Hest shock protein 70 (Hsp70) class of chaperones is highly conserved and present ubiquitously in all cellular organisms They play important role in folding of nascent polypeptides and translocation of precursor proteins to endoplasmic reticulum, mitochondria and chloroplast Hsp70 assists in assembly of proteins complexes as well as in disassembly e g uncoatmg of clathrin coated vesicles Chaperone function of Hsp70 is modulated by cochaperones of DnaJ class, Hip, Hop etc Hsp70 is a component of multi chaperone complex with Hsp90 and helps in maturation of kinases or transcription factors. Plasmodium falciparum is responsible for most severe form of human malaria Plasmodmm in its intraerythrocytic cycle presents an example of a cell with multiple, complex membrane bound structures both inside the parasite as well as m the infected erythrocyte cytosol Parasite deploys proteins in host erythrocyte cytosol, at erythrocyte plasma membrane or traffics them for secretion outside the infected cell in addition to trafficking of proteins to its own organelles like mitochondria, apicoplast, food vacuole, ER etc It is of interest to malaria biologists to understand these trafficking events and role of chaperones in regulating them This study was aimed at understanding the function(s) of Hsp70 in Plasmodium infected erythrocyte in protein maturation and trafficking events We have attempted to study Hsp70 chaperone present in Plasmodium infected erythrocytes We have largely focused on the cytosohc Hsp70, PfHsp70, in the parasite and systematically analyzed its expression, localization, abundance and complexes in the intraerythrocytic cycle To gain insight into its function, we have identified a subset of PfHsp70 interacting proteins, parasite Hsp90, Hsp70-3, Hsp60 and beta tubulin by coimmunoprecipitation experiments in conjunction with proteomic tools like 2DGE and mass spectrometry Parasite Hsp60 is a mitochondria-targeted protein and we have examined the involvement of PfHsp70 in translocation of Hsp60 precursor protein to parasite mitochondrion PfHsp70 and PfHsp90 were found to be present in a complex Geldanarnycm, a drug that affects Hsp70-Hsp90 complex, was used to investigate the role of PfHsp70 in parasite protein trafficking Since there are no known parasite derived chaperones in erythrocyte cytosol compartment, we have examined the possible "involvement of host Hsp70 in supporting transport and assembly of parasite proteins in erythrocyte cytosol Hsp70 in Plasmodium falciparum intraerythrocytic cycle P. falciparum genome codes for five Hsp70 homologs Two of these, pfHsp70-l and PfBiP are expressed in intraerythrocytic stage and have been localized to nucleocytoplasmic and endoplasmic reticulum fraction of the parasite respectively We have focused this study on PfHsp70 of the parasite We show that PfHsp70 is an abundant protein in the cytosol constituting about 2% of the total soluble pool It gets further induced during stress like heat shock and translocates to nuclear fraction indicating that PfHsp70 may be involved in protective function in the parasite nucleus during stress Nuclear translocation of mammalian Hsp70 during stress has been linked to its phosphorylation at Tyr524 We found PfHsp70 to be phosphorylated by in vivo phosphate labeling m the parasite Analysis of PfHsp70 by 2-dimensional gel electrophoresis on narrow gradient IPG strips indicated that it exists in four forms differing in their isoelectnc points (pi) Phosphatase treatment combined with analysis using a phosphorylation prediction tool,Proteomod (http //www biochem use ernet in/proteomod html) suggested that PfHsp70 is phosphorylated at three residues in the parasite The extent of phosphorylation of PfHsp70 may determine substrate specificity or subcellular localization or both Using 2DGE and mass spectrometry approach, we also identified chaperones like Hsp909 BiP, Hsp60, and protein disulphide isomerase (PDI) m P falciparum proteome In summary, PfHsp70 appears to be a highly abundant, cytosohc chaperone It is inducible by stress and multiply phosphorylated and is likely to participate in multiple processes in the parasite. PfHsp70 complexes and interacting proteins in the parasite To gam insight into the functions of Hsp70, we looked for PfHsp70 interacting proteins in the parasite We used gel filtration chromatography to resolve and enrich PfHsp70 complexes and also employed coimmunoprecipitation approach to identify interacting proteins We found parasite Hsp90, Hsp70-3, Hsp60 and beta-tubulin interact with PfHsp70 Fractionation of parasite lysate indicated that PfHsp70 is present in two major complexes of 200 kDa and 450 kDa We find that PfHsp90 interacts with PfHsp70 and both are present in 450 kDa complex Our analysis indicated that 450-kDa complex is like Hsp70-Hsp90 multichaperone complex described in mammalian cells while 200 kDa complex is likely to be an Hsp70-cochaperone complex Smaller complex appears to be a precursor for multichaperone complex Use of an Hsp90 inhibitor, geldanamycin (GA), to study the function of this multi chaperone showed that GA inhibits parasite growth Maturation of four phosphoproteins interacting with PfHsp70 was affected by GA implicating them in regulation of parasite growth GA appeared to mediate its effects by inhibiting H§p^0 phosphorylation Amongst the other three interacting proteins, PfHsp70-3 is amoveJ/Hsp70 homolog that was found at the protein level for the first time in this study PfHsp60 is mitochondria-targeted protein in the parasite and it is likely that cytoshc PfHsp70 helps in translocation of PfHsp60 to mitochondria from cytosol Tubuhn is a cytoskeletal protein and its interaction with PfHsp70 suggests possible role of PfHsp70 in cytoskeleton organization during invasion, growth or cell division In all, we find that Hsp70 in the parasite exist in a multi chaperone complex with Hsp90 which might be responsible for maturation of signaling molecules important for growth The smaller complex of PfHsp70 is a precursor of multi chaperone complex and is likely to be an Hsp70- co chaperone complex Role of Hsp70 in protein translocation and trafficking Cytosolic Hsp70 aids in translocation of precursor proteins from cytosol to mitochondria (or chloroplast) We found a mitochondnal chaperone, PfHsp60, interact with PfHsp70 and we examined the possibility that PfHsp60 translocation is assisted by cytosolic PfHsp70 We found that PfHsp60 had a cleavable, N-thermal targeting sequence Examination of PfHsp60 forms present in mitochondnal and cytosolic fraction of the parasite showed that mitochondnal form was more acidic in pi than cytosolic form as expected after targeting sequence cleavage Cytosolic PfHsp60 interacted with both PfHsp70 and PfHsp90 Interestingly, while mitochondnal PfHsp60 appeared to be in a chaperonm like complex, as expected, cytosolic form was present in smaller ohgomeric complex of about 450 kDa This suggested that PfHsp60 precursor form could be bound to multichperone complex All these experiments together strongly indicated that PfHsp60 precursor interacts with cytosolic Hsp70 and Hsp90 before former's translocation into mitochondria This interaction might be required to keep the precursor in the transport competent state P falciparum lives inside a vacuole in the infected cells but it deploys a number of proteins to host cell cytosol and to the plasma membrane To examine the involvement of multichaperone complex in trafficking, we studied the effect of GA on targeting of two parasite proteins, knob associated histidme-rich protein (KAHRP) and glycogen synthase kinase (GSK) KAHRP is indispensable for the formation of cytoadherence complexes called knobs at erythrocyte plasma membrane We found that KAHRP transport to erythrocyte plasma membrane was blocked in GA-treated parasites and it appeared all over the infected cell Further analysis showed that GA caused block in KAHRP transport at some step beyond its exit from parasite ER The targeting of GSK to membranous inclusions in the infected RBC cytosol was not severely affected m the GA-treated parasites suggesting that GSK transport may not be regulated by multi chaperone complex It also indicated that parasite may be using different pathways for trafficking of proteins to the host compartment In summary, PfHsp70 and PfHsp90 interact with PfHsp60 precursor in the cytosol They probably help keep the precursor in transport competent form before arrival at the translocase complex of mitochondria The multi chaperone complex may also be important for trafficking of at least one parasite protein, KAHRP, to the host cell compartment Analysis of erythrocyte Hsp70 in Plasmodium falciparum infected cells The remodeled plasma membrane of parasite-infected erythrocytes is important for the cytoadherence property of the infected cells Knobs, supramolecular complexes on the infected cell surface, formed by parasite proteins, PfEMPl, KAHRP, and PfEMP3 are responsible for cytoadherence of infected cells to vascular endothehum or placenta KAHRP transport is BFA-sensitive inside the parasite while PfEMP proteins undergo vesicle mediated trafficking in the erythrocyte cytosol The involvement of molecular chaperones has been implicated in the trafficking and assembly of knob components in the erythrocyte cytosol There is no evidence for the presence of bona fide parasite derived chaperones in the host compartment The chaperones of the erythrocyte origin, Hsp70, Hsp90, Hip and Hop were readily detected in the host cytosol, on the other hand By analyzing localization, abundance and biochemical characteristics of the host chaperones of erythrocyte origin, we examined if host chaperones are being utilized by the parasite for its functions Localization experiment showed that while PfHsp70, PfHsp90 and PfBiP were present in the parasite compartment, host-Hsp70 was present in erythrocyte cytosol fraction Host~Hsp70 was about 60% as abundant as PfHsp70 and was potentially capable of facilitating chaperone function in the erythrocyte cytosol Though host-Hsp70 was soluble in unmfected cells, it was present in membrane bound, triton-insoluble complexes, containing KAHRP, in infected cells Since knobs are triton-insoluble complexes at the erythrocyte plasma membrane, we isolated erythrocyte ghost (plasma membrane) fraction and could detect both Hsp70 and KAHRP Hsp70 association with erythrocyte plasma membrane was specific as it could be crosshnked to KAHRP in ghost fraction of infected cells Host-hsp70 was present in purified cytoskeleton fraction containing knobs from infected cells along with cochaperone Hop All these evidences suggest that parasite may be exploiting host-Hsp70 in erythrocyte cytosol compartment Summary This study gives insight into some functions performed by PfHsp70 in mtraerythrocytic cycle of malarial parasite PfHsp70 is an abundant cytosohc chaperone in the parasite It gets induced during stress and translocates to the nucleus It is also phosphorylated at three sites Analysis of Pfhsp70 complexes shows that it is present in bimodal complexes (450 kDa and 200 kDa), which are in equilibrium PfHsp70 and PfHsp90 interact and are part of 450 kDa multichaperone complex This multichaperone complex appears to regulate trafficking of one parasite protein to host cytosol compartment In addition, PfHsp70 and PfHsp90 are also bound to mitochondria-targeted PfHsp60 precursor in the cytosol probably keeping them m a transport competent state In addition to PfHsp90 and PfHsp60, PfHsp70 interacts with a novel Hsp70 homolog of the parasite, PfHsp70-3, and cytoskeletal protein, beta-tubuhn Examination of chaperones available in erythrocyte cytosol, showed that parasite chaperones were absent while host chaperone (Hsp70) was present and exhibited altered properties during parasite infection It was associated with membrane-bound, triton-insoluble complexes on the infected cell plasma membrane suggesting that host-Hsp70 might be involved in trafficking and/or assembly of parasite proteins In all, PfHsp70, as part of multichaperone complex, appears to be regulating translocation and trafficking of parasite proteins to organellar locations or outside the parasite Host-Hsp70, in erythrocyte cytosol, might also be engaged in specific chaperone function upon infection

MSc (Biochemistry) / Department of Biochemistry / In 2017 malaria cases were estimated at 219 million and of these 435 000 resulted in death. Malaria is transmitted by female Anopheles mosquitoes which thrive in tropical and sub-tropical areas. Malaria is caused by five species from the genus Plasmodium, namely P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi. P. falciparum causes the most severe form of the disease. P. falciparum has a complex life cycle in the human and mosquito hosts exposing the parasite to environmental changes, resulting in upregulation of heat shock proteins (Hsps). These Hsps facilitate protein folding and protein disaggregation. Hsp70 is a molecular chaperone whose function is to facilitate protein folding. P. falciparum Hsp70-x is the only member of this family of proteins that is exported to the erythrocyte cytosol by the parasite. PfHsp70-x has been implicated in the development of malaria pathogenesis. This is largely due to its association with P. falciparum erythrocyte membrane protein 1 (PfEMP1), an important virulent factor that is exposed to the exterior of the infected erythrocyte. In tumour cells, cell surface- bound Hsp70 is known to sensitize the tumour cells to cytolytic attack that is mediated by NK cells. Cell surface bound Hsp70 is thought to recruit NK cells and Granzyme B (GrB) via its 14 amino acid sequence, TKDNNLLGRFELSG, known as the TKD motif. Both PfHsp70-x and human Hsp70 (hHsp70) contain the TKD motif. Thus, this study sought to investigate the role of Hsp70 in facilitating the selective targeting of malaria parasite-infected erythrocytes by GrB. To this end, recombinant hHsp70 and PfHsp70-x were successfully expressed in E. coli and purified. Using slot blot and ELISA, it was observed that both PfHsp70-x and hHsp70 directly interact with GrB. PfHsp70-x showed greater affinity for GrB than hHsp70. In addition, using parasites cultured at the erythrocyte stage it was noted that GrB exhibits potent antiplasmodial activity (IC50 of 0.5μM). In addition, the findings suggest that GrB interacts with both Hsp70s (of parasite and human origin) resident in the infected erythrocyte. This makes GrB a promising antimalarial agent. / NRF

PhD (Biochemistry) / Department of Biochemistry / Malaria is a parasitic disease that accounts for more than 660 thousand deaths annually, mainly in children. Malaria is caused by five Plasmodium species P. ovale, P. vivax, P. malariae, P. falciparum and P. knowlesi. The most lethal cause of cerebral malaria is P. falciparum. The parasites have been shown to up-regulate some of their heat shock proteins (Hsp) in response to stress. Heat shock protein 70 (called DnaK in prokaryotes) is one of the most prominent groups of chaperones whose role is central to protein homeostasis and determines the fate of proteins. Six Hsp70 genes are represented on the genome of P. falciparum. The Hsp70 genes encode for proteins that are localised in different sub-cellular compartments. Of these two occur in the cytosol, PfHsp70-z and PfHsp70-1; two occur in the endoplasmic reticulum, PfHsp70-2 and PfHsp70-y; one in the mitochondria, PfHsp70-3 and one exported to the red blood cell cytosol, PfHsp70-x. PfHsp70-1 is a well characterized canonical Hsp70 involved in prevention of protein aggregation and facilitates protein folding. Little is known about PfHsp70-z. PfHsp70-z was previously shown to be an essential protein implicated in the folding of proteins possessing asparagine rich repeats. However, based on structural evidence PfHsp70-z belongs to the Hsp110 family of proteins and is thought to serve as a nucleotide exchange factor (NEF) of PfHsp70-1. The main aim of this study is to elucidate the functional roles of PfHsp70-z as a chaperone and its interaction with PfHsp70-1. In the current study, PfHsp70-z was cloned and expressed in E. coli JM109 cells. This was followed by its purification using nickel chromatography. The expression of PfHsp70-z in parasites cultured in vitro was investigated and its association with PfHsp70-1 was explored using a co-immuno precipitation assay. PfHsp70-z expression in malaria parasites is up regulated by heat stress and the protein is heat stable based on investigations conducted using Circular Dichroism. Furthermore, the direct interaction between recombinant forms of PfHsp70-z and PfHsp70-1 were investigated using slot blot and surface plasmon resonance assays. PfHsp70-z was observed to exhibit ATPase activity. In addition, the direct interaction between PfHsp70-z and PfHsp70-1 is promoted by ATP. Based on limited proteolysis and tryptophan fluorescence analyses, PfHsp70-z binds ATP to assume a unique structural conformation compared to the conformation of the protein bound to ADP or in nucleotide-free state. PfHsp70-z was able to suppress the heat-induced aggregation of malate dehydrogenase and luciferase in vitro. Interestingly, while ATP appears to modulate the conformation of PfHsp70-z, the chaperone function of PfHsp70-z was not influenced by ATP. Altogether, these findings suggest that Characterization of Heat Shock Protein 70-z (PfHsp70-z) from Plasmodium falciparum iii PfHsp70-z serves as an effective peptide substrate holding chaperone. In addition, PfHsp70-z may also serve as the sole nucleotide exchange factor of PfHsp70-1. The broad spectrum of functions of this protein, could explain this PfHsp70-z is an essential protein in malaria parasite survival. This is the first study to show that PfHsp70-z possess independent chaperone activity and that it interacts with its cytosolic counterpart, PfHsp70-1 in a nucleotide dependent fashion. Furthermore, the study shows that PfHsp70-z is a heat stable molecule and that it is capable of forming high order oligomers.

MSc (Biochemistry) / Department of Biochemistry / Malaria is a disease that claims about half a million lives annually, mainly children. There are 5 Plasmodium species that cause malaria; namely, P. falciparum, P. ovale, P. malariae, P. knowlesi and P. vivax. P. falciparum is the most virulent of them all. The parasite upregulates some heat shock proteins (Hsps) in response to stress it encounters during its life cycle. These Hsps play a major role in proteostasis. The drug resistance of P. falciparum to traditionally used remedies has led to a need for the development of novel drugs. Hsps have been implicated as antimalarial drug targets. Hsps act as molecular chaperones and some make complexes, which are important in facilitating protein folding. As an example, heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) form a functional complex through an adaptor protein, Hsp70-Hsp90 organizing protein (Hop). P. falciparum expresses six Hsp70s that are localized in different subcellular compartments. Amongst them, P. falciparum Hsp70-x (PfHsp70-x), is exported to the erythrocyte where it is implicated in host cell remodeling. PfHsp70-x possesses an ATPase domain, substrate binding domain and a C-terminal subdomain. PfHsp70-x possesses an EEVN motif on its C-terminus which is implicated in interactions with co-chaperones amongst them, Hop. Although some of the chaperone functions of PfHsp70-x have been reported, its interaction with human chaperones has not been investigated. The availability of PfHsp70-x in the infected erythrocyte cytosol presents a possibility that this protein may functionally cooperate with human Hsp90 via human Hop (human Hop). This hypothesis that PfHsp70-x interacts with human chaperones is strengthened by the absence of Hsp90 and Hop of parasite origin in the infected erythrocytes. The main aim of this study was to explore the chaperone activity of PfHsp70-x and its functional co-operation with human Hop. Recombinant PfHsp70-x (full length and EEVN deletion mutant) proteins were expressed in E. coli XL1 Blue cells and purified using nickel affinity chromatography. PfHsp70-x was found to be structurally comprised of mostly alpha helices and demonstrated heat stability based on circular dichroism (CD) spectrometry studies. It was established that the EEVN motif may be important for the ATPase activity of PfHsp70-x. However, it was established that the EEVN motif was not important in regulating the holdase chaperone (protein aggregation suppression) function of PfHsp70-x. Furthermore, PfHsp70-x and its mutant preferentially bound to asparagine-rich peptides. Parasite proteins have high asparagine repeat regions as compared to human proteins. In addition, preference for asparagine-rich proteins iii could signify that PfHsp70-x is biased towards binding proteins of parasitic origin. Surface plasmon resonance (SPR) analysis suggested that PfHsp70-x interacts with human Hop with relatively higher affinity compared to its EEVN minus derivative. In conclusion, the removal of the EEVN motif of PfHsp70-x does not affect the chaperone function of PfHsp70-x. However, the EEVN motif is essential for the interaction of PfHsp70-x with human Hop.

MSc (Biochemistry) / Department of Biochemistry / Plasmodium falciparum is a unicellular protozoan parasite that causes malaria in humans. The parasite is passed to humans through mosquito bites and migrates to the liver before it infects host erythrocytes. It is at the erythrocytic stage of development that the parasite causes malaria pathology. Malaria is characterized by the modification of host erythrocytes making them cytoadherent. This is as a result of formation of protein complexes (knobs) on the surface of the erythrocyte. The knobs that develop on the surface of the erythrocyte are constituted by proteins of host origin as well as some proteins that the parasite ‘exports’ to the host cell surface. Nearly 550 parasite proteins are thought to be exported to the infected erythrocyte. Amongst the exported proteins is P. falciparum heat shock protein 70-x (PfHsp70-x). Hsp70 proteins are known to maintain protein homeostasis. Thus, the export of PfHsp70-x may be important for maintaining protein homeostasis in the host cell. PfHsp70-x is not essential for parasite survival although is implicated in the development of parasite virulence. This is possibly through its role in facilitating the trafficking of parasite proteins to the erythrocyte as well as supporting the formation of protein complexes that constitute the knobs that develop on the surface of the infected erythrocyte. The main objective of the current study was to investigate protein interaction partners of PfHsp70-x. It is generally believed that PfHsp70-x interacts with various proteins of human and parasite origin. Potential candidate interactors include its protein substrates, Hsp70 co-chaperones such as Hsp40 members, and human Hsp70-Hsp90 organizing protein (hHop). The establishment of the PfHsp70-x interactome would highlight the possible role of PfHsp70-x in the development of malaria pathogenicity. Based on bioinformatics analysis, PfHsp70-x was predicted to interact with some exported P. falciparum Hsp40s, hHop and human Hsp90 (hHsp90). Recombinant forms of PfHsp70-x (full length and a truncated form that lacks the C-terminal EEVN motif implicated in co-chaperone binding) were expressed in E. coli BL21 Star (DE3) cells. Recombinant hHop and hHsp70 were expressed in E. coli JM109 (DE3) cells. The proteins were successfully purified using nickel affinity chromatography. Co-affinity chromatography using recombinant PfHsp70-x and immuno-affinity chromatography using PfHsp70-x specific antibody did not confirm the direct interaction of PfHsp70-x with human Hop. However, the direct interaction of hHop and PfHsp70-x has previously been validated in vitro and the current bioinformatics data support ii the existence of such a complex. PfHsp70-x was not stable in the cell lysate that was prepared and this could explain why its interaction with hHop could not be ascertained. However, taken together the evidence from a previous independent study, and the predicted interaction of PfHsp70-x with human chaperones suggests cooperation of chaperone systems which possibly facilitates the folding and function of parasite proteins that are exported to the infected erythrocyte. / NRF

MSc (Biochemistry) / Department of Biochemistry / Sustaining proteostasis is essential for the survival of the cell and altered protein regulation leads to many cellular pathologies. Heat shock proteins (Hsps) are involved in the regulation of the protein quality control. Hsps are a group of molecular chaperones that are upregulated in response to cell stress and some are produced constitutively. The Hsp70 family also known as DnaK in Escherichia coli (E. coli) is the most well-known group of molecular chaperones. Structurally, Hsp70s consist of a nucleotide binding domain (NBD) and a substrate binding domain (SBD) conjugated by a linker sub-domain. ATP binding and hydrolysis is central to the Hsp70 functional cycle. Hsp70s play a role in cytoprotection especially during heat stress in E. coli. Hsp70s from different organisms are thought to exhibit specialized cellular functions. As such E. coli Hsp70 (DnaK) is a molecular chaperone that is central to proteostasis in E. coli. On the other hand, Plasmodium falciparum Hsp70s are structurally amenable to facilitate folding of P. falciparum substrates. The heterologous production of P. falciparum proteins in E. coli towards drug discovery has been a challenge. There is need to develop tools that enhance heterologous expression and proper folding of P. falciparum proteins in an E. coli expression system. To this end, a chimeric Hsp70, KPf consisting of E. coli DnaK NBD and P. falciparum Hsp70-1 (PfHsp70-1) SBD was previously designed. KPf was shown to confer cytoprotection to E. coli DnaK deficient cells that were subjected to heat stress. In this study it was proposed that KPf has an advantage over E. coli DnaK and PfHsp70-1 in its function as a protein folding chaperone. Therefore, the main aim of this study was to characterize the chaperone function of KPf relative to the function of wild type E. coli and P. falciparum Hsp70s. The recombinant forms of KPf, DnaK and PfHsp70-1 proteins were successfully expressed and purified using nickel affinity chromatography. Circular Dichroism (CD) structural study demonstrated that KPf and PfHsp70-1 are predominantly α-helical and are also heat stable. Tertiary structure studies of PfHsp70-1 and KPf using tryptophan fluorescence revealed that both confirmations of recombinant proteins are perturbed by the presence of ATP more than ADP. Interestingly, the substrate binding capabilities of these proteins were comparable both in the absence or presence of nucleotides ATP/ADP. KPf is an independent chaperone, that exhibit nucleotide binding and hydrolysis. The current study has established unique structure-function features of KPf that distinguishes it from its “parental” forms, DnaK and PfHsp70-1. / NRF

Infections or diseases are not just stressful for the one who encounters it. The pathogens causing the same also have to deal with the hostile environment present in the host. The maintenance of physiological homeostatic balance is must for survival of all organisms. This becomes a challenging task for the protozoan parasites which often alternate between two different hosts during their life cycle and thereby encounter several environmental insults which they need to acclimatize against, in order to establish a productive infection. Since their discovery as proteins up-regulated upon heat shock, heat shock proteins have emerged as main mediators of cellular stress responses and are now also known to chaperone normal cellular functions. Parasites like Plasmodium falciparum have fully utilized the potential of these molecular chaperones. This is evident from the fact that parasite has dedicated about 2% of its genome for this purpose. During transmission from the insect vector to humans, the malaria parasite Plasmodium falciparum experiences a temperature rise of about 10oC, and the febrile episodes associated with asexual cycle further add to the heat shock which the parasite has to bear with. The exact mechanism by which the parasite responds to temperature stress remains unclear; however, the induction of chaperones such as PfHsp90 and PfHsp70 has been reported earlier. In other eukaryotes, there are three main factors which regulate heat shock response (HSR): heat shock factor (HSF), heat shock element (HSE) and HSF binding protein (HSBP). Bioinformatics analysis revealed presence of HSE and HSBP in P. falciparum genome; however, no obvious homolog of HSF could be identified. Either the HSF homologue in P. falciparum is highly divergent or the parasite has evolved alternate means to tackle temperature stress. Therefore, we decided to biochemically characterize HSBP and understand the heat shock response pathway in the parasite using transcriptomics and proteomics. The expression for PfHSBP was confirmed at both mRNA and protein level and it was found to translocate into the nucleus during heat shock. As previously reported for HSBP in other organisms, PfHSBP also exists predominantly in trimeric and hexameric form and it interacts with PfHsp70-1. Nearly 900 genes, which represent almost 17% of the parasite genome, were found to have HSE in their promoter region. HSE are represented by three repeating units of nGAAn pentamer and its inverted repeat nCTTn; however, the most abundant class of genes in P. falciparum possessed an atypical HSE which had only 2 continuous repeat units. Next, we were interested to find out if these HSE could actually bind to any parasite protein. Therefore, we performed EMSA analysis with the parasite nuclear extracts using HSE sequence as the oligonucleotide. We observed retarded mobility of the oligonucleotide suggesting that it was indeed able to recruit some protein from the nuclear extract. The importance of transcriptional regulation during heat shock was further confirmed when parasite culture subjected to heat shock in the presence of transcription inhibitor did not show induction in the levels of PfHsp70. These evidences suggest that parasite indeed possesses all the components of heat shock response pathway with either a divergent homologue of HSF or an alternate transcription factor which would have taken its role. Next, we performed global profiling of heat shock response using transcriptomic analysis and 2DDIGE based proteomic profiling. Overall, the parasite’s response to heat shock can be classified under 5 functional categories which aim at increasing the folding capacity of the cell, prevent protein aggregation, increase cytoadhesion, increase host cell remodelling and increase erythrocyte membrane rigidity. Out of the 201 genes found to be up-regulated upon heat shock, 36 were found to have HSE in their promoter region. This suggested that HSE-mediated protein up-regulation could be responsible for the induction of only 18% of total number of genes up-regulated upon heat shock. How would the parasite bring about up-regulation of rest of the heat shock responsive genes? It has been previously reported that genes for some of the heat shock proteins in P. falciparum possess G-box regulatory elements in their promoters and recently, it was shown that these elements served as the binding site for one of the transcription factors (PF13_0235) of AP2 family. Therefore, we looked for the status of this AP2 factor and its targets in our transcriptome data. Although, PF13_0235 was itself not up-regulated, we found up-regulation of its target genes which included another AP2 factor gene PF11_0404. The target genes of PF11_0404 were also up-regulated upon heat shock, thereby suggesting the functioning of an AP2 factor mediated response to heat shock. The next major challenge which the malaria parasite has to deal with is the remodelling of the erythrocyte as these cells do not have a cellular machinery which the parasite can take control of. The parasite remodels the erythrocyte with the help of its large repertoire of exported proteins and develops protrusions known as “knobs” on the erythrocyte surface. These protrusions are cytoadherent in nature and constitute the main virulence determinants of malaria. They also represent variable antigens that allow immune escape. Our lab has previously demonstrated an exported PfHsp40, termed as KAHsp40, to be involved in knob biogenesis. Apart from KAHsp40, there are 19 other PfHsp40s which possess the PEXEL motif required for protein export to erythrocytes. Although, Hsp40s work with an Hsp70 partner, none of the parasitic Hsp70s were known to be exported and was always a missing link in the field of malaria chaperone biology. A genomic re-annotation event could fill this gap by re-annotating the sequence for a pseudogene, PfHsp70-x and described it to contain a functional ORF. According to the re-annotated ORF sequence, PfHsp70-x possessed an ER signal peptide and thus could be targeted to the secretory pathway. Following validation of the re-annotation using a PCR-based approach, we confirmed the expression of this protein at the protein level by immunoblot analysis. Using various subcellular fractionation approaches and immunolocalization studies we established that PfHsp70-x indeed gets exported to the erythrocyte compartment; however, it did not contain the PEXEL motif required for protein export. It gets secreted into the vacuole around the parasite via the canonical ER-Golgi secretory pathway. Its trafficking from vacuole into the erythrocyte was mediated by a hexameric sequence which was present just after the signal peptide cleavage site and before the beginning of ATP-binding domain. In the erythrocyte compartment, it was found to interact with KAHsp40 and MAHRP1, proteins previously implicated in knob biogenesis. Most importantly, PfHsp70-x interacted with the major knob component PfEMP1; however, itself did not become part of knobs. Instead, it localized to the Maurer’s clefts in the erythrocyte compartment. Inside the parasite, PfHsp70-x was present in a complex with Plasmepsin V and PfHsp101. These proteins have been shown to be essential for host cell remodelling process. Plasmepsin V recognizes the PEXEL motif and brings about its cleavage and PfHsp101 specifically targets these PEXEL-cleaved exported proteins to the translocon in vacuolar membrane thereby facilitating their export into the erythrocyte. Thus, PfHsp70-x could also be involved in directing the export of knob constituents apart from just facilitating their assembly. Since, we found out that heat shock or the febrile episodes encountered during the asexual cycling of the parasite promote host cell remodelling; we wanted to find out if PfHsp70-x has any specific role under conditions of temperature stress. PfHsp70-x gene expression was not influenced upon heat shock, however, its export into the erythrocyte was inhibited and the protein got accumulated within the parasite compartment. Surprisingly, immunolocalization studies revealed that the accumulated pool of PfHsp70-x localized into the nucleus instead of ER thus suggesting an alternate role to be associated with PfHsp70-x under stress. Overall, our study addresses two major aspects of malaria pathogenesis. First, response to heat shock and second, remodelling of the host cell. We, for the first time describe global profiling of the parasite’s heat shock response and identify a novel P. falciparum specific heat shock protein member to be involved in malaria pathogenesis.