Background: Nephrotic syndrome (NS) occurs when the glomerular filtration barrier becomes abnormally permeable, leading to the clinical triad of proteinuria, massive oedema, and hypoalbuminaemia. Historically, NS has been thought to result from dysregulation of the immune system, although recent evidence suggests the glomerular podocyte plays a central role in disease pathogenesis. Children with NS are generally treated with an empiric course of glucocorticoid (Gc) therapy; a class of steroids which are activating ligands for the glucocorticoid receptor (GR) transcription factor. A major factor limiting the clinical utility of these agents is the marked variation observed in response to treatment. Although Gc-therapy has been the cornerstone of NS management for several decades, the mechanism of action, and target cell, remain poorly understood. Hypothesis and aims: The central hypothesis for this thesis states that glucocorticoids act directly on the podocyte to produce clinically useful effects without involvement of the immune system. Findings: Using a wild-type human podocyte cell line, I demonstrated that the basic GR-signalling mechanism is intact in the podocyte, and that glucocorticoids produce a direct, protective effect on the podocyte without immune cell involvement, by using electrical resistance across a podocyte monolayer as a surrogate marker for barrier integrity. To understand potential mechanisms underpinning this direct effect I defined the podocyte GR cistrome (using a combination of chromatin immunoprecipitation followed by massively parallel DNA sequencing and transcriptomic analysis) as well as total cell proteomics. Subsequent gene ontology analysis revealed that Gc treatment had prominent effects on podocyte motility, and these findings were validated with live-cell imaging. To gain mechanistic insight, I investigated the role of the pro-migratory small GTPase regulator Rac1, and demonstrated that treatment with Gc reduced Rac1 activity. Furthermore, the Rac1 inhibitor EHT 1864 had a direct, protective effect on the podocyte. To create a model to study the role of podocyte GR in vivo I generated a mouse line with a podocyte-specific GR deletion. Impact: Gc exposure produces potentially clinically-relevant effects directly on the podocyte, and Gc-induced podocyte hypomobility may underlie the clinical efficacy of these agents. Future animal studies investigating the consequences of GR deletion in the podocyte and the anti-proteinuric effects of Rac1 inhibition are warranted.

Aims Angiotensin II (Ang II) stimulates cardiac remodelling and fibrosis in the mechanically overloaded myocardium. Although Rho GTPases regulate several cellular processes, including myocardial remodelling, involvement in mediating mechanical stretch-induced regulation of angiotensinogen (Ao), the precursor to Ang II, remains to be determined. We, therefore, examined the role and associated signalling mechanisms of Rho GTPases (Rac1 and RhoA) in regulation of Ao gene expression in a stretch model of neonatal rat cardiac fibroblasts (CFs). Methods and resultsCFs were plated on deformable stretch membranes. Equiaxial mechanical stretch caused significant activation of both Rac1 and RhoA within 25 min. Rac1 activity returned to control levels after 4 h, whereas RhoA remained at a high level of activity until the end of the stretch period (24 h). Mechanical stretch initially caused a moderate decrease in Ao gene expression, but was significantly increased at 824 h. RhoA had a major role in mediating both the stretch-induced inhibition of Ao at 4 h and the subsequent upregulation of Ao expression at 24 h. β1 integrin receptor blockade by Tac β1 expression impaired acute (2 and 15 min) stretch-induced Rac1 activation, but increased RhoA activity. Molecular experiments revealed that Ao gene expression was inhibited by Rac1 through both JNK-dependent and independent mechanisms, and stimulated by RhoA through a p38-dependent mechanism. Conclusion These results indicate that stretch-induced activation of Rac1 and RhoA differentially regulates Ao gene expression by modulating p38 and JNK activation.

No description available.

No description available.

Les évènements moléculaires en amont et en aval de la petite GTPase Rac1 menant à la migration cellulaire sont encore mal compris. La première partie du projet consiste à utiliser une approche protéomique non-biaisée pour tenter d’identifier les partenaires de Rac. Pour ce faire, nous avons développé une méthode de purification efficace et rapide de manière à maintenir les complexes protéiques transitoires intacts. Dans un deuxième temps, nous avons identifié des sites de phosphorylation sur la RacGEF atypique Dock5 en aval des intégrines. Afin de mieux comprendre le rôle de la phosphorylation de cette protéine, nous avons criblé une banque de kinases ce qui nous a permis d’identifier 14 kinases pouvant phosphoryler la région PXXP de Dock5. D’après nos résultats, ceci aurait comme effet de diminuer l’interaction entre Dock5 et ses partenaires contenant des domaines SH3. Ainsi, la phosphorylation de Dock5 régulerait la formation de complexes et le recrutement de Dock5 par des protéines adaptatrices. / The molecular events upstream and downstream of Rac leading to cell migration and still to date not fully understood allthough more than 20 effectors have been identified for this GTPase. The first part of our project is to use a non-biased proteomic approach to try to identify novel binding partners of Rac1. In order to do so, we developped a novel purification strategy that enabled us to purify Rac and its binding partners in a timely manner. The second part of our project is to understand the role of Dock5 phosphorylation downstream of the integrins. We identified phosphorylated residues in the PXXP region of the atypical RacGEF upon fibronectin stimulation and found 14 kinases able to phosphorylate this region. According to our results, Dock5 phosphorylation does not affect its GEF activity but diminishes its interaction with various SH3 domain-containing proteins. Thus, our data suggest that Dock5 phosphorylation would regulate complex formation and recruitment of this protein by adaptor proteins.

Les évènements moléculaires en amont et en aval de la petite GTPase Rac1 menant à la migration cellulaire sont encore mal compris. La première partie du projet consiste à utiliser une approche protéomique non-biaisée pour tenter d’identifier les partenaires de Rac. Pour ce faire, nous avons développé une méthode de purification efficace et rapide de manière à maintenir les complexes protéiques transitoires intacts. Dans un deuxième temps, nous avons identifié des sites de phosphorylation sur la RacGEF atypique Dock5 en aval des intégrines. Afin de mieux comprendre le rôle de la phosphorylation de cette protéine, nous avons criblé une banque de kinases ce qui nous a permis d’identifier 14 kinases pouvant phosphoryler la région PXXP de Dock5. D’après nos résultats, ceci aurait comme effet de diminuer l’interaction entre Dock5 et ses partenaires contenant des domaines SH3. Ainsi, la phosphorylation de Dock5 régulerait la formation de complexes et le recrutement de Dock5 par des protéines adaptatrices. / The molecular events upstream and downstream of Rac leading to cell migration and still to date not fully understood allthough more than 20 effectors have been identified for this GTPase. The first part of our project is to use a non-biased proteomic approach to try to identify novel binding partners of Rac1. In order to do so, we developped a novel purification strategy that enabled us to purify Rac and its binding partners in a timely manner. The second part of our project is to understand the role of Dock5 phosphorylation downstream of the integrins. We identified phosphorylated residues in the PXXP region of the atypical RacGEF upon fibronectin stimulation and found 14 kinases able to phosphorylate this region. According to our results, Dock5 phosphorylation does not affect its GEF activity but diminishes its interaction with various SH3 domain-containing proteins. Thus, our data suggest that Dock5 phosphorylation would regulate complex formation and recruitment of this protein by adaptor proteins.

La GTPase Rac1 est une protéine de signalisation intracellulaire qui joue notamment un rôle clé dans la prolifération cellulaire. Notre laboratoire a montré que la toxine CNF1, produite par les Escherichia coli pathogènes, catalyse l’activation de Rac1. Nous avons également identifié le rôle de la E3 ubiquitine-ligase HACE1, un suppresseur de tumeur avéré, dans la régulation par ubiquitylation de Rac1 actif. S’il est prouvé que la forme activée de Rac1 est une cible d’HACE1, le mode d’interaction de ces deux protéines reste à définir ainsi que le rôle de ces interactions dans l’infection et le cancer. L’objectif de mon travail a été de caractériser les interactions moléculaires entre HACE1 et Rac1. Nous avons testé l’hypothèse que des mutations ponctuelles d’HACE1 identifiées dans les cancers pourraient interférer avec son interaction avec Rac1 et sa capacité de contrôle de la croissance cellulaire. J’ai ainsi pu mettre en évidence que 13 mutations somatiques d’HACE1 issues de tumeurs séquencées altèrent sa fonction de contrôle de la croissance cellulaire. De plus, l’étude de ces mutations nous a permis d’identifier un groupe d’acides aminés, situés sur les ankyrin-repeats 5 à 7 d’HACE1, qui contrôle l’interaction d’HACE1 avec Rac1 et de ce fait son ubiquitylation. Enfin dans cette étude nous précisons le rôle du domaine intermédiaire d’HACE1 (MID) dans la spécificité d’interaction de la ligase avec la forme active de Rac1. In fine, la caractérisation de mutants d’interaction entre HACE1 et Rac1 ainsi que l’effet de la toxine CNF1 sur cet axe de signalisation doit nous renseigner sur l’importance de cette voie de régulation dans le cancer et l’infection. / The small GTPase Rac1 plays a key role in various intracellular signaling pathways including cell proliferation. Our laboratory has shown that the CNF1 toxin, produced by pathogenic Escherichia coli, catalyzes the activation of Rac1. We also identified the role of the E3 ubiquitin-ligase HACE1, a tumor suppressor, in the regulation by ubiquitylation of active Rac1. If the activated form of Rac1 is proved to be a target of HACE1, the mode of interaction between these two proteins remains to be define as well as the role of these interactions in infection and cancer. The aim of my work was to characterize the molecular interactions between HACE1 and Rac1. We tested the hypothesis that HACE1 point mutations identified in cancers could interfere with its interaction with Rac1 and its ability to control cell growth. We showed that 13 cancer-associated somatic mutations of HACE1, led to a defective control of cell proliferation. Moreover, the study of these mutations allowed us to identify a group of amino acids, located on the ankyrin-repeats 5 to 7 of HACE1, which controls the interaction of HACE1 with Rac1 and thus its ubiquitylation. We also identified a role for the intermediate domain of HACE1 (MID) in conferring the specificity of association of HACE1 to the active form of Rac1. Ultimately, the characterization of interaction mutants between HACE1 and Rac1 as well as the effect of the CNF1 toxin on this signaling axis will give us more insight on this regulatory pathway in cancer and infection.

HACE1 est une E3 ubiquitine ligase qui contrôle notamment l’activité de la petite GTPase Rac1 en catalysant son ubiquitination dégradative. Rac1 contrôle de nombreux processus cellulaires tels que l’adhérence, la migration et la prolifération. Aussi, la perte d’expression d’HACE1 dues à des altérations génétiques ou épigénétiques est associée à des pathologies humaines tels que le cancer, des syndromes neurodégénératifs et des maladies développementales. Pourtant, malgré l’importance de HACE1 en physiopathologie, rien n’est connu sur la régulation post-traductionnelle de son activité. Au cours de ce travail, nous avons montré que la serine 385 de HACE1 est phosphorylée par les kinases PAKs de groupe I en réponse à l’activation de Rac1 et de Cdc42. Nous montrons que le mutant phospho-mimetic HACE1(S385E) présente une activité réduite d’ubiquitination de Rac1. De plus, nous mettons en évidence un rôle centrale de la régulation de la Ser-385 par phosphorylation dans l’oligomérisation de HACE1, définissant ainsi les bases moléculaires de la relation entre structure et fonction de HACE1. En parallèle, nous avons déterminé que la perte d’expression d’HACE1 altère la cohésion des jonctions entre cellules épithéliales. Cet effet de dissociation s’apparente à une transition épithelio-mésenchymateuse (EMT) caractérisée par un échange d’expression de la E-cadhérine par la N-cadhérine régulé au niveau transcriptionnel. L’ensemble de ce travail a donc permis de mettre en évidence un mode inédit de régulation par phosphorylation de l’activité de HACE1 contrôlée par les kinases PAK du groupe I, ainsi qu’un rôle majeur de HACE1 dans la régulation de la cohésion cellulaire et l’EMT. / The E3 ubiquitin ligase HACE1 is a key regulator of cellular homeostasis best-characterized for its ability to control the activity of the Rho GTPase Rac1. This GTPase is encoded by an essential gene whose product controls a wide array of cellular processes such as cell adhesion, migration and proliferation. Accordingly, the repression of HACE1 expression due to genetic and epigenetic alterations has been associated with numerous pathologies, including cancer, neurodegenerative and developmental diseases. However, nothing is known about the posttranslational regulation of HACE1 activity. Here, we unveiled that HACE1 gets phosphorylated at serine Ser-385 by Group-I Pak kinases in response to Rac1/Cdc42 activation. Mechanistically, we define that the phospho-mimetic mutant HACE1(S385E) displays a lower capacity to ubiquitinate Rac1 in cells. In addition, our work attributes to the phosphorylation of Ser-385 a pivotal role in the state of HACE1 oligomerization, which sets the basis for deciphering the relationship between HACE1 structure and activity. In parallel, we have found that the loss of HACE1 expression leads to the disruption of epithelial monolayer cohesion characterized by disrupted of cell-cell junctions. Accordingly, we determined that loss of HACE1 results in the acquisition of epithelial-mesenchymal transition (EMT) features, including a transcriptionally regulated switch of expression between E-cadherin and N-cadherin. Altogether, this work reveals a phospho-mediated regulation of HACE1 activity that is under the control of Group I PAKs and implicates HACE1 in the balance between epithelium integrity versus EMT.

Des produits efficaces pour stimuler le système immunitaire auraient de très nombreuses applications dans le domaine de la santé publique : lutte contre la résistance aux antibiotiques, épidémies, protection des personnels de secours et des populations en cas de crise sanitaire ou d’attentat biologique. Aujourd’hui, les rares produits immunostimulants efficaces sont coûteux, dangereux, et donc réservés au traitement de pathologies graves ciblées comme les cancers, aplasies ou infections virales chroniques. La petite protéine G Rac1 fut identifiée comme une potentielle cible thérapeutique. Activée par de nombreux pathogènes, elle contrôle l’inflammation et la mise en place des défenses de l’hôte. CNF1 est une toxine bactérienne activant fortement Rac1, ce qui conduit à sa dégradation protéasomale. Le maintien d’un pool de Rac1 activé permettrait d’augmenter les défenses immunitaires. En utilisant CNF1 comme un outil permettant de diminuer celui-ci, un test cellulaire fut mis au point pour cribler 17 680 composés. A l’aide de différents paramètres de tri, un ensemble de molécules fut identifié comme capable de prévenir la dégradation de Rac1 médiée par CNF1.De manière inattendue, la plupart des composés semblent posséder un effet anti-inflammatoire, en baissant la sécrétion de cytokines de cellules stimulées. Le potentiel thérapeutique et en recherche de tels composés doit être évalué. En parallèle, deux autres composés montrent une protection anti-toxines large spectre (CNF1, toxine diphtérique, toxine de Shiga, toxine B de C. difficile). De même, leur mécanisme d’action, encore inconnu, pourrait permettre le traitement d’infections variées. / Immune system boosters could have many applications in public health: fighting antibiotics resistance, epidemics, protection of health care staff and populations during health crisis or biological warfare. Currently, the rare efficient immune stimulants are costly and dangerous; hence, they are earmarked to severe and targeted pathologies such as cancers, aplasia, or chronic viral infections. The small G protein Rac1 was identified as a potential therapeutic target. Once activated by various pathogens, it controls inflammation and host defenses establishment. CNF1 is a bacterial toxin that strongly activating Rac1, leading to its proteasomal degradation. Maintaining an activated Rac1 pool could enhance immune defenses. By using CNF1 as a tool to reduce it, a cellular bioassay was developed and optimized to screen 17 680 compounds. Through various filters, a group of molecules was identified to prevent CNF1-mediated Rac1 depletion. Unexpectedly, most of them seems to possess anti-inflammatory properties, down-regulating cytokines production from stimulated cells. The therapeutic potential of such compounds must be now evaluated. In parallel, two other molecules show a broad-spectrum anti-toxins protection (CNF1, diphtheria toxin, Shiga toxin, toxin B from C. difficile). Their unknown mode of action may allow the treatment of various infections.

<p>The endothelial receptor tyrosine kinase (RTK) Tie1 was discovered over 20 years ago, yet its precise function and mode of action remain enigmatic. To shed light on Tie1’s role in endothelial cell biology, we investigated a potential threonine phosphorylation site within the juxtamembrane domain of Tie1. Expression of a non-phosphorylatable mutant of this site (T794A) in zebrafish (Danio rerio) significantly disrupted vascular development, resulting in fish with stunted and poorly branched intersomitic vessels. Similarly, T794A-expressing human umbilical vein endothelial cells formed significantly shorter tubes with fewer branches in three-dimensional Matrigel cultures. However, mutation of T794 did not alter Tie1 or Tie2 tyrosine phosphorylation or downstream signaling in any detectable way, suggesting that T794 phosphorylation may regulate a Tie1 function independent of its activity as a kinase. Although T794 is within a consensus Akt phosphorylation site, we were unable to identify a physiological activator of Akt that could induce T794 phosphorylation, suggesting that Akt is not the physiological Tie1-T794 kinase. However, the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for angiogenesis and capillary morphogenesis, was found to associate with phospho-T794 but not the non-phosphorylatable T794A mutant. Pharmacological activation of Rac1 induced downstream activation of p21-activated kinase (PAK1) and T794 phosphorylation in vitro, and inhibition of PAK1 abrogated T794 phosphorylation. Our results provide the first demonstration of a signaling pathway mediated by Tie1 in endothelial cells, and they suggest that a novel feedback loop involving Rac1/PAK1-mediated phosphorylation of Tie1 on T794 is required for proper angiogenesis.</p> / Dissertation