Skeleton binding protein 1 localizes to the Maurer's cleft and interacts with PfHSP70-1 and PfHSP70-x in Plasmodium falciparum gametocyte-infected erythrocytes.

Plasmodium falciparum accounts for the majority of malaria deaths, due to pathology provoked by the ability of infected erythrocytes to adhere to vascular endothelium within deep tissues. The parasite recognizes endothelium by trafficking and displaying protein ligands on the surface of asexual stage infected erythrocytes, such as members of the large family of pathogenic proteins, P. falciparum erythrocyte membrane protein 1 (PfEMP1). Parasite-encoded skeleton binding protein 1 (SBP1) plays an important role in the transport of these binding-related surface proteins, via cleft-like membranous structures termed Maurer's clefts, which are present within the cytoplasm of infected erythrocytes. Erythrocytes infected with gametocyte stages accumulate in the extravascular compartment of bone marrow; and it was suggested that their surface-expressed adhesion molecule profile and protein trafficking mechanisms might differ from those in asexual stage parasites. Protein trafficking mechanisms via Maurer's clefts have been well investigated in asexual stage parasite-infected erythrocytes; but little is known regarding the gametocyte stages. In this study, we characterized SBP1 during gametocyte maturation and demonstrated that SBP1 is expressed and localizes to dot-like Maurer's cleft structures in the cytoplasm of gametocyte-infected erythrocytes. Co-immunoprecipitation and mass spectrometry assays indicated that SBP1 interacts with the molecular chaperones PfHSP70-1 and PfHSP70-x. Localization analysis suggested that some PfHSP70-1 and/or PfHSP70-x localize in a dot-like pattern within the cytoplasm of immature gametocyte-infected erythrocytes. These findings suggest that SBP1 may interact with HSP70 chaperones in the infected erythrocyte cytoplasm during the immature gametocyte stages.

Human granzyme B binds Plasmodium falciparum Hsp70-x and mediates antiplasmodial activity in vitro.

Cell surface-bound human Hsp70 (hHsp70) sensitises tumour cells to the cytolytic attack of natural killer (NK) cells through the mediation of apoptosis-inducing serine protease, granzyme B (GrB). hHsp70 is thought to recruit NK cells to the immunological synapse via the extracellularly exposed 14 amino acid sequence, TKDNNLLGRFELSG, known as the TKD motif of Hsp70. Plasmodium falciparum-infected red blood cells (RBCs) habour both hHsp70 and an exported parasite Hsp70 termed PfHsp70-x. Both PfHsp70-x and hHsp70 share conserved TKD motifs. The role of PfHsp70-x in facilitating GrB uptake in malaria parasite-infected RBCs remains unknown, but hHsp70 enables a perforin-independent uptake of GrB into tumour cells. In the current study, we comparatively investigated the direct binding of GrB to either PfHsp70-x or hHsp70 in vitro. Using ELISA, slot blot assay and surface plasmon resonance (SPR) analysis, we demonstrated a direct interaction of GrB with hHsp70 and PfHsp70-x. SPR analysis revealed a higher affinity of GrB for PfHsp70-x than hHsp70. In addition, we established that the TKD motif of PfHsp70-x directly interacts with GrB. The data further suggest that the C-terminal EEVN motif of PfHsp70-x augments the affinity of PfHsp70-x for GrB but is not a prerequisite for the binding. A potent antiplasmodial activity (IC50 of 0.5 µM) of GrB could be demonstrated. These findings suggest that the uptake of GrB by parasite-infected RBCs might be mediated by both hHsp70 and PfHsp70-x. The combined activity of both proteins could account for the antiplasmodial activity of GrB at the blood stage.

Structures of the Plasmodium falciparum heat-shock protein 70-x ATPase domain in complex with chemical fragments identify conserved and unique binding sites.

Plasmodium falciparum invades erythrocytes and extensively modifies them in a manner that increases the virulence of this malaria parasite. A single heat-shock 70 kDa-type chaperone, PfHsp70-x, is among the parasite proteins exported to the host cell. PfHsp70-x assists in the formation of a key protein complex that underpins parasite virulence and supports parasite growth during febrile episodes. Previous work resolved the crystallographic structures of the PfHsp70-x ATPase and substrate-binding domains, and showed them to be highly similar to those of their human counterparts. Here, 233 chemical fragments were screened for binding to the PfHsp70-x ATPase domain, resulting in three crystallographic structures of this domain in complex with ligands. Two binding sites were identified, with most ligands binding proximal to the ATPase nucleotide-binding pocket. Although amino acids participating in direct ligand interactions are conserved between the parasite and human erythrocytic chaperones, one nonconserved residue is also present near the ligand. This work suggests that PfHsp70-x features binding sites that may be exploitable by small-molecule ligands towards the specific inhibition of the parasite chaperone.

Structure of the substrate-binding domain of Plasmodium falciparum heat-shock protein 70-x.

The malaria parasite Plasmodium falciparum extensively modifies erythrocytes that it invades by exporting a large complement of proteins to the host cell. Among these exported components is a single heat-shock 70 kDa class protein, PfHsp70-x, that supports the virulence and growth rate of the parasite during febrile episodes. The ATP-binding domain of PfHsp70-x has previously been resolved and showed the presence of potentially druggable epitopes that differ from those on human Hsp70 chaperones. Here, the crystallographic structure of the substrate-binding domain (SBD) of PfHsp70-x is presented in complex with a hydrophobic peptide. The PfHsp70-x SBD is shown to be highly similar to the counterpart from a human erythrocytic Hsp70 chaperone. The binding of substrate at the interface between ß-sandwich and α-helical subdomains of this chaperone segment is also conserved between the malaria parasite and humans. It is hypothesized that the parasite may partly exploit human chaperones for intra-erythrocytic trafficking and maintenance of its exported proteome.

The Plasmodium falciparum Hsp70-x chaperone assists the heat stress response of the malaria parasite.

Plasmodium falciparum is the most lethal of human-infective malaria parasites. A hallmark of P. falciparum malaria is extensive remodeling of host erythrocytes by the parasite, which facilitates the development of virulence properties such as host cell adhesion to the endothelial lining of the microvasculature. Host remodeling is mediated by a large complement of parasite proteins exported to the erythrocyte; among them is a single heat shock protein (Hsp)70-class protein chaperone, P. falciparum Hsp70-x (PfHsp70-x). PfHsp70-x was previously shown to assist the development of virulent cytoadherence characteristics. Here, we show that PfHsp70-x also supports parasite growth under elevated temperature conditions that simulate febrile episodes, especially at the beginning of the parasite life cycle when most of host cell remodeling takes place. Biochemical and biophysical analyses of PfHsp70-x, including crystallographic structures of its catalytic domain and the J-domain of its stimulatory Hsp40 cochaperone, suggest that PfHsp70-x is highly similar to human Hsp70 chaperones endogenous to the erythrocyte. Nevertheless, our results indicate that selective inhibition of PfHsp70-x function using small molecules may be possible and highlight specific sites of its catalytic domain as potentially of high interest. We discuss the likely roles of PfHsp70-x and human chaperones in P. falciparum biology and how specific inhibitors may assist us in disentangling their relative contributions.-Day, J., Passecker, A., Beck, H.-P., Vakonakis, I. The Plasmodium falciparum Hsp70-x chaperone assists the heat stress response of the malaria parasite.

Structural and biochemical characterization of Plasmodium falciparum Hsp70-x reveals functional versatility of its C-terminal EEVN motif.

Plasmodium falciparum, the main agent of malaria expresses six members of the heat shock protein 70 (Hsp70) family. Hsp70s serve as protein folding facilitators in the cell. Amongst the six Hsp70 species that P. falciparum expresses, Hsp70-x (PfHsp70-x), is partially exported to the host red blood cell where it is implicated in host cell remodeling. Nearly 500 proteins of parasitic origin are exported to the parasite-infected red blood cell (RBC) along with PfHsp70-x. The role of PfHsp70-x in the infected human RBC remains largely unclear. One of the defining features of PfHsp70-x is the presence of EEVN residues at its C-terminus. In this regard, PfHsp70-x resembles canonical eukaryotic cytosol-localized Hsp70s which possess EEVD residues at their C-termini in place of the EEVN residues associated with PfHsp70-x. The EEVD residues of eukaryotic Hsp70s facilitate their interaction with co-chaperones. Characterization of the role of the EEVN residues of PfHsp70-x could provide insights into the function of this protein. In the current study, we expressed and purified recombinant PfHsp70-x (full length) and its EEVN minus form (PfHsp70-xT ). We then conducted structure- function assays towards establishing the role of the EEVN motif of PfHsp70-x. Our findings suggest that the EEVN residues of PfHsp70-x are important for its ATPase activity and chaperone function. Furthermore, the EEVN residues are crucial for the direct interaction between PfHsp70-x and human Hsp70-Hsp90 organizing protein (hHop) in vitro. Hop facilitates functional cooperation between Hsp70 and Hsp90. However, it remains to be established if PfHsp70-x and hHsp90 cooperate in vivo.