Heat shock proteins (HSPs) form a large family that has a vital role in proteostasis in living organisms (Hartl et al., 2011). Cellular housekeeping tasks that HSPs perform include folding of newly synthesized polypeptides, refolding or destroying misfolded proteins, assembling protein complexes, and dissociating protein aggregates (Saibil, 2013). Genes of several HSPs are transcribed in response to stressors, and account for a significant proportion of total cellular proteins (Murshid et al., 2018). Members of the heat shock protein family are classified on the basis of their molecular weights (in kDa) into Hsp10, 40, 60, 70, 90 etc. (Lindquist, 1986).
The 70 kDa HSPs are widely expressed conserved molecular chaperones termed DnaK in prokaryotes and HSP70 in eukaryotes (Hu et al., 2022). While expression of some HSP70s is stress-induced, cellular expression of HSC70 (heat shock cognate) is constitutive (Brocchieri et al., 2008). HSP70s regulate the dynamic equilibrium of synthesis, folding, degradation and translocation of proteins. All these processes appear to be reliant on the ability of Hsp70s to bind with hydrophobic peptidic fragments in an ATP-controlled manner. HSP70s perform their diverse functions through recruitment of co-chaperones and collaboration with other chaperone systems to expand their activity spectrum (Behl et al., 2019; Behl & Mishra, 2019; Daugaard et al., 2007). The molecular structures of HSP70 proteins are conserved. A flexible linker connects their N-terminal nucleotide binding domain (NBD) with a C-terminal substrate binding domain (SBD). SBD is subdivided into SBDα and SBDβ followed by a C-terminal tail containing the co-chaperone recognition motif ‘EEVD’ (Mayer & Bukau, 2005) (Fig. 1A). While SBDβ interacts with the substrates, SBDα forms a LID that controls substrate entry.
Growing evidence suggests that P. falciparum (Pf) heat shock proteins may function as cytoprotective elements in the parasite's life cycle (Przyborski et al., 2015). Since the parasite experiences physiological changes through different growth stages, expression of HSP70 in response to stress is an important survival strategy (Sharma & Masison, 2009) that helps to maintain proteostasis and evade the host immune response. Pf, the most fatal of all malaria causative species has four true Hsp70 genes PfHsp70-1, 2, 3 and x. It was discovered in 2012 that PfHsp70-x is the sole member that is exported to the host erythrocyte cytoplasm (Külzer et al., 2012). While PfHsp70-2 and 3 localize to endoplasmic reticulum and mitochondria of the parasite respectively, PfHsp70-1 is found in both nucleus and cytoplasm (Shonhai et al., 2007). Compartmentalization of Hsp70 protein homologs in eukaryotic cells facilitates efficient performance of their specialized cellular functions (Shonhai et al., 2007). PfHsp70-1 and PfHsp70-x share maximum sequence identity (73%) suggesting that the two proteins are closely related. The ER homolog (PfHsp70-2) is 54% and 46% identical to PfHsp70-x (exported) and PfHsp70-3 (mitochondrial) homologs respectively. Importantly, PfHsp70-1 has received significant attention and is considered a possible target for combating malaria (Przyborski et al., 2015).
Phosphatidylinositol 3 phosphate (PI3P) is a well-known signalling molecule that is involved in protein trafficking and autophagy in many organisms (Balla, 2013; Mayinger, 2012). PI3P is produced in Pf via its only phosphatidylinositol 3 kinase (PI3K) that is essential for parasite survival. It is known to be involved in trafficking of haemoglobin to the digestive vacuole (DV) in Plasmodia, which is a functional counterpart of lysosomes and functions to degrade haemoglobin and detoxify heme (Vaid et al., 2010). Also, PfHsp70-1 is reported to bind to PI3P and stabilize the structure of parasite DV (Lu et al., 2020). Another study reported that PfKelch13 mutations led to PI3P build up in parasitized erythrocytes, which was strongly correlated with artemisinin resistance in Plasmodium falciparum (Mbengue et al., 2015). Additionally, heat induced destabilization of DVs occurred in PI3P deficient parasites before their death.
PfHSP70-1, 2 and 3 are considered to be essential for parasite survival, and are expressed in different organelles within the parasite where PI3P concentration is high (Ali et al., 2021; Iershov et al., 2019; Nascimbeni et al., 2017). Considering the critical role of PI3P in Pf as a lipid modulator and its binding with PfHSP70-1, we performed in silico analysis of the binding pocket of PI3P on all PfHSP70s. An understanding of the potential of PfHSP70 homologs to bind with PI3P would provide important insights into malaria pathogenesis. Deatailed information about these molecular interactions and the residues involved may assist in developing targeted therapeutic strategies against Pf. By disrupting the PfHSP70-PI3P interaction, it may be possible to perturb critical cellular processes within the parasite and infected red blood cells (iRBCs), thereby compromising its survival and virulence.