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HIF1α et HIF2α sont des protéines largement impliquées dans le développement de pathologies posant des problèmes majeurs de santé publique, comme le cancer. Leur activité, qui est régulée prioritairement par leur stabilité via le système ubiquitine-protéasome, coordonne de nombreux processus cellulaires susceptibles de favoriser le développement de ces maladies. Un enjeu récent de la recherche thérapeutique est d’identifier des partenaires protéiques pouvant réguler les protéines HIFα, afin de mettre au point des thérapies ciblées. Les protéines de choc thermique (HSPs) sont une classe de protéines dont une des fonctions essentielles est de réguler l’homéostasie protéique dans la cellule, en interagissant avec le protéasome. Certaines d’entre elles, HSP27 et HSP90, ont la faculté de pouvoir réguler spécifiquement la stabilité de nombreuses protéines souvent elles-mêmes impliquées dans l’apparition de ces pathologies. L’objectif de ce travail était de savoir si ces deux HSPs peuvent contrôler la stabilité de la protéine HIF2α. Nos résultats suggèrent qu’HSP27 pourrait stimuler la dégradation de HIF2α en favorisant son ubiquitination. Ce résultat est surprenant, en raison du rôle connu d’HSP27 dans la progression tumorale. Il est donc nécessaire de le confirmer et d’en préciser les processus biologiques sous-jacents. D’autre part, nos autres résultats semblent confirmer qu’HIF2α est une protéine cliente d’HSP90. De plus, nous montrons pour la première fois que l’inhibition d’HSP90 par le 17-DMAG diminue la production de VEGF dépendante de HIF2α. Des travaux récents suggèrent qu’HIF2α a un rôle prédominant dans la progression tumorale, et peut constituer une cible globale de choix dans plusieurs types de cancer. Il conviendrait d’évaluer la capacité des inhibiteurs d’HSP90 à supprimer des fonctions de HIF2α nouvellement décrites, comme son rôle dans la maintenance des cellules souches cancéreuses. / Both HIF1α and HIF2α proteins are highly involved in the development of pathologies, such as cancers, which are prime public health issues. These proteins are primarily controlled at the protein level by ubiquitin-dependant degradation, and regulate numerous cellular processes which are likely to favor the development of these diseases. A recent issue in therapeutic research is to identify partners that might regulate the expression and the activity of the HIFα proteins, with the aim to elaborate targeted therapies. Heat shock proteins (HSPs) form a family of proteins whose main function is to regulate protein homeostasis in cells, which they achieve through interaction with the ubiquitin-proteasome pathway. HSP27 and HSP90 are able to specifically control the stability of certain client proteins involved in those pathologies. In the present work, we sought to determine whether these HSPs could regulate the expression of the HIF2α protein. Our results suggest that HSP27 may induce ubiquitin and proteasome-dependant degradation of HIF2α, which is quite intriguing given the well-known role of HSP27 in tumor promotion. This needs to be confirmed and the underlying biological significance of such a regulation remains to be defined. Our results also may confirm that HIF2α is an HSP90 client protein. Moreover, we show for the first time that inhibition of HSP90 by 17-DMAG decreases the HIF2α-dependant VEGF production. Recent studies emphasize HIF2α as a major promoter of tumor progression, and suggest that HIF2α may constitute an attractive global target in several cancer types. Therefore, the ability of HSP90 inhibitors to disrupt newly described HIF2α functions, such as cancer stem cell maintenance, should be evaluated.

Her2-positiver Brustkrebs, der Subtyp des Mammakarzinoms, bei dem eine Überexpression des epidermalen Wachstumsfaktor-Rezeptors-2 (ErbB2/Her2) vorliegt, hat für die betroffenen Frauen eine besonders schlechte Prognose. Eine positive Korrelation zwischen der ErbB2-Expression in Brusttumoren und der Expression des Makrophagen-Migration-inhibierenden Faktors (MIF), einem inzwischen gut bekannten tumorfördernden Protein, konnte bereits gezeigt werden. Ferner konnte gezeigt werden, dass MIF durch das Hitzeschockprotein 90 (Hsp90) in einem Mausmodell des Her2-positiven Brustkrebses stabilisiert wird. Der zugrundeliegende Mechanismus war bisher unverstanden. In dieser Doktorarbeit konnte erstmalig demonstriert werden, dass Hitzeschockfaktor-1 (HSF-1), der Transkriptionsfaktor der stressinduzierten Hitzeschockproteine (HSP), einschließlich Hsp90, in ErbB2-überexprimierenden SK-BR-3-Brustkrebszellen konstitutiv durch ErbB2 aktiviert wird. Durch eine Behandlung mit dem ErbB2-Inhibitor CP724.714 konnte die aktivierende Serin326-Phosphorylierung von HSF-1 verhindert werden. Als Folge wird, wie durch Western-Blot-Analysen gezeigt, die HSP-Maschinerie inhibiert und tumorfördernde Hsp90-Klienten wie MIF, Akt, mutiertes p53, ErbB2 und HSF-1 destabilisiert. Außerdem konnte die unterbleibende HSF-1-Aktivierung durch quantitative PCR-Analysen und Immunfluoreszenzmikroskopie bestätigt werden. Die mechanistischen Untersuchungen konnten die hier erstmalig beschriebene ErbB2-Akt-HSF-1-Achse aufdecken, über die HSF-1 in SK-BR-3-Zellen reguliert wird. Ferner konnte eine ErbB2-Inhibition sogar die HSF-1-Aktivierung durch einen Hitzeschock unterbinden. Unsere Ergebnisse zeigen zum ersten Mal, dass die ErbB-2-Überexpression in SK-BR-3-Zellen eine konstitutive HSF-1-Aktivierung bewirkt, mit der Folge, dass tumorfördernde Hsp90-Klienten wie MIF, HSF-1 selbst, ErbB2 und mutiertes p53 stabilisiert werden. Die neu entdeckte ErbB2-Akt-HSF-1-Hsp90-Klienten-Achse legt möglicherweise neue Angriffspunkte für zusätzliche Pharmaka bei der Therapie Her2-positiven Brustkrebses offen.

<p>Apoptosis is a cellular suicide program that can be initiated by various genotoxic and cytotoxic stimuli. In many cases, such cell damaging agents promote cell death through the intrinsic apoptotic pathway by triggering mitochondrial cytochrome <italic>c</italic> release and subsequent caspase activation. Cytosolic cytochrome <italic>c</italic> is directly responsible for initiating formation of the caspase-activating apoptosome, which plays a crucial role in the apoptotic process. Given the importance of cellular fate, apoptosis is tightly controlled by a balance between survival and death signals. It has been shown that activated cell survival pathways, including the mitogen-activated protein kinase (MAPK) cascade and the PI3K/Akt signaling, enhance cell viability by conferring resistance to apoptotic cell death. However, the underlying mechanism(s) that lead to inhibition of functional apoptosome formation (and caspase activation) has yet to be elucidated. In the studies that are described in this dissertation, I have investigated the regulation of apoptosis downstream of mitochondrial cytochrome <italic>c</italic> release with the goal of understanding how survival signaling can alter the apoptotic program, contributing to human malignancies. </p><p>First, we describe a mechanism for the inhibition of cytochrome <italic>c</italic>-induced caspase activation by MAPK signaling, identifying a novel mode of apoptotic regulation exerted through Apaf-1 phosphorylation by the 90-kDa ribosomal S6 kinase (Rsk). We have found that recruitment of 14-3-3&epsilon; to phosphorylated Ser268 impedes the ability of cytochrome <italic>c</italic> to nucleate apoptosome formation and activate downstream caspases. High endogenous levels of Rsk in PC3 prostate cancer cells or Rsk activation in other cell types promoted 14-3-3&epsilon; binding to Apaf-1 and rendered the cells insensitive to cytochrome <italic>c</italic>, suggesting a role for Rsk signaling in apoptotic resistance of prostate cancers and other cancers with elevated Rsk activity. These results identify a novel locus of apoptosomal regulation wherein MAPK signaling promotes Rsk-catalyzed Apaf-1 phosphorylation and consequent binding of 14-3-3&epsilon;, resulting in decreased cellular responsiveness to cytochrome <italic>c</italic>. </p><p>In the second part, we examine how apoptosis is inhibited by oncogenic tyrosine kinase signaling by using leukemogenic tyrosine kinase-induced leukemia model systems. We have demonstrated that protein phosphatase 5 (PP5) is responsible for Hsp90&beta; hypophosphorylation, which can contribute to impaired cell death in leukemia expressing oncogenic tyrosine kinases. Loss of PP5 results in an increase of Hsp90&beta; phosphorylation, raising leukemic cells' responsiveness to imatinib, a BCR-ABL kinase inhibitor. Further we have discovered that acetylation regulates PP5 activity on Hsp90&beta;. Mutational study showed that K144 acetylation on PP5, which was diminished in leukemic conditions, inhibited PP5 binding to Hsp90&beta;, causing Hsp90&beta; hyperphosphorylation and subsequently potentiating cells to apoptosis. These studies reveal a molecular mechanism by which agents enhancing PP5 acetylation may be a potential treatment for leukemias. Collectively, this work provides new insight into mechanisms of regulation of apoptosome formation/function, helping us understand how the evasion of apoptotic cell death contributes to cancer cell survival. Further, this finding implicates cytochrome <italic>c</italic>-induced apoptotic signaling in the context of cancer cell responsiveness to chemotherapeutic treatments.</p> / Dissertation

Microtubular cytoskeleton is involved in many processes in plant cells, including cell division, growth and development. Other proteins enable its functions by modulation of its dynamics and organization and by mediation of functional and structural interaction with other cell structures. Identification of the mediating proteins and the functions of these interactions under specific conditions were the main aims of the thesis. Membrane proteins interacting with microtubules were identified using biochemical methods. Surprisingly, the identified proteins co-sedimenting with microtubules were not members of the "classical" microtubule associated proteins (MAPs). There were enzymes, chaperones and plant specific proteins among them. For further studies, the identified microtubule-associated heat-shock protein 90 (Hsp90_MT) was chosen. Recombinant Hsp90_MT binds directly to microtubules and tubulin dimers in vitro. The ATP-binding pocket is not responsible for this association. In BY-2, Hsp90_MT co-localizes with phragmoplast and cortical microtubules and is involved in microtubule recovery after their depolymerization during cold treatment. In plants, Hsp90 is involved in cell cycle progression, its inhibition causes cell-cycle arrest in G1 phase. Based on literature search for animal proteins...

Systemic scleroderma (SSc) is a systemic connective tissue disease affecting skin and internal organs. The pathogenesis of SSc is characterized by inflammation, vasculopathy and fibrosis. No agent has been proven effective in the treatment of SSc. There is a lack of suitable biomarkers for monitoring the disease activity or the response to the treatment of SSc. Therefore our aim was to analyse the extracellular levels of S100A4, Hsp90 (Heat shock protein 90) and IL-35 (interleukin-35) in SSc. S100A4 and Hsp90 have been initially studied in tumours; in some of them considered as suitable prognostic markers and candidates for future therapies. We have recently described the profibrotic role of S100A4 and Hsp90 in the pathogenesis of SSc. Our results showed that inactivation of S100A4 and Hsp90 effectively prevented the development of experimental skin fibrosis. This was consequently confirmed by the analysis of S100A4 and Hsp90 in the peripheral blood of patients with SSc, where significant associations with disease activity and organ involvement were detected. IL-35 may become another potential biomarker of SSc. We detected increased expression of IL-35 in the affected skin, dermal fibroblasts and in serum of patients with SSc. Moreover, the main profibrotic mediator transforming growth factor...

Faculty of Science School of Anatomical Sciences 9901061h karrenthomson@yahoo.co.uk / Clomiphene citrate (CC), a synthetic estrogen, is an efficient superovulator used in infertility treatment. However pregnancy rates resulting from CC treatment are low. Research has suggested that this may be due to an aberrant effect on implantation; CC binds to estrogen receptors (ER) and may affect estrogen responsive gene expression and thus implantation. This study investigates the effect of CC on ERa, 90kDa heat shock protein (Hsp90) and Hoxa10 expression in the rat uterus. Hsp90 binds to ERa in the absence of ligand and is involved in inducing a high affinity ligand binding conformation in the ER and in transactivation of the ER. Hoxa10 has been shown to be essential for uterine receptivity to implantation. CC (0.25mg) was given to ovariectomized rats, either alone or prior to a hormonal regime known to induce uterine receptivity for implantation. Expression of ERa, Hsp90 and Hoxa10 was determined by Western blotting, fluorescence immunocytochemistry and reverse transcription polymerase chain reaction. The single dose CC treated rats were compared to the controls as well as to ovariectomized rats treated with 0.5mg 17b estradiol (E2). The CC treated pseudopregnant rats (CCPPPE treated) were compared to 5½ day pregnant and pseudopregnant rats without CC (PPPE treated), to determine CCs effect at implantation. E2 upregulated ERa and Hsp90 expression in the rat uterus compared to controls (p<0.05). The finding for ERa was unexpected as other studies have shown that E2 decreases ERa levels a few hours after administration in the uterus. The present study therefore suggests a biphasic effect of E2 on ERa expression in the rat uterus. The effect of E2 on Hsp90 and ERa also proposes a balance between the levels of these two proteins in the uterus, to keep ERa in its optimal state and suggests that too high and too low a concentration of Hsp90 may both be inhibitory to ERa functioning. No significant difference was found in ERa and Hsp90 expression between the non-receptive (vehicle treated) and the receptive (PPPE treated) rat uteri, suggesting that these two genes are not markers for receptivity. However E2 is known to induce implantation of donor blastocysts in progesterone (P4) primed uteri. Therefore it is still essential for ERa to be present at implantation. It is of interest that CC downregulated ERa levels both in ii the absence of ovarian hormones and at implantation in the rat uterus. It is therefore proposed that this antiestrogenic effect would render the uterus less sensitive to the E2 required to induce implantation, thus accounting for low pregnancy rates with CC use. Although CC did not alter the expression of Hsp90 in this study, the reduction in ERa levels in response to CC may also upset the balance in the expression of these two genes, which may affect the transcriptional activity of ERa, and further prevent implantation. No clear results were obtained for Hoxa10 expression with the Western blots. However based on the ICC results, CC did not appear to affect Hoxa10 expression. Since P4 and not E2 is known to have the predominant effect on Hoxa10 expression, it is likely that E2 analogs, such as CC, would also not affect Hoxa10 expression to a significant degree. Future work will aim to separate the different uterine compartments and to determine the effects of CC on the expression of other implantation specific genes in the uterus.

<p>GRP94, the endoplasmic reticulum Hsp90, is a metazoan-restricted chaperone essential for early development in mammals, yet dispensable for mammalian cell viability. These data suggest that GRP94 is required for important developmental processes relying on cell-cell communication and cell-cell interaction. Consistent with this hypothesis, loss of GRP94 expression in mouse is embryonic lethal yet tissue culture cells expressing no GRP94 are viable. To date, functional studies of GRP94 have relied on cell-autonomous model systems, the use of which has lead to discoveries of proteins that GRP94 chaperones also called client proteins. These systems give limited insight into the essential role(s) played by GRP94 in metazoan biology. The dichotomy that GRP94 is necessary for metazoan life, but dispensable for cellular viability suggests that the chaperone is required for the functional expression of secretory and/or membrane proteins that enable cells to function in the context of tissues.</p><p>To explore this hypothesis, the <italic>Drosophila</italic> ortholog of <italic>GRP94</italic>, <italic>Gp93</italic>, was identified and <italic>Gp93</italic> mutants were created using imprecise P-element excision. <italic>Gp93</italic> was found to be an essential gene in <italic>Drosophila</italic>. Loss of zygotic <italic>Gp93</italic> expression is late larval lethal and causes prominent defects in the larval midgut, the sole endoderm-derived larval tissue. <italic>Gp93</italic> mutant larvae display pronounced defects in the midgut epithelium, with aberrant copper cell structure, markedly reduced gut acidification, atypical septate junction structure, depressed gut motility, and deficits in intestinal nutrient uptake. The metabolic consequences of the loss of <italic>Gp93</italic>-expression are profound; <italic>Gp93</italic> mutant larvae exhibit a starvation-like metabolic phenotype, including suppression of insulin signaling and extensive mobilization of amino acids and triglycerides. The defects in copper cell structure/function accompanying loss of <italic>Gp93</italic> expression resemble those reported for mutations in <italic>labial</italic>, an endodermal homeotic gene required for copper cell specification, and &alpha;-spectrin, thus suggesting an essential role for Gp93 in the functional expression of secretory/integral membrane protein-encoding labial protein target genes and/or integral membrane protein(s) that interact with the spectrin cytoskeleton.</p><p>The creation of <italic>Gp93</italic> mutant <italic>Drosophila</italic> has allowed for the study of GRP94 function <italic>in vivo</italic> and will be of upmost importance to future studies examining the function of this chaperone in all aspects of metazoan biology. This dissertation focuses on the morphological and physiological defects that accompany loss of <italic>Gp93</italic> expression in <italic>Drosophila</italic> larvae. It will also outline future studies utilizing this model.</p> / Dissertation

Zebrafish, Danio rerio, have become an important model for developmental studies and have several advantages over other model systems. These advantages include (1) the easy accessibility of zebrafish embryos for direct observation of their development and (2) their suitability for systematic mutagenesis studies for the identification of genes regulating the development of various tissues and organs, including the skeletal system. Recently, it has been reported that glutamate receptors are expressed in many types of bone cells and regulate bone physiological functions. In the present study, we have examined the effects of a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist¡Xdextromethorphan¡Xon the development of the axial skeleton in zebrafish embryos by using calcein stain. Our results revealed that dextromethorphan significantly attenuates the formation of the axial skeleton and that it is inhibited on pretreatment with glutamate. Moreover, immunohistochemical analysis revealed protein level expression of the NMDA subunit NR1 in the axial region of zebrafish. Our results also indicate that attenuation of NMDA receptor activity-induced change in the axial skeleton may be related to heat-shock protein and extracellular signal-regulated kinase (ERK) signalings. In conclusion, we suggest that the NMDA receptor plays an important role in the development of the axial skeleton. However, further studies are required on the cellular mechanisms of glutamate regulated bone formation.

Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Human Genetics. Title from title-page of electronic thesis. Bibliography: leaves 166-195.