Polymyxin B inhibits the chaperone activity of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> Hsp70

Heat shock protein 70 (Hsp70) is a molecular chaperone that plays an important role in cellular proteostasis. Hsp70s are also implicated in the survival and pathogenicity of malaria parasites. The main agent of malaria, Plasmodium falciparum, expresses six Hsp70s. Of these, two (PfHsp70-1 and PfHsp70-z) localize to the parasite cytosol. Previously conducted gene knockout studies suggested that PfHsp70-z is essential, and it has been demonstrated that small-molecule inhibitors targeting PfHsp70-1 cause parasite death. For this reason, both PfHsp70-1 and PfHsp70-z are potential antimalarial targets. Two cyclic lipopeptides, colistin and polymyxin B (PMB), have been shown to bind another heat shock protein, Hsp90, inhibiting its chaperone function. In the current study, we investigated the effect of PMB on the structure–function features of PfHsp70-1 and PfHsp70-z. Using surface plasmon resonance analysis, we observed that PMB directly interacts with both PfHsp70-1 and PfHsp70-z. In addition, using circular dichroism spectrometric analysis combined with tryptophan fluorescence measurements, we observed that PMB modulated the secondary and tertiary structures of Hsp70. Furthermore, PMB inhibited the basal ATPase activity and chaperone function of the two Hsp70s. Our findings suggest that PMB associates with Hsp70 to inhibit its function. In light of the central role of Hsp70 in cellular proteostasis and its essential role in the development of malaria parasites in particular, our findings expand the library of small-molecule inhibitors that target this medically important class of molecular chaperones.     

Plasmodium falciparum Hop (PfHop) Interacts with the Hsp70 Chaperone in a Nucleotide-Dependent Fashion and Exhibits Ligand Selectivity

Heat shock proteins (Hsps) play an important role in the development and pathogenicity of malaria parasites. One of the most prominent functions of Hsps is to facilitate the folding of other proteins. Hsps are thought to play a crucial role when malaria parasites invade their host cells and during their subsequent development in hepatocytes and red blood cells. It is thought that Hsps maintain proteostasis under the unfavourable conditions that malaria parasites encounter in the host environment. Although heat shock protein 70 (Hsp70) is capable of independent folding of some proteins, its functional cooperation with heat shock protein 90 (Hsp90) facilitates folding of some proteins such as kinases and steroid hormone receptors into their fully functional forms. The cooperation of Hsp70 and Hsp90 occurs through an adaptor protein called Hsp70-Hsp90 organising protein (Hop). We previously characterised the Hop protein from Plasmodium falciparum (PfHop). We observed that the protein co-localised with the cytosol-localised chaperones, PfHsp70-1 and PfHsp90 at the blood stages of the malaria parasite. In the current study, we demonstrated that PfHop is a stress-inducible protein. We further explored the direct interaction between PfHop and PfHsp70-1 using far Western and surface plasmon resonance (SPR) analyses. The interaction of the two proteins was further validated by co-immunoprecipitation studies. We observed that PfHop and PfHsp70-1 associate in the absence and presence of either ATP or ADP. However, ADP appears to promote the association of the two proteins better than ATP. In addition, we investigated the specific interaction between PfHop TPR subdomains and PfHsp70-1/ PfHsp90, using a split-GFP approach. This method allowed us to observe that TPR1 and TPR2B subdomains of PfHop bind preferentially to the C-terminus of PfHsp70-1 compared to PfHsp90. Conversely, the TPR2A motif preferentially interacted with the C-terminus of PfHsp90. Finally, we observed that recombinant PfHop occasionally eluted as a protein species of twice its predicted size, suggesting that it may occur as a dimer. We conducted SPR analysis which suggested that PfHop is capable of self-association in presence or absence of ATP/ADP. Overall, our findings suggest that PfHop is a stress-inducible protein that directly associates with PfHsp70-1 and PfHsp90. In addition, the protein is capable of self-association. The findings suggest that PfHop serves as a module that brings these two prominent chaperones (PfHsp70-1 and PfHsp90) into a functional complex. Since PfHsp70-1 and PfHsp90 are essential for parasite growth, findings from this study are important towards the development of possible antimalarial inhibitors targeting the cooperation of these two chaperones.     

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 Hsp70-Hsp40 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 PfHsp70-PfHsp40 partnership in the malaria parasite, and furthermore that the plasmodial and human Hsp70-Hsp40 chaperones possess unique attributes that are differentially modulated by small molecules.     

Characterisation of the <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> 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 Hsp70–Hsp90 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 co-immunoprecipitation 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.     

15-deoxyspergualin primarily targets the trafficking of apicoplast proteins in Plasmodium falciparum

15-Deoxyspergualin, an immunosuppressant with tumoricidal and antimalarial properties, has been implicated in the inhibition of a diverse array of cellular processes including polyamine synthesis and protein synthesis. Endeavoring to identify the mechanism of antimalarial action of this molecule, we examined its effect on Plasmodium falciparum protein synthesis, polyamine biosynthesis, and transport. 15-Deoxyspergualin stalled protein synthesis in P. falciparum through Hsp70 sequestration and subsequent phosphorylation of the eukaryotic initiation factor eIF2alpha. However, protein synthesis inhibition as well as polyamine depletion were invoked only by high micromolar concentrations of 15-deoxyspergualin, in contrast to the submicromolar concentrations sufficient to inhibit parasite growth. Further investigations demonstrated that 15-deoxyspergualin in the malaria parasite primarily targets the hitherto underexplored process of trafficking of nucleus-encoded proteins to the apicoplast. Our finding that 15-deoxyspergualin kills the malaria parasite by interfering with targeting of nucleus-encoded proteins to the apicoplast not only exposes a chink in the armor of the malaria parasite, but also reveals new realms in our endeavors to study this intriguing biological process.     

Activation of the herpes simplex virus type-1 origin-binding protein (UL9) by heat shock proteins.

Heat shock proteins participate in the initiation of DNA replication of different organisms by facilitating the assembly of initiation complexes. We have examined the effects of human heat shock proteins (Hsp40 and Hsp70) on the interaction of the herpes simplex virus type-1 initiator protein (UL9) with oriS, one of the viral origins of replication. Hsp40 and Hsp70 act substoichiometrically to increase the affinity of UL9 for oriS. The major contributor to this effect is Hsp40. Heat shock proteins also stimulate the ATPase activity of UL9 with oriS and increase opening of the origin. In contrast, heat shock proteins have no effect on the origin-independent activities of UL9 suggesting that their role is not merely in refolding denatured protein. These observations are consistent with a role for heat shock proteins in activating UL9 to efficiently initiate viral origin-dependent DNA replication. The action of heat shock proteins in this capacity is analogous to their role in activating the initiator proteins of other organisms.     

Screening for small molecule modulators of Hsp70 chaperone activity using protein aggregation suppression assays: inhibition of the plasmodial chaperone PfHsp70-1

Plasmodium falciparum heat shock protein 70 (PfHsp70-1) is thought to play an essential role in parasite survival and virulence in the human host, making it a potential antimalarial drug target. A malate dehydrogenase based aggregation suppression assay was adapted for the screening of small molecule modulators of Hsp70. A number of small molecules of natural (marine prenylated alkaloids and terrestrial plant naphthoquinones) and related synthetic origin were screened for their effects on the protein aggregation suppression activity of purified recombinant PfHsp70-1. Five compounds (malonganenone A-C, lapachol and bromo-β-lapachona) were found to inhibit the chaperone activity of PfHsp70-1 in a concentration dependent manner, with lapachol preferentially inhibiting PfHsp70-1 compared to another control Hsp70. Using growth inhibition assays on P. falciparum infected erythrocytes, all of the compounds, except for malonganenone B, were found to inhibit parasite growth with IC(50) values in the low micromolar range. Overall, this study has identified two novel classes of small molecule inhibitors of PfHsp70-1, one representing a new class of antiplasmodial compounds (malonganenones). In addition to demonstrating the validity of PfHsp70-1 as a possible drug target, the compounds reported in this study will be potentially useful as molecular probes for fundamental studies on Hsp70 chaperone function.     

Discovery and development of heat shock protein 90 inhibitors

Heat shock protein 90 (Hsp90) is an important target in cancer because of its role in maintaining transformation and has recently become the focus of several drug discovery and development efforts. While compounds with different modes of action are known, the focus of this review is on those classes of compounds which inhibit Hsp90 by binding to the N-terminal ATP pocket. These include natural product inhibitors such as geldanamycin and radicicol and synthetic inhibitors comprised of purines, pyrazoles, isoxazoles and other scaffolds. The synthetic inhibitors have been discovered either by structure-based design, high throughput screening and more recently using fragment-based design and virtual screening techniques. This review will discuss the discovery of these different classes, as well as their development as potential clinical agents.     

Plant Hsp90 family with special reference to rice

Hsp90 family represents a group of highly conserved and strongly expressed proteins present in almost all biological species. Heat shock proteins in the range of 90 kDa have been detected in a range of plant species andhsp90 genes have been cloned and characterized in selected instances. However, the expression characteristics of plant Hsp90 are poorly understood. Work on expression characteristics of rice Hsp90 is reviewed in this paper. Experimental evidence is provided for indicating that while the rice 87 kDa protein is transiently synthesized within initial 2 h of heat shock, high steady-state levels of this protein are retained even under prolonged high temperature stress conditions or recovery following 4 h heat shock. It is further shown that fifteen different wild rices accumulate differential levels of these proteins in response to heat shock treatment.     

15-Deoxyspergualin modulates Plasmodium falciparum heat shock protein function

Heat shock proteins are essential for the survival of all cells. The C-terminal EEVD motif of Hsp70 has previously been implicated in binding 15-deoxyspergualin (DSG), an immunosuppressant with antimalarial activity whose mechanism of action is uncertain. We report the cloning, overexpression, and characterization of three members of the heat shock family, PfHsp70-1 (an Hsp70 protein with a C-terminal EEVD motif), PfHsp70-2 (an Hsp70 protein without the EEVD motif), and PfHsp70 interacting protein. The chaperone activity of PfHsp70-1, and PfHsp70-2 was enhanced by ATP and by PfHip. Interestingly, while binding of protein substrates to PfHsp70-1, PfHsp70-2 and PfHip was unaffected in the presence of DSG, the ATP enhanced chaperone activity of PfHsp70-1 but not PfHsp70-2 was stimulated further by DSG. Our finding suggests that the binding partner of DSG in the parasite cellular milieu is PfHsp70-1 and paves the way for the elucidation of the mechanism of antimalarial action of DSG.     

The treatment of Plasmodium falciparum-infected erythrocytes with chloroquine leads to accumulation of ferriprotoporphyrin IX bound to particular parasite proteins and to the inhibition of the parasite's 6-phosphogluconate dehydrogenase

Ferriprotoporphyrin IX (FPIX) is a potentially toxic product of hemoglobin digestion by intra-erythrocytic malaria parasites. It is detoxified by biomineralization or through degradation by glutathione. Both processes are inhibited by the antimalarial drug chloroquine, leading to the accumulation of FPIX in the membranes of the infected cell and their consequent permeabilization. It is shown here that treatment of Plasmodium falciparum-infected erythrocytes with chloroquine also leads to the binding of FPIX to a subset of parasite proteins. Parasite enzymes such as aldolase, pyrimidine nucleaside monophosphate kinase and pyrimidine 5'-nucleotidase were inhibited by FPIX in vitro, but only the activity of 6-phosphogluconate dehydrogenase was reduced significantly in cells after drug treatment. Additional proteins were extracted from parasite cytosol by their ability to bind FPIX. Sequencing of these proteins identified heat shock proteins 90 and 70, enolase, elongation factor 1-alpha, phoshoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, L-lactate dehydrogenase and gametocytogenesis onset-specific protein. The possible involvement of these proteins in the antimalarial mode of action of chloroquine is discussed. It is concluded that drug-induced binding of FPIX to parasite glycolytic enzymes could underlie the demonstrable inhibition of glycolysis by chloroquine. The inhibition of 6-phosphogluconate dehydrogenase could explain the reduction of the activity of the hexose monophosphate shunt by the drug. Inhibition of both processes is deleterious to parasite survival. Binding of FPIX to other proteins is probably inconsequential to the rapid killing of the parasite by chloroquine.     

Three-dimensional structure of heat shock protein 90 from <Emphasis Type="Italic">Plasmodium falciparum</Emphasis>: molecular modelling approach to rational drug design against malaria

We have recently implicated heat shock protein 90 from Plasmodium falciparum (PfHsp90) as a potential drug target against malaria. Using inhibitors specific to the nucleotide binding domain of Hsp90, we have shown potent growth inhibitory effects on development of malarial parasite in human erythrocytes. To gain better understanding of the vital role played by PfHsp90 in parasite growth,we have modeled its three dimensional structure using recently described full length structure of yeast Hsp90.S equence similarity found between PfHsp90 and yeast Hsp90 allowed us to model the core structure with high confidence. The superimposition of the predicted structure with that of the template yeast Hsp90 structure reveals an RMSD of 3.31 Angstrom. The N-terminal and middle domains showed the least RMSD (1.76 Angstrom) while the more divergent C-terminus showed a greater RMSD (2.84 Angstrom) with respect to the template. The structure shows overall conservation of domains involved in nucleotide binding, ATPase activity, co-chaperone binding as well as inter-subunit interactions. Important co-chaperones known to modulate Hsp90 function in other eukaryotes are conserved in malarial parasite as well. An acidic stretch of amino acids found in the linker region, which is uniquely extended in PfHsp90 could not be modeled in this structure suggesting a flexible conformation. Our results provide a basis to compare the overall structure and functional pathways dependent on PfHsp90 in malarial parasite. Further analysis of differences found between human and parasite Hsp90 may make it possible to design inhibitors targeted specifically against malaria.     

Heat shock protein 90 from neglected protozoan parasites

Significant advances have been made in our understanding of heat shock protein 90 (Hsp90) in terms of its structure, biochemical characteristics, post-translational modifications, interactomes, regulation and functions. In addition to yeast as a model several new systems have now been examined including flies, worms, plants as well as mammalian cells. This review discusses themes emerging out of studies reported on Hsp90 from infectious disease causing protozoa. A common theme of sensing and responding to host cell microenvironment emerges out of analysis of Hsp90 in Malaria, Trypanosmiasis as well as Leishmaniasis. In addition to their functional roles, the potential of Hsp90 from these infectious disease causing organisms to serve as drug targets and the current status of this drug development endeavor are discussed. Finally, a unique and the only known example of a split Hsp90 gene from another disease causing protozoan Giardia lamblia and its evolutionary significance are discussed. Clearly studies on Hsp90 from protozoan parasites promise to reveal important new paradigms in Hsp90 biology while exploring its potential as an anti-infective drug target. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

The Membrane-Associated Transient Receptor Potential Vanilloid Channel Is the Central Heat Shock Receptor Controlling the Cellular Heat Shock Response in Epithelial Cells

The heat shock response (HSR) is a highly conserved molecular response to various types of stresses, including heat shock, during which heat-shock proteins (Hsps) are produced to prevent and repair damages in labile proteins and membranes. In cells, protein unfolding in the cytoplasm is thought to directly enable the activation of the heat shock factor 1 (HSF-1), however, recent work supports the activation of the HSR via an increase in the fluidity of specific membrane domains, leading to activation of heat-shock genes. Our findings support the existence of a plasma membrane-dependent mechanism of HSF-1 activation in animal cells, which is initiated by a membrane-associated transient receptor potential vanilloid receptor (TRPV). We found in various non-cancerous and cancerous mammalian epithelial cells that the TRPV1 agonists, capsaicin and resiniferatoxin (RTX), upregulated the accumulation of Hsp70, Hsp90 and Hsp27 and Hsp70 and Hsp90 respectively, while the TRPV1 antagonists, capsazepine and AMG-9810, attenuated the accumulation of Hsp70, Hsp90 and Hsp27 and Hsp70, Hsp90, respectively. Capsaicin was also shown to activate HSF-1. These findings suggest that heat-sensing and signaling in mammalian cells is dependent on TRPV channels in the plasma membrane. Thus, TRPV channels may be important drug targets to inhibit or restore the cellular stress response in diseases with defective cellular proteins, such as cancer, inflammation and aging.     

Exploring the Trypanosoma brucei Hsp83 Potential as a Target for Structure Guided Drug Design

Human African trypanosomiasis is a neglected parasitic disease that is fatal if untreated. The current drugs available to eliminate the causative agent Trypanosoma brucei have multiple liabilities, including toxicity, increasing problems due to treatment failure and limited efficacy. There are two approaches to discover novel antimicrobial drugs - whole-cell screening and target-based discovery. In the latter case, there is a need to identify and validate novel drug targets in Trypanosoma parasites. The heat shock proteins (Hsp), while best known as cancer targets with a number of drug candidates in clinical development, are a family of emerging targets for infectious diseases. In this paper, we report the exploration of T. brucei Hsp83 – a homolog of human Hsp90 – as a drug target using multiple biophysical and biochemical techniques. Our approach included the characterization of the chemical sensitivity of the parasitic chaperone against a library of known Hsp90 inhibitors by means of differential scanning fluorimetry (DSF). Several compounds identified by this screening procedure were further studied using isothermal titration calorimetry (ITC) and X-ray crystallography, as well as tested in parasite growth inhibitions assays. These experiments led us to the identification of a benzamide derivative compound capable of interacting with TbHsp83 more strongly than with its human homologs and structural rationalization of this selectivity. The results highlight the opportunities created by subtle structural differences to develop new series of compounds to selectively target the Trypanosoma brucei chaperone and effectively kill the sleeping sickness parasite.     

Hsp90 Interaction with INrf2(Keap1) Mediates Stress-induced Nrf2 Activation

INrf2(Keap1) functions as an adapter for Cul3/Rbx1-mediated degradation of Nrf2. In response to stress, Nrf2 is released from INrf2 and translocates inside the nucleus leading to activation of cytoprotective proteins critical in protection against adverse effects including cancer. We demonstrate here a novel role of heat shock protein 90 (Hsp90) in control of the INrf2 and Nrf2 activation. Hsp90 interacted with INrf2 that leds to stabilization of INrf2 during heat shock stress. Domain mapping showed the requirement of INrf2-NTR and the Hsp90-CLD region for interaction of Hsp90 with INrf2. Heat shock and antioxidants induced Hsp90, and casein kinase 2 (CK2) phosphorylated INrf2Thr55. This led to increased Hsp90-INrf2 interaction, dissociation of the Rbx1/Cul3·INrf2·Nrf2 complex, and activation of Nrf2. Inhibitors of CK2 and Hsp90, and mutation of INrf2Thr55 abolished the Hsp90-INrf2 interaction and downstream signaling. INrf2 is released from Hsp90 once the heat shock or antioxidant stress subsidized, thereby allowing INrf2 to interact with Nrf2 and facilitate Nrf2 ubiquitination and degradation. The results together demonstrate a novel role for the stress-induced Hsp90-INrf2 interaction in regulation of Nrf2 activation and induction of cytoprotective proteins.     

Heat shock protein 90 associates with monarch-1 and regulates its ability to promote degradation of NF-kappaB-inducing kinase

Monarch-1/NLRP12 is expressed in myeloid cells and functions as a negative regulator of inflammation by inducing proteasome-mediated degradation of NF-kappaB-inducing kinase. Monarch-1 is a member of the CATERPILLER gene family, also known as the nucleotide-binding domain leucine-rich repeat gene family. This family shares strong structural homology to major immune regulators expressed in lower organisms, including plants. In plants, these disease-resistance proteins (R proteins) sense pathogenic insult and initiate a protective response to limit pathogen growth. To perform this role, many R proteins require the highly conserved chaperone molecule, heat shock protein (Hsp) 90. Using a two-dimensional gel/mass spectrometry system, we detected the association of the nucleotide-binding domain leucine-rich repeat protein Monarch-1 with heat shock proteins. Further analysis indicates that analogous to plant R proteins, Hsp90 is required for Monarch-1 activity. In human monocytes, Monarch-1 associates with Hsp90, and these complexes are sensitive to treatment with specific Hsp90 inhibitors. Disruption of these complexes results in rapid degradation of Monarch-1 via the proteasome and prevents Monarch-1-induced proteolysis of NF-kappaB-inducing kinase. This demonstrates that Hsp90 is a critical regulator of Monarch-1 anti-inflammatory activity.