Un certain nombre d’études épidémiologiques font état d’une augmentation de l’infertilité masculine durant ces cinquante dernières années, en particulier dans les pays industrialisés, mais aussi d’une augmentation des malformations de l’appareil reproducteur masculin telles que la cryptorchidie (absence de migration des testicules dans les bourses) ou l’hypospadias (malformation du pénis), et des cancers testiculaires. Des données expérimentales suggèrent que ces anomalies du tractus génital mâle sont liées. Ces symptômes forment le syndrome de dysgénésie testiculaire. Les causes d’apparition ce syndrome semblent être d’origine environnementale. En effet, les évolutions relativement rapides de ce syndrome suggèrent des facteurs dynamiques, en lien avec le mode de vie ou l’environnement. Une des hypothèses est que, l’exposition durant la vie fœtale ou néonatale à des composés présents dans l’environnement capables d’interférer avec le système hormonal (perturbateurs endocriniens environnementaux, PEEs), serait responsable de l’augmentation de l’incidence de ces pathologies. Au banc des principaux accusés, les molécules qui possèdent des activités de type estrogénique ou antiandrogénique. A ce jour, les mécanismes d’action à l’origine du syndrome de dysgénésie testiculaire sont encore mal connus. Certaines études suggèrent des mécanismes de type épigenétique dans les effets à long terme des PEEs. L’objectif de notre travail était d’identifier et caractériser les mécanismes d’action de type épigenétique impliqué dans l’infertilité mâle. Pour cela, nous avons utilisé un modèle expérimental (rats nouveau-nés) reposant sur une exposition développementale à un estrogène (estradiol benzoate). Ce modèle induit chez le rat adulte un phénotype d’hypospermatogenèse liée à une à apoptose chronique des cellules germinales testiculaires. Nous montrons que ce phénotype est lié à l’altération de deux voies, impliquant en amont des effecteurs épigénétiques. La première voie implique la famille des miR-29s. Ainsi, nous observons une augmentation de l’expression des miR-29a, b, c qui provoque une diminution de deux de ses cibles: la protéine antiapoptotique MCL-1 et les enzymes de méthylation de l’ADN DNMTs. La chute des DNMTs entraine une hypométhylation globale (estimée à travers le gène Line-1) et spécifique du facteur de choc thermique HSF1. Ceci provoque une réexpression de ces facteurs entrainant l’apoptose des cellules germinales adultes. La deuxième voie implique le miR-18a. L’augmentation de son expression provoque une chute de l’expression de sa cible HSF2 qui régule la protéine de choc thermique HSP70/HSPA2. Le faible taux d’HSPA2 est une autre explication de l’apoptose des cellules germinales dans notre modèle. Nous montrons aussi que ce phénotype est irréversible lorsque l’exposition à lieu chez le nouveau-né alors qu’il est réversible quand l’exposition à lieu à l’âge adulte. Ces données suggèrent que l’exposition néonatale à l’estradiol benzoate induit une programmation développementale de l’hypospermatogenèse.Enfin, les anomalies tissulaires d’expression des miRNAs se retrouvent au niveau sanguin, suggérant leur utilisation potentielle comme biomarqueurs. Nous avons validé cet aspect chez l’homme en montrant que l’expression des miR29s et du miR-18a était plus élevée chez les patients oligo- ou azoospermiques que les chez patients normospermiques.En conclusion, nos résultats indiquent que l’hypospermatogenèse due à une apoptose chronique des cellules germinales observée chez l’animal adulte après exposition néonatale à l’EB met en jeu une modification d’expression de plusieurs effecteurs épigénétiques clés: miR-29s, miR-18a et DNMTs. De plus, les miR-29s et miR-18a pourraient être de nouveaux biomarqueurs circulants non invasifs de la stérilité masculine dans le contexte d’une oligo ou azoospermie chez l'homme. / Epidemiological studies have reported an increase in male infertility over the past fifty years, especially in industrialized countries, but also an increase in malformations of the male reproductive tract such as cryptorchidism (no migration of the testes into the scrotum) and hypospadias (malformation of the penis), and testicular cancers. Experimental data suggest that these abnormalities of the male genital tract are related. These symptoms form the testicular dysgenesis syndrome. The causes of the occurrence of this syndrome appear to be environmental in origin. Indeed, the relatively rapid evolution of this syndrome suggests dynamic factors related to lifestyle or environment. One hypothesis is that exposure during fetal or neonatal life to compounds present in the environment can interfere with the hormonal system (environmental endocrine disruptors), would be responsible for the increased incidence of these pathologies. Bench of the main accused, molecules that have estrogenic or anti-androgenic activity types. To date, the mechanisms of action behind the testicular dysgenesis syndrome are poorly understood. Some studies suggest that epigenetic mechanisms are at playThe objective of our work was to identify and characterize the epigenetic mechanisms of action involved in male infertility induced by neonatal exposure to xenoestrogen. For this, we used an experimental model based on a developmental exposure to estrogen (estradiol benzoate). This model induced in adult rats a hypospermatogenesis phenotype due to chronic apoptosis of germ cells.We show that this phenotype is related to an alteration of two pathways, involving upstream effectors epigenetic. The first pathway involves the family of miR- 29s. Thus, we observe an up-regulation of miR -29a, b, c, which causes a decrease in two of his targets: the anti-apoptotic protein MCL- 1 and the enzymes of DNA methylation DNMTs. Falling DNMTs leads to a global hypomethylation (estimated through the Line -1 gene) and to specific hypomethylation of the heat shock factor, HSF1. This causes a re-expression of factors that induce apoptosis in adult germ cells. The second pathway involves up-regulation of miR -18a that causes a down-regulation of its target HSF2 which regulates the heat shock protein HSP70/HSPA2. The down-regulation of HSPA2 is another explanation of germ cell apoptosis in our model. We also show that this phenotype is irreversible when the estrogen exposure takes place in the newborn whereas it is reversible when exposure takes place in adulthood, suggesting that neonatal exposure to estradiol benzoate induced a developmental programming of hypospermatogenesis.Finally, abnormal tissue expressions of miRNAs are found in the blood, suggesting their potential use as biomarkers. We validated this aspect in humans showing that the expression of miR29s and miR-18a was higher in patients with decrease or no sperm counts compared to normal sperm count. In conclusion, our results indicate that hypospermatogenesis due to chronic germ cell apoptosis observed in adult animals after neonatal exposure to EB involves a change in expression of several key epigenetic effectors: miR-29, miR-18a and DNMTs. In addition, miR-29 and miR-18a could be new non invasive circulating biomarkers of men infertility.

No description available.

Mitochondria are essential eukaryotic organelles, acting as the sites for numerous crucial metabolic and signalling pathways. The biogenesis of mitochondria requires efficient targeting of several hundreds of proteins from the cytosol, to their varied functional locations within the organelle. The translocation of localized proteins across the inner membrane, and their subsequent folding is achieved by the ATP-dependent function of mitochondrial Hsp70 (mtHsp70). It is a bonafide member of the Hsp70 chaperone family, which are involved in a multitude of functions, together aimed at protein quality control and maintenance of cellular homeostasis. These varied functions of Hsp70 proteins require binding to exposed hydrophobic patches in substrate polypeptides thus preventing non-productive associations. The interaction with substrates occurs through the substrate-binding domain (SBD) and is regulated by the ATPase activity of the nucleotide-binding domain (NBD), through a series of conformational changes. Conversely, substrate binding to the SBD also stimulates ATP hydrolysis, and thereby the core activities of the two domains are regulated by mutual allosteric signalling. This mechanism of bidirectional inter-domain communication is indispensable for Hsp70 function, which is characterized by cycles of substrate binding and release, coupled to cycles of ATP binding and hydrolysis. The process of allosteric regulation in Hsp70 proteins has been comprehensively investigated, especially in the bacterial homolog, DnaK. However, the in vivo functional significance of inter-domain communication in the eukaryotic mtHsp70 system and the mechanism of its regulation remain unexplored. Furthermore, the complex physiological implications of impairment in allosteric communication and their correlation with diverse disease conditions, including Myelodysplastic syndrome (MDS), and Parkinson’s disease (PD), are yet to be elucidated. Based on this brief introduction, the primary research objectives set out in the present thesis were to: 1. uncover the regulation of ligand-modulated allosteric communication between the two domains of mtHsp70; and its in vivo significance in the context of protein import into the organelle. (Chapter 2) 2. understand the role of mtHsp70 in progression of Parkinson’s disease; and to study the modulation of α-synuclein toxicity by the protein quality control function of the mtHsp70 chaperone network. (Chapters 3 and 4) We have employed a battery of genetic and biochemical approaches to investigate the above questions using the Saccharomyces cerevisiae mtHsp70 protein, Ssc1; an essential protein that is involved in a plethora of critical functions in this eukaryotic model system. Objective 1: Structural studies, primarily in bacterial DnaK, have yielded mechanistic insights into its interactions with ligands and cochaperones, as well as conformational transitions in different ligand-bound states. In recent years, the availability of crystal structures of full-length DnaK and detailed information from NMR studies and single-molecule resolution spectroscopic analyses (both DnaK and eukaryotic Hsp70s), have significantly contributed to our understanding of the inter-domain interface, critical residues and contacts, and the energetics of the entire process of ligand-modulated conformational changes. Although eukaryotic mtHsp70s have a high degree of conservation with DnaK, they possess significant differences in their conformational and biochemical properties. They are essential for a vast repertoire of physiological functions, which are distinctly different from their bacterial counterpart. Using a combined in vivo and in vitro approach, we have uncovered specific structural elements within mtHsp70s, which are required for allosteric modulation of the chaperone cycle and maintenance of in vivo functions of the protein. Foremost, we demonstrate that a conserved SBD loop, L4,5 plays a critical role in inter-domain communication, and multiple mutations in this loop result in significant growth and protein translocation defects. The mutants are associated with a specific set of altered biochemical properties, which are indicative of impaired inter-domain communication. Using the loop L4,5 mutant, E467A as a template for genetic screening, we report a series of intragenic suppressor mutations, which are capable of correcting a distinct subset of the altered properties, and thereby leading to restoration of in vivo functions, including growth, preprotein import and mitochondria biogenesis. The suppressors modify the altered conformational landscape associated with E467A, and also provide us with information regarding unique aspects governing the regulation of allosteric communication, especially in physiological contexts. Strikingly, they reveal that restoration of communication in the NBD to SBD direction is sufficient for function, when the protein is primed in a high ATPase activity state. In this unique scenario, the requirement for ATPase stimulation upon substrate binding is rendered unnecessary, thereby making conformational changes in the SBD to NBD direction, dispensable for function. Further, we provide evidence to show that loop L4,5 functions synergistically with the linker region, working in tandem for organization of the inter-domain interface and propagation of communication. Together, our analyses provide the first insights into regulation of allosteric inter-domain communication in vivo and their implications in mitochondrial protein translocation and organelle biogenesis. Objective 2: Point mutations in the loop L4,5 have been associated with Myelodysplastic syndrome. Additionally, a mutation isolated in clinical cases of Parkinson’s disease was found to be impaired in allosteric communication. These observations further highlight the importance of efficient inter-domain communication in mtHsp70 in the complex physiological scenario of eukaryotic cells. Independent clinical screens of PD patients have revealed unique point mutations in the mtHsp70 and a strong association of the gene locus with the disease progression. This is also correlated with decreased mtHsp70 levels in affected neurons and the interactions of this protein with established PD-candidate proteins like α-synuclein and Dj-1. Further, mitochondrial dysfunction is a common phenomenon associated with neurodegenerative disorders. To understand the specific role of mtHsp70 in PD, we have developed a yeast model for studying the disease variants in isolation from other players of the multifactorial disease, and in complete absence of the wild type protein. We generated two analogous PD-mutations in Ssc1, R103W and P486S; which recapitulated the symptoms of mitochondrial dysfunction in affected neurons, including cell death, inner membrane depolarization, increased generation of ROS, and respiratory incompetence. At the molecular level, we observed an increased aggregation propensity of R103W, while P486S exhibited futile enhanced interaction with J-protein cochaperone partners thereby resulting in loss of chaperoning activity and impaired mitochondrial protein quality control. Remarkably, these altered biochemical properties mimicked similar defects in the human mtHsp70 variants, therefore, affirming the involvement of mtHsp70 in PD progression. To further investigate the relevance of impaired mitochondrial protein quality control in PD, we have explored whether mtHsp70 can act as a genetic modifier of α-synuclein toxicity. It is known that α-synuclein can act as an unfolded substrate for the Hsp70 chaperone system and also deposits as intracellular aggregates in PD-affected brains. Intriguingly, it is known to translocate into mitochondria under conditions of neuronal stress in spite of lacking a canonical mitochondrial signal sequence. Utilizing our yeast-PD model, we find that targeting of α-synuclein A30P disease variant into mitochondria leads to a severe mitochondrial dysfunction phenotype in the wild type Ssc1 background, but not the P486S mutant background. This results in multiple cellular manifestations, which are reversed upon overexpression of the Ssc1 chaperone. Significantly, increasing the J-protein cochaperone availability also leads to reversal of the mutant-associated defects. However, the simultaneous overexpression of both together does not additively improve the protective effects; highlighting the importance of the relative availability of chaperone and cochaperone proteins in preventing aggregation. Our analyses further reveal that while both the wild type and P486S Ssc1 proteins are equally capable of delaying aggregation of α-synuclein, only the wild-type chaperone is better able to prevent aggregation in the presence of its J-protein cochaperone, leading to accumulation of soluble oligomeric species. These observations raised the intriguing possibility, that the reduced chaperoning ability of the proline to serine PD-mutant is, in fact, a compensatory adaptation, favoring the aggregation of α-synuclein over its more toxic soluble oligomeric form. We verify this hypothesis with the aggregation kinetics of A30P α-synuclein, whose intrinsically lower aggregation tendency results in a pronounced delay in aggregation with the wild-type chaperone, thereby strongly favoring the toxic oligomeric species and correlating with the observed lethality in yeast cells. In conclusion, our study provides a model of α-synuclein aggregation-related toxicity and its modulation by the extent of protein quality control within the mitochondrial matrix, through the action of the mtHsp70 chaperone network.

Ionizing radiation (IR) leads to fibrosing alveolitis (FA) after a lag period of several weeks to months. In a rat model, FA starts at 8 weeks after IR. Before that, at 5.5 weeks after IR, the transcription factors Sp1 (stimulating protein 1) and AP-1 (activator protein 1) are inactivated. To find genes/proteins that were down-regulated at that time, differentially expressed genes were identified in a subtractive cDNA library and verified by quantitative RT-PCR (reverse transcriptase polymerase chain reaction), western blotting and immunohistochemistry (IH). The mRNA of the molecular chaperone HSP90AB1 (heat shock protein 90 kDa alpha, class B member 1) was down-regulated 5.5 weeks after IR. Later, when FA manifested, HSP90ab1 protein was down-regulated by more than 90% in lung cells with the exception of mast cells. In most mast cells of the normal lung, both HSP90ab1 and HSP70, another major HSP, show a very low level of expression. HSP70 was massively up-regulated in all mast cells three months after irradiation whereas HSP90AB1 was up-regulated only in a portion of mast cells. The strong changes in the expression of central molecular chaperones may contribute to the well-known disturbance of cellular functions in radiation-damaged lung tissue. (J Histochem Cytochem 62:355–368, 2014)

L'érythropoïèse inefficace joue un rôle central dans la physiopathologie de l'anémie des β-TM. Ses caractéristiques sont triple: accélération de la différenciation érythroïde, arrêt de maturation au stade d'érythroblaste polychromatophile et mort par apoptose à ce stade de différenciation. Les mécanismes précis de cette apoptose et de l'arrêt de la maturation n'ont pas encore été élucidés. Il a été montré, au cours de l'érythropoïèse physiologique, que la protéine chaperonne Hsp70, en se localisant dans le noyau des érythroblastes en cours de différenciation, protège GATA-1 (facteur de transcription érythroïde majeur) de sa destruction par la caspase-3. Cette enzyme clé de l'apoptose est en effet activée physiologiquement au cours de la différenciation érythroïde et peut cliver GATA-1. Notre travail se base sur l'hypothèse suivante : Hsp70 pourrait, au cours de l'érythropoïèse des β-TM, être séquestrée dans le cytoplasme des érythroblastes matures (stade d'une intense hémoglobinisation) afin d'exercer son rôle de chaperonne des chaînes d'-globine libres. Cela aurait comme conséquence néfaste l'absence de localisation nucléaire d'Hsp70 et, en conséquence, la destruction de GATA-1 à l'origine de l'arrêt de maturation et de la mort cellulaire. Nous avons montré dans ce travail qu'Hsp70 était localisée principalement dans le cytoplasme des érythroblastes matures dans la moelle de patients β-TM, avec un défaut d'expression nucléaire. Par ailleurs, GATA-1 n'est plus exprimé dans ces cellules. Nous avons confirmé ces résultats dans un système de culture cellulaire érythroïde humaine en milieu liquide reproduisant les étapes de la différenciation érythroïde terminale. Une intéraction physique directe entre Hsp70 et l'-globine a été identifiée par techniques de microscopie confocale, d'immunoprécipitation et de double hybride. Enfin, la transduction dans les érythroblastes de β-TM d'un mutant d'Hsp70-S400A, principalement nucléaire, ou d'un mutant de GATA-1 non clivable par la caspase-3 corrige l'érythropoïèse inefficace.Une modélisation mathématique du complexe Hsp70/-globine nous a permis de préciser les domaines impliqués dans l'intéraction, ce qui ouvre la voie à une possibilité de criblage de petites molécules permettant la rupture de ce complexe afin de ramener Hsp70 dans le noyau avec un espoir thérapeutique pour améliorer l'érythropoïèse inefficace des β-TM.

Protein folding is the process in which polypeptides in their non-native states attain the unique folds of their native states. Adverse environmental conditions and genetic predisposition challenge the folding process and accelerate the production of proteotoxic misfolded proteins. Misfolded proteins are selectively recognized and removed from the cell by processes of protein quality control (PQC). In PQC molecular chaperones of the Heat shock protein 70 kDa (Hsp70) family play important roles by recognizing and facilitating the removal of misfolded proteins. Hsp70 function is dependent on cofactors that regulate the intrinsic ATPase activity of the chaperone. In this thesis I have used yeast genetic, cell biological and biochemical experiments to gain insight into the regulation of Hsp70 function in PQC by nucleotide-exchange factors (NEFs). Study I shows that the NEF Fes1 is a key factor essential for cytosolic PQC. A reverse genetics approach demonstrated that Fes1 NEF activity is required for the degradation of misfolded proteins associated with Hsp70 by the ubiquitin-proteasome system. Specifically, Fes1 association with Hsp70-substrate complexes promotes interaction of the substrate with downstream ubiquitin E3 ligase Ubr1. The consequences of genetic removal of FES1 (fes1Δ) are the failure to degrade misfolded proteins, the accumulation of protein aggregates and constitutive induction of the heat-shock response. Taken the experimental data together, Fes1 targets misfolded proteins for degradation by releasing them from Hsp70. Study II describes an unusual example of alternative splicing of FES1 transcripts that leads to the expression of the two alternative splice isoforms Fes1S and Fes1L. Both isoforms are functional NEFs but localize to different compartments. Fes1S is localized to the cytosol and is required for the efficient degradation of Hsp70-associated misfolded proteins. In contrast, Fes1L is targeted to the nucleus and represents the first identified nuclear NEF in yeast. The identification of distinctly localized Fes1 isoforms have implications for the understanding of the mechanisms underlying nucleo-cytoplasmic PQC. Study III reports on the mechanism that Fes1 employs to regulate Hsp70 function. Specifically Fes1 carries an N-terminal domain (NTD) that is conserved throughout the fungal kingdom. The NTD is flexible, modular and is required for the cellular function of Fes1. Importantly, the NTD forms ATP-sensitive complexes with Hsp70 suggesting that it competes substrates of the chaperone during Fes1-Hsp70 interactions. Study IV reports on methodological development for the efficient assembly of bacterial protein-expression plasmids using yeast homologous recombination cloning and the novel vector pSUMO-YHRC. The findings support the notion that Fes1 plays a key role in determining the fate of Hsp70-associated misfolded substrates and thereby target them for proteasomal degradation. From a broader perspective, the findings provide information essential to develop models that describe how Hsp70 function is regulated by different NEFs to participate in protein folding and degradation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>

The purpose of this study was to establish whether curcumin protects renal proximal tubule cells against ischemic injury, determine whether this postulated cytoprotective effect is mediated through the upregulation of HSP70, and investigate whether the mechanism by which curcumin induces HSP70 expression and confers its protective effect is through activation of the Unfolded Protein Response. LLC-PK1 cells were cultured on collagen-coated filters to mimic conditions of in vivo renal proximal tubule cells and induce cell polarization. Injury with and without curcumin treatment was studied by using chemically-induced ATP-depletion which mimics renal ischemic injury. Cell injury was assessed using a TUNEL assay in order to evaluate DNA cleavage associated with ischemia-induced apoptosis and actin staining used to assess cytoskeletal disruption. Renal ischemic damage was further investigated by determining detachment of the Na-K ATPase from the basolateral membrane, which represents loss of cell polarity. Cells were incubated with curcumin in a dose- and time-response fashion and subsequent levels of HSP70 expression were assessed. Cells were then incubated with AEBSF, an inhibitor of the Unfolded Protein Response (UPR) and HSP70 and BiP/GRP78 (an ER resident chaperone that is upregulated by the UPR) expression levels were evaluated. Results demonstrated that treatment with curcumin during two hours of injury results in significantly less injury-related apoptosis and cytoskeletal disruption compared to control injured cells. It was demonstrated that curcumin induces HSP70 in both a dose- and time-response fashion. Moreover, curcumin treatment resulted in profound stabilization of Na-K ATPase on the basolateral membranes as there was significantly less Na-K ATPase detachment in cells treated with curcumin during two hours of injury compared to control injured cells. Finally, treatment with AEBSF inhibited HSP70 upregulation in curcumin-treated cells as well as inhibiting the GRP78 over-expression otherwise demonstrated in curcumin-treated cells. Protection of proximal tubule cells against renal ischemic injury by curcumin was therefore indicated to be mediated by the activation of the UPR through which HSP70 is upregulated. Curcumins activation of the UPR and induction of HSP70 explains the stabilization of Na-K ATPase on the cytoskeleton and also provides a potential mechanism explaining many of curcumins therapeutic and protective qualities.

PhD (Biochemistry) / Department of Biochemistry / The main malaria agent, Plasmodium falciparum expresses an Hsp70 (PfHsp70-1) which plays a significant role in parasite survival. PfHsp70-1 is distinct in that it possesses glycine-glycine-methionine-proline (GGMP) tetrapeptide repeats in its C-terminal domain. To date, the GGMP motif of PfHsp70-1 has not been studied. The motif is positioned within the C-terminal lid segment of PfHsp70-1. The motif is also about seven residues upstream the terminal EEVD residues that are responsible for the interaction of PfHsp70-1 with its functional regulators (co-chaperones). P. falciparum Hsp70/Hsp90 organizing protein (PfHop) constitutes one of the functional regulators of PfHsp70-1. PfHop allows PfHsp70-1 and its chaperone partner, PfHsp90 to form a functional partnership. Given the proximity of the GGMP repeats to the C-terminus of PfHsp70-1, it was postulated in this study that the GGMP repeat residues may regulate attachment of PfHop to PfHsp70-1. Hence, this study hypothesized that the GGMP repeat motif is important for the interaction between PfHop and PfHsp70-1 as well as the chaperone activity of PfHsp70-1. Two variants in which the N-terminal and the C-terminal GGMP repeats were conservatively substituted were generated. E. coli Hsp70 (DnaK) lacks a GGMP motif. Thus, the GGMP motif of PfHsp70-1 was introduced into E. coli DnaK in order to generate a third GGMP variant. Recombinant forms of PfHsp70-1, DnaK, and their GGMP variants were heterologously expressed in E. coli XL1 Blue cells. The proteins were purified to homogeneity by using a combination of Ni-NTA affinity chromatography, ion exchange, and size exclusion chromatography. Purified proteins were then biophysically characterized using CD spectroscopy and tryptophan fluorescence. Findings from this study revealed that there were minimal secondary structural differences between PfHsp70-1, DnaK and their GGMP variants. In order to investigate the chaperone function of PfHsp70-1, DnaK and the GGMP variants, a complementation assay in E. coli dnak756 cells whose Hsp70 is functionally compromised was conducted. The PfHsp70-1 GGMP variants were able to suppress the thermosensitivity of the E. coli cells. However, the Investigation of the role of GGMP motif of Plasmodium falciparum Hsp70-1 on the chaperone function of the protein and its interaction with a co-chaperone, PfHop ii DnaK-G variant failed to confer cytoprotection to the E. coli dnak756 cells. To further validate the findings from the complementation assay, the ability of the recombinant proteins to suppress aggregation of heat stressed Malate dehydrogenase (MDH) was elucidated. PfHsp70-1 had better MDH aggregation suppression capabilities than its GGMP variants. Overall, findings from the MDH aggregation suppression assay suggest that the GGMP repeats may contribute towards substrate binding. Substrate binding might be dependent on the specific positioning of a particular repeat in the GGMP motif of PfHsp70-1. Furthermore, the ATPase activity of PfHsp70-G632 and PfHsp70-G648 was significantly reduced compared to PfHsp70-1 (wild type). However, PfHsp70-G632 had the lowest ATPase activity. Interestingly, the ATPase activity of PfHsp70-G632 was enhanced in the presence of synthetic Hsp70 model peptide substrates. Slot blot and ELISA approaches confirmed that the GGMP mutations partially abrogated the interaction of PfHsp70-1 with PfHop. Altogether, the findings suggest that the GGMP motif of PfHsp70-1 has marginal effects on the structure of PfHsp70-1. In conclusion, this study provides the first direct evidence that the GGMP motif is important for the chaperone function of PfHsp70-1 as well as its interaction with PfHop. / NRF

Nutrient and growth factor receptors internalize through dathrin coated pits. The signal sequences which mediate the association between receptors and the coated pit reside in receptor cytoplasmic tail domains. These signal sequences have been extensively investigated in nutrient receptors, and a minimal functional sequence has been identified consisting of a tyrosine residue in an exposed b turn. Protein-protein contacts between internalization signal sequences and components of the coated pit machinery have been proposed to mediate rapid internalization. In vitro evidence suggests the AP-2 adaptor may be that protein component. The signal sequences of growth factor receptors are less well understood. However, a growth factor- and temperature- dependent binding between the epideimal growth factor receptor and the AP-2 adaptor has been observed. We identified Hsc70 as a cytosolic ligand for the cytoplasmic tail of the transferrin receptor. The binding was mapped to the internalization signal sequence of the receptor tail. Mutations within the signal sequence which inhibit internalization result in alteration of signal sequence secondary structure and reduction in stimulation of the Hsc70 ATPase. Co-immunoprecipitation analysis showed a population of transferrin receptors which are bound to Hsc70, suggesting an association in vivo. We also showed binding of Hsc70 to the epidermal growth factor receptor by co-immunoprecipitation analysis. This binding was increased by treatment with EGF. The binding was transient, and occured prior to the binding of the receptor to AP-2 adaptors. Other agents which induce EGF receptor clustering and internalization also stimulate the transient increase in Hsc70 binding and the later AP-2 binding, suggesting a role in early endocytosis. These data support the hypothesis that Hsc70 is associated with the receptors for transferrin and epidermal growth factor in vitro and in vivo. We propose a role for the 70 kD heat shock protein in the assembly/disassembly of protein complexes involved in receptor signalling and/or internalization.

No description available.