Eva-Rachele Pesce

The Plasmodium falciparum heat shock protein 40, Pfj4, associates with heat shock protein 70 and shows similar heat induction and localisation patterns.

Human cerebral malaria is caused by the protozoan parasite Plasmodium falciparum, which establishes itself within erythrocytes. The normal body temperature in the human host could constitute a possible source of heat stress to the parasite. Molecular chaperones belonging to the heat shock protein (Hsp) class are thought to be important for parasite subsistence in the host cell, as the expression of some members of this family has been reported to increase upon heat shock. In this paper we investigated the possible functions of the P. falciparum heat shock protein DnaJ homologue Pfj4, a type II Hsp40 protein. We analysed the ability of Pfj4 to functionally replace Escherichia coli Hsp40 proteins in a dnaJ cbpA mutant strain. Western analysis on cellular fractions of P. falciparum-infected erythrocytes revealed that Pfj4 expression increased upon heat shock. Localisation studies using immunofluorescence and immuno-electron microscopy suggested that Pfj4 and P. falciparum Hsp70, PfHsp70-1, were both localised to the parasites nucleus and cytoplasm. In some cases, Pfj4 was also detected in the erythrocyte cytoplasm of infected erythrocytes. Immunoprecipitation studies and size exclusion chromatography indicated that Pfj4 and PfHsp70-1 may directly or indirectly interact. Our results suggest a possible involvement of Pfj4 together with PfHsp70-1 in cytoprotection, and therefore, parasite survival inside the erythrocyte.

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

Abstract 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 IC50 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.

Heat shock protein 40 (Hsp40) plays a key role in the virus life cycle

The heat shock proteins (Hsps) are a diverse subset of molecular chaperones that generally promote the proper folding of proteins after translation and also prevent their aggregation during cellular stress. Paradoxically, cellular chaperones might perform important antiviral functions for host cells, yet, at the same time, might be beneficial for virus replication. Among them, Hsp40 is a specialized co-chaperone that has recently received much attention for its crucial role in both constitutive cellular functions and virus pathogenicity. The aim of this review is to raise awareness of its importance in the life cycles of a wide range of viruses.

Selective modulation of plasmodial Hsp70s by small molecules with antimalarial activity

Abstract Plasmodial heat shock protein 70 (Hsp70) chaperones represent a promising new class of antimalarial drug targets because of the important roles they play in the survival and pathogenesis of the malaria parasite Plasmodium falciparum. This study assessed a set of small molecules (lapachol, bromo-β-lapachona and malonganenones A, B and C) as potential modulators of two biologically important plasmodial Hsp70s, the parasite-resident PfHsp70-1 and the exported PfHsp70-x. Compounds of interest were assessed for modulatory effects on the steady-state basal and heat shock protein 40 (Hsp40)-stimulated ATPase activities of PfHsp70-1, PfHsp70-x and human Hsp70, as well as on the protein aggregation suppression activity of PfHsp70-x. The antimalarial marine alkaloid malonganenone A was of particular interest, as it was found to have limited cytotoxicity to mammalian cell lines and exhibited the desired properties of an effective plasmodial Hsp70 modulator. This compound was found to inhibit plasmodial and not human Hsp70 ATPase activity (Hsp40-stimulated), and hindered the aggregation suppression activity of PfHsp70-x. Furthermore, malonganenone A was shown to disrupt the interaction between PfHsp70-x and Hsp40. This is the first report to show that PfHsp70-x has chaperone activity, is stimulated by Hsp40 and can be specifically modulated by small molecule compounds.

Plasmodial Hsp40 and Hsp70 chaperones: current and future perspectives

Plasmodium falciparum displays a large and remarkable variety of heat shock protein 40 family members (PfHsp40s). The majority of the PfHsp40s are poorly characterized, and although the functions of some of them have been suggested, their exact mechanism of action is still elusive and their interacting partners and client proteins are unknown. The P. falciparum heat shock protein 70 family members (PfHsp70s) have been more extensively characterized than the PfHsp40s, with certain members shown to function as molecular chaperones. However, little is known about the PfHsp70-PfHsp40 chaperone partnerships. There is mounting evidence that these chaperones are important not only in protein homoeostasis and cytoprotection, but also in protein trafficking across the parasitophorous vacuole (PV) and into the infected erythrocyte. We propose that certain members of these chaperone families work together to maintain exported proteins in an unfolded state until they reach their final destination. In this review, we critically evaluate what is known and not known about PfHsp40s and PfHsp70s.

Isolation of a Latimeria menadoensisheat shock protein 70 (Lmhsp70) that has all the features of an inducible gene and encodes a functional molecular chaperone

Molecular chaperones facilitate the correct folding of other proteins, and heat shock proteins form one of the major classes of molecular chaperones. Heat shock protein 70 (Hsp70) has been extensively studied, and shown to be critically important for cellular protein homeostasis in almost all prokaryotic and eukaryotic systems studied to date. Since there have been very limited studies conducted on coelacanth chaperones, the main objective of this study was to genetically and biochemically characterize a coelacanth Hsp70. We have successfully isolated an Indonesian coelacanth (L. menadoensis) hsp70 gene, Lmhsp70, and found that it contained an intronless coding region and a potential upstream regulatory region. Lmhsp70 encoded a typical Hsp70 based on conserved structural and functional features, and the predicted upstream regulatory region was found to contain six potential promoter elements, and three potential heat shock elements (HSEs). The intronless nature of the coding region and the presence of HSEs suggested that Lmhsp70 was stress-inducible. Phylogenetic analyses provided further evidence that Lmhsp70 was probably inducible, and that it branched as a clade intermediate between bony fish and tetrapods. Recombinant LmHsp70 was successfully overproduced, purified and found to be functional using ATPase activity assays. Taken together, these data provide evidence for the first time that the coelacanth encodes a functional molecular chaperone system.

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 concentration-dependent 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.

PFB0595w is a Plasmodium falciparum J protein that co-localizes with PfHsp70-1 and can stimulate its in vitro ATP hydrolysis activity

Heat shock proteins, many of which function as molecular chaperones, play important roles in the lifecycle and pathogenesis of the malaria parasite, Plasmodium falciparum. The P. falciparum heat shock protein 70 (PfHsp70) family of chaperones is potentially regulated by a large complement of J proteins that localize to various intracellular compartments including the infected erythrocyte cytosol. While PfHsp70-1 has been shown to be an abundant cytosolic chaperone, its regulation by J proteins is poorly understood. In this study, we characterized the J protein PFB0595w, a homologue of the well-studied yeast cytosolic J protein, Sis1. PFB0595w, similarly to PfHsp70-1, was localized to the parasite cytosol and its expression was upregulated by heat shock. Additionally, recombinant PFB0595w was shown to be dimeric and to stimulate the in vitro ATPase activity of PfHsp70-1. Overall, the expression, localization and biochemical data for PFB0595w suggest that it may function as a cochaperone of PfHsp70-1, and advances current knowledge on the chaperone machinery of the parasite.

Hsp40 Co-chaperones as Drug Targets: Towards the Development of Specific Inhibitors

The heat shock protein 40 (Hsp40/DNAJ) family of co-chaperones modulates the activity of the major molecular chaperone heat shock protein 70 (Hsp70) protein group. Hsp40 stimulates the basal ATPase activity of Hsp70 and hence regulates the affinity of Hsp70 for substrate proteins. The number of Hsp40 genes in most organisms is substantially greater than the number of Hsp70 genes. Therefore, different Hsp40 family members may regulate different activities of the same Hsp70. This fact, along with increasing knowledge of the function of Hsp40 in diseases, has led to certain Hsp40 isoforms being considered promising drug targets. Here we review the role of Hsp40 in human disease and recent developments towards the creation of Hsp40-specific inhibitors.