma on the Hsp70 mechanism of action
Use of a chimeric Hsp70 to enhance the quality of recombinant Plasmodium falciparum S-adenosylmethionine decarboxylase protein produced in Escherichia coli
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.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.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
Comparative characterization of plasmodium falciparum Hsp70-1 relative to E. coli DnaK reveals the functional specificity of the parasite chaperone
CITATION: Lebepe, Charity Mekgwa et al. 2020. Comparative characterization of plasmodium falciparum Hsp70-1 relative to E. coli DnaK reveals the functional specificity of the parasite chaperone. Biomolecules, 10(6): 856, doi:10.3390/biom10060856.The original publication is available at: https://www.ncbi.nlm.nih.govHsp70 is a conserved molecular chaperone. How Hsp70 exhibits specialized functions across species remains to be understood. Plasmodium falciparum Hsp70-1 (PfHsp70-1) and Escherichia coli DnaK are cytosol localized molecular chaperones that are important for the survival of these two organisms. In the current study, we investigated comparative structure-function features of PfHsp70-1 relative to DnaK and a chimeric protein, KPf, constituted by the ATPase domain of DnaK and the substrate binding domain (SBD) of PfHsp70-1. Recombinant forms of the three Hsp70s exhibited similar secondary and tertiary structural folds. However, compared to DnaK, both KPf and PfHsp70-1 were more stable to heat stress and exhibited higher basal ATPase activity. In addition, PfHsp70-1 preferentially bound to asparagine rich peptide substrates, as opposed to DnaK. Recombinant P. falciparum adenosylmethionine decarboxylase (PfAdoMetDC) co-expressed in E. coli with either KPf or PfHsp70-1 was produced as a fully folded product. Co-expression of PfAdoMetDC with heterologous DnaK in E. coli did not promote folding of the former. However, a combination of supplementary GroEL plus DnaK improved folding of PfAdoMetDC. These findings demonstrated that the SBD of PfHsp70-1 regulates several functional features of the protein and that this molecular chaperone is tailored to facilitate folding of plasmodial proteins.Publisher's versio
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
Heterologous expression of plasmodial proteins for structural studies and functional annotation
Malaria remains the world's most devastating tropical infectious disease with as many as 40% of the world population living in risk areas. The widespread resistance of Plasmodium parasites to the cost-effective chloroquine and antifolates has forced the introduction of more costly drug combinations, such as CoartemÂź. In the absence of a vaccine in the foreseeable future, one strategy to address the growing malaria problem is to identify and characterize new and durable antimalarial drug targets, the majority of which are parasite proteins. Biochemical and structure-activity analysis of these proteins is ultimately essential in the characterization of such targets but requires large amounts of functional protein. Even though heterologous protein production has now become a relatively routine endeavour for most proteins of diverse origins, the functional expression of soluble plasmodial proteins is highly problematic and slows the progress of antimalarial drug target discovery. Here the status quo of heterologous production of plasmodial proteins is presented, constraints are highlighted and alternative strategies and hosts for functional expression and annotation of plasmodial proteins are reviewed
The Role of Non-Canonical Hsp70s (Hsp110/Grp170) in Cancer
Although cancers account for over 16% of all global deaths annually, at present, no reliable therapies exist for most types of the disease. As protein folding facilitators, heat shock proteins (Hsps) play an important role in cancer development. Not surprisingly, Hsps are among leading anticancer drug targets. Generally, Hsp70s are divided into two main subtypes: canonical Hsp70 (Escherichia coli Hsp70/DnaK homologues) and the non-canonical (Hsp110 and Grp170) members. These two main Hsp70 groups are delineated from each other by distinct structural and functional specifications. Non-canonical Hsp70s are considered as holdase chaperones, while canonical Hsp70s are refoldases. This unique characteristic feature is mirrored by the distinct structural features of these two groups of chaperones. Hsp110/Grp170 members are larger as they possess an extended acidic insertion in their substrate binding domains. While the role of canonical Hsp70s in cancer has received a fair share of attention, the roles of non-canonical Hsp70s in cancer development has received less attention in comparison. In the current review, we discuss the structure-function features of non-canonical Hsp70s members and how these features impact their role in cancer development. We further mapped out their interactome and discussed the prospects of targeting these proteins in cancer therapy
The Role of Non-Canonical Hsp70s (Hsp110/Grp170) in Cancer
Although cancers account for over 16% of all global deaths annually, at present, no reliable therapies exist for most types of the disease. As protein folding facilitators, heat shock proteins (Hsps) play an important role in cancer development. Not surprisingly, Hsps are among leading anticancer drug targets. Generally, Hsp70s are divided into two main subtypes: canonical Hsp70 (Escherichia coli Hsp70/DnaK homologues) and the non-canonical (Hsp110 and Grp170) members. These two main Hsp70 groups are delineated from each other by distinct structural and functional specifications. Non-canonical Hsp70s are considered as holdase chaperones, while canonical Hsp70s are refoldases. This unique characteristic feature is mirrored by the distinct structural features of these two groups of chaperones. Hsp110/Grp170 members are larger as they possess an extended acidic insertion in their substrate binding domains. While the role of canonical Hsp70s in cancer has received a fair share of attention, the roles of non-canonical Hsp70s in cancer development has received less attention in comparison. In the current review, we discuss the structure-function features of non-canonical Hsp70s members and how these features impact their role in cancer development. We further mapped out their interactome and discussed the prospects of targeting these proteins in cancer therapy
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