L'α-Synuclein (αSyn) fibrillaire, impliqué dans la maladie de Parkinson et d’autres synucleinopathies, peut se propager entre cellules de manière « prion-like » et cette propagation est liée à la progression de la maladie. Durant cette étude, nous nous sommes tournés vers les chaperons moléculaires impliqués dans l’agrégation de l’αSyn ou bien dans sa toxicité afin de trouver des candidats capables d’interférer avec la propagation. Nous avons ensuite testé l’effet des chaperons capables de se lier aux fibres d’αSyn sur l’internalisation des fibres d’αSyn par les cellules Neuro-2a. Nous démontrons que l’interaction avec l’αSyn agrégeant avec αB-crystallin (αBc) ou Carboxyl terminus of Hsc70-interacting protein (CHIP) a mené à la formation de fibres qui sont moins internalisées par les cellules. Enfin, en passant par une stratégie de pontage chimique optimisé couplé à la spectrométrie de masse, nous avons identifié les zones d’interaction entre l’αSyn fibrillaire et soit αBc, soit CHIP. Ces résidus issus des chaperons, se trouvant à proximité des fibres d’αSyn dans les complexes, pourraient être développés dans des mini-chaperons peptidiques, capables d’enrober la surface des fibres et ainsi de bloquer la liaison à la membrane et l’internalisation des fibres. De surcroît, des polypeptides issus des partenaires précédemment identifiés d’αSyn ont été testé pour leur liaison aux fibres et leur effet sur la propagation des fibres. / Fibrillar α-Synuclein (αSyn) is the molecular hallmark of Parkinson’s Disease and other synucleinopathies. Its prion-like propagation between cells is linked to disease progression. In this study, we looked to molecular chaperones previously implicated in αSyn fibrillation and/or toxicity to identify proteins capable of binding αSyn fibrillar aggregates in order to target their propagation. We further assessed the effect of the fibril-binding chaperones on internalization of αSyn fibrils by Neuro-2a cells. We demonstrate that the interaction of aggregating αSyn with αB-crystallin (αBc) or Carboxyl terminus of Hsc70-interacting protein (CHIP) led to the formation of fibrils that are less internalized by cells. Finally, using an optimized chemical cross-linking and mass spectrometry strategy, we identified the interaction areas between fibrillar αSyn and either αBc or CHIP. These chaperone residues, located proximally to αSyn fibrils, could be subsequently developed into peptidic mini chaperones, capable of coating the fibril surface and thereby blocking fibrillar cell binding and internalization. Furthermore, polypeptides derived from previously identified αSyn binding partners were tested for their binding to αSyn fibrils and subsequent effect on fibril propagation.

BACKGROUND: Scythe/BAT3 is a member of the BAG protein family whose role in apoptosis has been extensively studied. However, since the developmental defects observed in Bat3-null mouse embryos cannot be explained solely by defects in apoptosis, we investigated whether BAT3 is also involved in cell-cycle progression. METHODS/PRINCIPAL FINDINGS: Using a stable-inducible Bat3-knockdown cellular system, we demonstrated that reduced BAT3 protein level causes a delay in both G1/S transition and G2/M progression. Concurrent with these changes in cell-cycle progression, we observed a reduction in the turnover and phosphorylation of the CDK inhibitor p21, which is best known as an inhibitor of DNA replication; however, phosphorylated p21 has also been shown to promote G2/M progression. Our findings indicate that in Bat3-knockdown cells, p21 continues to be synthesized during cell-cycle phases that do not normally require p21, resulting in p21 protein accumulation and a subsequent delay in cell-cycle progression. Finally, we showed that BAT3 co-localizes with p21 during the cell cycle and is required for the translocation of p21 from the cytoplasm to the nucleus during the G1/S transition and G2/M progression. CONCLUSION: Our study reveals a novel, non-apoptotic role for BAT3 in cell-cycle regulation. By maintaining a low p21 protein level during the G1/S transition, BAT3 counteracts the inhibitory effect of p21 on DNA replication and thus enables the cells to progress from G1 to S phase. Conversely, during G2/M progression, BAT3 facilitates p21 phosphorylation by cyclin A/Cdk2, an event required for G2/M progression. BAT3 modulates these pro- and anti-proliferative roles of p21 at least in part by regulating cyclin A abundance, as well as p21 translocation between the cytoplasm and the nucleus to ensure that it functions in the appropriate intracellular compartment during each phase of the cell cycle. / Dissertation

Molecular chaperones have emerged in recent years as major players in many aspects of cell biology. Molecular chaperones are also known as heat shock proteins (HSPs) since many were originally discovered due to their increased synthesis in response to heat shock. They were initially identified when Drosophila salivary gland cells were exposed to a heat shock at 37°C for 30 min and then returned to their normal temperature of 25°C for recovery. A “puffing” of genes was found to have occurred in the chromosome of recovering cells, which was later shown to be accompanied by an increase in the synthesis of proteins with molecular masses of 70 and 26 kDa. These proteins were hence named “heat shock proteins”. The first identification of a function for HSPs was the discovery in Escherichia coli that five proteins synthesized in response to heat shock were involved in λ phage growth. The products of the groEL and groES genes were found to be essential for phage head assembly while the dnaK, dnaJ and grpE gene products were essential for λ phage replication. It was later shown that GroEL and GroES are part of a chaperonin system for protein folding in the prokaryotic cytosol while DnaK is a member of the Hsp70 family that works in conjunction with the DnaJ (Hsp40) co-chaperone and the nucleotide exchange factor GrpE to promote phage replication by dissociating the DnaB helicase from the phage-encoded P protein. Since then, a large number of other proteins collectively referred to as HSPs have been discovered. However, heat shock is not the only signal that induces synthesis of heat shock proteins. Stress of any kind, such as nutrient deprivation, chemical treatment and oxidative stress among others causes increased production of HSPs and therefore, they are also known as stress proteins. The term “molecular chaperone” was originally used to describe the function of nucleoplasmin, a Xenopus oocyte protein that promotes nucleosome assembly by binding tightly to histones and donating the bound histone to chromatin. However, since then, chaperones have been defined as “a family of unrelated classes of proteins that mediate the correct assembly of other proteins, but are not themselves components of the final functional structure”. This view of molecular chaperones, though undoubtedly correct, doesn’t capture the multifaceted roles they have since been discovered to play in cellular processes. In recent years, molecular chaperones have been shown to perform other functions in addition to the maintenance of protein homeostasis: translocation of proteins across organelle membranes, quality control in the endoplasmic reticulum, turnover of misfolded proteins as well as signal transduction. As a result, many chaperones are also essential under non-stress conditions and play crucial roles in cell growth and development, cell-cell communication and regulation of gene expression. Heat shock protein 90 (Hsp90) is one of the most abundant and highly conserved molecular chaperones in organisms ranging from bacteria to all branches of eukarya. It has been shown to be essential for cell viability in Saccharomyces cerevisiae, Schizosaccharomyces pombe and Drosophila melanogaster. Although the bacterial homolog HtpG is dispensable under normal conditions, it is important for cell survival during heat shock. In addition to its role as general chaperone in protein folding following stress, Hsp90 has a more specialized role as a chaperone for several protein kinases and transcription factors. Many Hsp90 client proteins are signaling proteins involved in regulation of cell growth and survival. These proteins are critically dependent on Hsp90 for their maturation and conformational maintenance resulting in a key role for Hsp90 in these processes. Recent reports have also highlighted a role for Hsp90 in linking the expression of genetic and epigenetic variation in response to environmental stress with morphological development in Drosophila melanogaster and Arabidopsis thaliana. In Candida albicans, Hsp90 augments the development of drug resistance, implicating a role for Hsp90 in the evolution of infectious diseases. The malarial parasite, Plasmodium falciparum, is the causative agent of the most lethal form of human malaria. The parasite life cycle involves two hosts: an invertebrate mosquito vector and a vertebrate human host. As the parasite moves from the mosquito to the human body, it experiences an increase in temperature resulting in a severe heat shock. The mechanisms by which the parasite adapts to changes in temperature have not been deciphered. Our laboratory has been interested in investigating the role of heat shock proteins during acclimatization of the parasite to such temperature fluctuations. Heat shock proteins of the Hsp40, Hsp60, Hsp70 and Hsp90 families have been characterized in the parasite and are being examined in our laboratory. This thesis pertains to understanding the functional role of Plasmodium falciparum Hsp90 (PfHsp90) during adaptation of the parasite to fluctuations in environmental temperature. The parasite expresses a single gene for cytosolic Hsp90 on chromosome 7 (PlasmoDB accession no.: PF07_0029) coding for a protein of 745 amino acids with a pI of 4.94 and Mw of 86 kDa. Eukaryotic Hsp90 regulates several protein kinases and transcription factors involved in cell growth and differentiation pathways resulting in a crucial role for Hsp90 in developmental processes. A role for PfHsp90 in parasite development, therefore, seems likely. Indeed, PfHsp90 has previously been implicated in parasite development from the ring stage to the trophozoite stage during the intra-erythrocytic cycle. Pharmacological inhibition of PfHsp90 function using geldanamycin (GA), a specific inhibitor of Hsp90 activity, abrogates stage progression. These experiments suggest that PfHsp90 may play a critical role in parasite development. This is further substantiated by the fact that several pathogenic protozoan parasites such as Leishmania donovani, Trypanosoma cruzi, Toxoplasma gondii and Eimeria tenella depend on Hsp90 function during different stages of their life cycles. It appears, therefore, that a principal role of Hsp90 in protozoan parasites may be the regulation of their developmental cycles. However, the precise functions of PfHsp90 during the intra-erythrocytic cycle of the malarial parasite are not clear. In this study we have carried out a functional analysis of PfHsp90 in the malarial parasite. We have examined the role of PfHsp90 in parasite development during repeated exposure to febrile temperatures. We have investigated its involvement in parasite development during a commonly used synchronization protocol involving cyclical changes in temperature. We have examined the interaction of GA with the Hsp90 multi-chaperone complex from P. falciparum as well as the human host. Finally, we have carried out a systems level analysis of chaperone networks in the malarial parasite as well as its human host using an in silico approach. We have analyzed the protein-protein interactions of PfHsp90 in the chaperone network and predicted putative cellular processes likely to be regulated by parasite chaperones, particularly PfHsp90.

Small heat shock proteins (sHSPs) are a ubiquitous family of molecular chaperones that play a vital role in maintaining protein homeostasis in cells. They are the first line of defence against the detrimental effects of cellular stress conditions like fluctuations in temperature, pH, oxidative and osmotic potentials, heavy metal toxicity, drought and anoxia. Many sHSPs are also constitutively expressed during developmental stages of different plant tissues. Members of this family are ATP-independent chaperones, with monomeric masses varying from 12-40 kDa. A characteristic feature of sHSPs is their ability to assemble into large oligomers, ranging from dimers to 48-mers. Under stress conditions, these oligomers dissociate and/or undergo drastic conformational changes to facilitate their binding to misfolded substrate proteins in the cell. This interaction prevents the substrate from aggregating during stress. When physiological conditions are restored, the substrates are transferred to other ATP-dependent heat shock proteins for refolding. Thus sHSPs do not refold their substrates, but instead prevent them from aggregating and maintain them in a „folding-competent‟ state. The clientele of sHSPs includes proteins with a wide range of molecular masses, secondary structures and pIs. This promiscuity has led to sHSPs occupying key positions in the protein quality control network. As molecular chaperones that protect proteins, sHSPs prevent disease. Concomitantly, mutations in sHSPs have also been linked to various human diseases. Till date, high resolution crystal structures are available only for 3 sHSP oligomers. This insufficiency of structural information has hindered our understanding of the mechanism of chaperone function, the link between the oligomeric status and chaperone activity, identification of substrate binding sites and the role of the flexible terminal segments in mediating both the oligomerization and chaperone function. We undertook structural and functional characterization of plant and bacterial sHSPs in order to address some of these questions. Chapter 1 of this thesis gives an overview of the sHSP family, with special emphasis on the oligomeric assemblies of sHSPs of known structures. We highlight what we know about this family through mutational studies, what is as yet unknown, and why it is important to study this family. Chapter 2 describes our efforts at structural and functional characterization of 5 sHSPS in rice, each targeted to a different organelle. We probed the role played by the N-terminal region in mediating oligomer assembly and in the chaperone activity of the protein. Rice sHSPs displayed a wide range of hydrodynamic radii, from 4 nm to 14 nm, suggesting that their oligomeric assemblies are likely to be diverse. In chapter 3, we discuss our attempts at the structural characterization of a bacterial sHSP, Aggregation suppressing protein A, or AgsA from Salmonella enterica. We obtained a high resolution crystal structure of the dimer of the core sHSP domain. We compared this dimer with other known sHSP dimers, reported the deviations that we observed and analysed the structure to account for these differences. We used this dimer structure to successfully obtain solutions for low resolution X-ray diffraction data for oligomers of different truncated constructs of AgsA. We observed that a C-terminal truncated construct formed an octahedral 24¬mer (4.5 Å resolution), whereas a construct truncated at both termini formed a triangular bipyramidal 18-mer (7.7 Å resolution), an assembly hitherto unobserved for any sHSP. A similar 18-mer was obtained when the C-terminal truncated construct was incubated with a dipeptide prior to crystallisation (6.7 Å resolution). The cryo-EM map of the wild type protein (12 Å resolution) could be fitted with a different 18-mer. The low resolution of the data pre-empted an atomic-level description of the interfaces of the assemblies. However, our work highlights the structural plasticity of this protein and probes the sensitivity of the oligomeric assembly to minor differences in construct length.

Die erfolgreiche Synthese und Faltung von Proteinen ist eine Voraussetzung der Zellfunktion und ein Versagen der Proteinhomöostase führt zu Krankheit oder Tod. In der Zelle sichern molekulare Chaperone die korrekte Faltung der Proteine oder tragen zur Entsorgung unwiederbringlich fehlgefalteter Proteinsubstrate bei. Unter diesen Chaperonen sind kleine Hitzeschockproteine (sHsp) ein ATP-unabhängiger Teil des Proteostasenetzwerks. In dieser Arbeit habe ich das bisher wenig erforschte sHsp HSP-17 aus C. elegans untersucht. Im Gegensatz zu anderen sHsps zeigte HSP-17 nur eine geringe Aktivität beim Verhindern der Aggregation von Proteinsubstraten. Stattdessen konnte ich in vitro zeigen, dass HSP-17 die Aggregation von Modellsubstraten fördert, was hier für Metazoen-sHsps erstmals gezeigt wurde. HSP-17 kopräzipitiert mit Substraten und modifiziert deren Aggregate möglicherweise. HSP-17 kolokalisiert in vivo mit Aggregaten, und seine aggregationsfördernde Aktivität konnte ich für das physiologische Substrat KIN-19 und heterolog exprimierte polyQ-Peptide validieren. Durch ex vivo Analysen konnte ich zeigen, dass die Aktivität von HSP-17 für die Fitness relevant ist  In einem zweiten Projekt habe ich zur Entwicklung eines neuen Modelles für Aß-Pathologie in C. elegans beigetragen, welches substöchiometrische Markierungen verwendet, um eine zeitnahe Visualisierung der Aß-Aggregation in spezifischen Zelltypen zu ermöglichen. Das Modell spiegelt bekannte Phänotypen der Aß-Proteotoxizität aus Menschen und bestehenden C. elegans Aß-Stämmen wider. Interessanterweise zeigt eine Untergruppe der Neuronen, die IL2-Neuronen, eine höhere Anfälligkeit für die Aggregation und Proteotoxizität von Aß1-42. Eine gezielte Reduktion von Aß1-42 in IL2 Neuronen führt zu einer systemischen Reduktion der Pathologie. Somit bietet das Modell eine neue Plattform, um die Bedeutung molekularer Chaperone, wie z. B. der sHsps, für Amyloidosen zu untersuchen, auch im Hinblick auf menschliche Erkrankungen. / Successful synthesis and folding of proteins is a prerequisite for cellular function and failure of protein homeostasis leads to disease or death. Within the cell, molecular chaperones ensure correct protein folding or aid in the disposal of terminally misfolded protein substrates. Among these chaperones, small heat shock proteins (sHsps) are ATP-independent members of the proteostasis network. In this work, I analyzed the so far under-researched C. elegans sHsp HSP-17. Unlike other sHsps, HSP-17 exhibited only weak activity in preventing aggregation of protein substrates. Instead, I could show in vitro that HSP-17 can promote the aggregation of protein substrates, which is the first demonstration for metazoan sHsps. HSP-17 co-precipitates with substrates and potentially modifies the aggregates.  HSP-17 colocalizes with aggregates and pro-aggregation activity is present in vivo, which I demonstrated for the physiological substrate KIN-19 and heterologously expressed amyloidogenic polyQ peptides. By physiological, biochemical and proteomic analysis I showed that HSP-17 activity is relevant for organismal fitness In a second project, I contributed to the development and characterization of a novel model of Aß pathology in C. elegans. This new AD model employs sub-stoichiometric labeling to allow live visualization of Aß aggregation in distinct cell types. The model mirrors known phenotypes of Aß proteotoxicity in humans and existing C. elegans Aß strains. Interestingly, a subset of neurons, the IL2 neurons, is shown to be more vulnerable to Aß proteotoxicity and targeted depletion of Aß in these neurons systemically ameliorates pathology. Thereby, the model presents a new platform to assess the relevance of molecular chaperones such as sHsps in amyloidosis with a perspective on human disease.

Indiana University-Purdue University Indianapolis (IUPUI) / Disruptions of the endoplasmic reticulum (ER) cause perturbations in protein folding and result in a cellular condition known as ER stress. ER stress and the accumulation of unfolded protein activate the unfolded protein response (UPR) which is a cellular attempt to remedy the toxic accumulation of unfolded proteins. The UPR is implemented through three ER stress sensors PERK, ATF6, and IRE1. Phosphorylation of the α-subunit of eIF2 by PERK during ER stress represses protein synthesis and also induces preferential translation of ATF4, a transcriptional activator of stress response genes. Early UPR signaling involves translational and transcriptional changes in gene expression that is geared toward stress remedy. However, prolonged ER stress that is not alleviated can trigger apoptosis. This dual signaling nature of the UPR is proposed to mimic a 'binary switch' and the regulation of this switch is a key topic of this thesis. Adaptive gene expression aimed at balancing protein homeostasis encompasses the first phase of the UPR. In this study we show that the PERK/eIF2~P/ATF4 pathway facilitates both the synthesis of ATF6 and trafficking of ATF6 from the ER to the Golgi where ATF6 is activated. Liver-specific depletion of PERK significantly lowers expression of survival genes, leading to reduced expression of protein chaperones. As a consequence, loss of PERK in the liver sensitizes cells to stress which ultimately leads to apoptosis. Despite important roles in survival, PERK signaling is often extended to the vii activation of other downstream transcription factors such as CHOP, a direct target of ATF4-mediated transcription. Accumulation of CHOP is a hallmark of the second phase in the binary switch model where CHOP is shown to be required for full activation of apoptosis. Here the transcription factor ATF5 is found to be induced by CHOP and that loss of ATF5 improves the survival of cells following changes in protein homeostasis. Taken together this study highlights the importance of UPR signaling in determining the balance between cell survival and cell death. A topic that is important for understanding the more complex pathological conditions of diseases such as diabetes, cancer, and neurodegeneration.

No description available.

Background: Heat-shock proteins of the 70 kDa family (Hsp70s) are essential chaperones required for key cellular functions. In eukaryotes, four subfamilies can be distinguished according to their function and localisation in different cellular compartments: cytosol, endoplasmic reticulum, mitochondria and chloroplasts. Generally, multiple cytosol-type Hsp70s can be found in metazoans that show either constitutive expression and/or stress-inducibility, arguing for the evolution of different tasks and functions. Information about the hsp70 copy number and diversity in microbial eukaryotes is, however, scarce, and detailed knowledge about the differential gene expression in most protists is lacking. Therefore, we have characterised the Hsp70 gene family of Paramecium caudatum to gain insight into the evolution and differential heat stress response of the distinct family members in protists and to investigate the diversification of eukaryotic hsp70s focusing on the evolution of heat-inducibility. Results: Eleven putative hsp70 genes could be detected in P. caudatum comprising homologs of three major Hsp70-subfamilies. Phylogenetic analyses revealed five evolutionarily distinct Hsp70-groups, each with a closer relationship to orthologous sequences of Paramecium tetraurelia than to another P. caudatum Hsp70-group. These highly diverse, paralogous groups resulted from duplications preceding Paramecium speciation, underwent divergent evolution and were subject to purifying selection. Heat-shock treatments were performed to test for differential expression patterns among the five Hsp70-groups as well as for a functional conservation within Paramecium. These treatments induced exceptionally high mRNA up-regulations in one cytosolic group with a low basal expression, indicative for the major heat inducible hsp70s. All other groups showed comparatively high basal expression levels and moderate heat-inducibility, signifying constitutively expressed genes. Comparative EST analyses for P. tetraurelia hsp70s unveiled a corresponding expression pattern, which supports a functionally conserved evolution of the Hsp70 gene family in Paramecium. Conclusions: Our analyses suggest an independent evolution of the heat-inducible cytosol-type hsp70s in Paramecium and in its close relative Tetrahymena, as well as within higher eukaryotes. This result indicates convergent evolution during hsp70 subfunctionalization and implies that heat-inducibility evolved several times during the course of eukaryotic evolution.

The formation and deposition of α-Synuclein fibrils in the human brain is at the origin of Parkinson's disease. The objective of my thesis was to document the role of two molecular chaperones on the assembly of α-Syn into fibrils: Hsc70, a constitutively expressed human heat shock protein, and Ssa1p, its yeast equivalent. The aim was to expand the catalogue of known effects of molecular chaperones on the PD implicated protein, which could have therapeutic significance. We showed that Hsc70 inhibits the assembly of α-Syn into fibrils, by binding with high affinity to the soluble form of α-Syn. We documented that Hsc70 binds preferentially to α-Syn fibrils and that this binding has a cytoprotective effect, as it renders the fibrils less toxic to cultured mammalian cells. Similarly to Hsc70, Ssa1p inhibits the assembly of α-Syn into fibrils, and has a higher affinity for fibrils than for the soluble form of α-Syn. On the other hand, binding of Ssa1p to α-Syn fibrils does not have a cytoprotective effect, almost certainly due to differences in the amino acid sequences of the peptide binding sites of the two molecular chaperones, which mean that Ssa1p has a lower affinity than Hsc70 for α-Syn fibrils. We stabilized the complex between Ssa1p and α-Syn using chemical cross-linkers, to then map the interaction site between the two proteins. This is indispensable if a "mini" Ssa1p, comprised of only what is necessary and sufficient of Ssa1p, is to be used as a therapeutic agent to decrease the toxicity of α-Syn fibrils. A therapeutic agent based on exogenous protein Ssa1p is less likely to trigger an autoimmune response than for example the endogenous protein Hsc70.

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.