Inheritance of one mutant von Hippel-Lindau (VHL) allele gives rise to the development of the autosomal dominant VHL disease, which affects approximately 1 in 36 000 individuals. The VHL tumour suppressor protein plays a critical role in the E3 ubiquitin ligase-mediated destruction of hypoxia-inducible factor (HIF) and the promotion of fibronectin extracellular matrix assembly. A failure in either process is associated with oncogenic progression. Work included in this thesis provides evidence that these tumour suppressor functions are mutually exclusive. Additionally, post-translational modification of VHL by NEDD8 is shown to act as a ‘molecular switch’, altering VHL protein associations and providing a mechanism of pathway segregation. As a result of HIF stabilization, the expression of a homophilic adhesion molecule E-cadherin is significantly down-regulated in primary renal clear-cell carcinoma (RCC) upon VHL loss. E-cadherin down-regulation is shown to increase the invasive potential and is of prognostic value in RCC. Finally, VHL and SOCS1 are shown to dimerize and negatively regulate the JAK2-STAT5 signalling cascade. Defects in this dimerization are shown to underlie Chuvash polycythemia and provide a molecular understanding of the phenotypic observations associated with VHL-related polycythemias.

Inheritance of one mutant von Hippel-Lindau (VHL) allele gives rise to the development of the autosomal dominant VHL disease, which affects approximately 1 in 36 000 individuals. The VHL tumour suppressor protein plays a critical role in the E3 ubiquitin ligase-mediated destruction of hypoxia-inducible factor (HIF) and the promotion of fibronectin extracellular matrix assembly. A failure in either process is associated with oncogenic progression. Work included in this thesis provides evidence that these tumour suppressor functions are mutually exclusive. Additionally, post-translational modification of VHL by NEDD8 is shown to act as a ‘molecular switch’, altering VHL protein associations and providing a mechanism of pathway segregation. As a result of HIF stabilization, the expression of a homophilic adhesion molecule E-cadherin is significantly down-regulated in primary renal clear-cell carcinoma (RCC) upon VHL loss. E-cadherin down-regulation is shown to increase the invasive potential and is of prognostic value in RCC. Finally, VHL and SOCS1 are shown to dimerize and negatively regulate the JAK2-STAT5 signalling cascade. Defects in this dimerization are shown to underlie Chuvash polycythemia and provide a molecular understanding of the phenotypic observations associated with VHL-related polycythemias.

The ability of proteins to engage in nuclear-cytoplasmic shuttling is required for their proper function. The nuclear export of the von Hippel Lindau (VHL) tumour suppressor protein is necessary for the proteasomal degradation of the hypoxia inducible factor alpha (HIFα). Studies have identified that the nuclear export of VHL and other proteins encoding a Transcription-Dependent Nuclear Export Motif (TD-NEM) is independent of the classical CRM1 nuclear export pathway but requires ongoing transcription. Furthermore, the eukaryotic elongation factor 1 alpha (eEF1A) was identified as a mandatory component of the TD-NEM-mediated nuclear export machinery. In this study, we have uncovered the ability of eEF1A to mediate the nuclear export of proteins by accessing the nuclear compartment in its inactive, GDP-bound form. Although previously thought of as a strictly cytoplasmic protein, work conducted in this thesis has shown that eEF1A is a nuclear-cytoplasmic shuttling protein and this ability is required for the effective export of proteins encoding a TD-NEM.

The ability of proteins to engage in nuclear-cytoplasmic shuttling is required for their proper function. The nuclear export of the von Hippel Lindau (VHL) tumour suppressor protein is necessary for the proteasomal degradation of the hypoxia inducible factor alpha (HIFα). Studies have identified that the nuclear export of VHL and other proteins encoding a Transcription-Dependent Nuclear Export Motif (TD-NEM) is independent of the classical CRM1 nuclear export pathway but requires ongoing transcription. Furthermore, the eukaryotic elongation factor 1 alpha (eEF1A) was identified as a mandatory component of the TD-NEM-mediated nuclear export machinery. In this study, we have uncovered the ability of eEF1A to mediate the nuclear export of proteins by accessing the nuclear compartment in its inactive, GDP-bound form. Although previously thought of as a strictly cytoplasmic protein, work conducted in this thesis has shown that eEF1A is a nuclear-cytoplasmic shuttling protein and this ability is required for the effective export of proteins encoding a TD-NEM.

The ability of proteins to engage in nuclear-cytoplasmic shuttling is required for their proper function. The nuclear export of the von Hippel Lindau (VHL) tumour suppressor protein is necessary for the proteasomal degradation of the hypoxia inducible factor alpha (HIFα). Studies have identified that the nuclear export of VHL and other proteins encoding a Transcription-Dependent Nuclear Export Motif (TD-NEM) is independent of the classical CRM1 nuclear export pathway but requires ongoing transcription. Furthermore, the eukaryotic elongation factor 1 alpha (eEF1A) was identified as a mandatory component of the TD-NEM-mediated nuclear export machinery. In this study, we have uncovered the ability of eEF1A to mediate the nuclear export of proteins by accessing the nuclear compartment in its inactive, GDP-bound form. Although previously thought of as a strictly cytoplasmic protein, work conducted in this thesis has shown that eEF1A is a nuclear-cytoplasmic shuttling protein and this ability is required for the effective export of proteins encoding a TD-NEM.

Indiana University-Purdue University Indianapolis (IUPUI) / The transcription factor and tumor suppressor protein p53 critically regulates cell survival or death in response to cellular stress. p53 can activate genes involved in a wide variety of processes, including apoptosis, cell cycle arrest, angiogenesis, metabolism, and senescence. Mutations in p53 are common in cancer and alter its interactions with other proteins, but there are other mechanisms and posttranslational modifications that can alter these interactions as well. In some tumors, such as renal cell carcinoma, p53 is commonly inactive even though mutations to TP53 are rare. This suggests that there are other biochemical mechanisms of inhibition, which we explore in this study. Mutations in the DNA-binding domain of p53 result in conformational changes that enable p53 to interact with and inhibit its family member p73, thereby promoting cell survival instead of apoptosis. In contrast, it has been reported that wild-type p53 does not bind to p73. We found that JNK-mediated phosphorylation of Thr81 in the proline-rich domain (PRD) of p53 enabled wild-type p53 to form a complex with p73. The dimerization of wild-type p53 with p73 facilitated the expression of apoptotic target genes such as PUMA and BAX, as well as the induction of apoptosis. In addition to the apoptotic function of p53, the tumor suppressor also plays a major role in the inhibition of angiogenesis. Here we also report a new mechanism where the Mdm2 oncoprotein can indirectly inactive p53 through the regulation of the tumor suppressor VHL. In response to hypoxia, VHL can bind p53, which results in activation of several anti-angiogenic targets of p53 such as THBS1 and COL18A1. Mdm2 regulates the VHL-p53 interaction by conjugating nedd8 to VHL within a region that is important for the VHL-p53 interaction, blocking the induction of anti-angiogenic genes and resulting in a proangiogenic phenotype. Due to its positive regulation of major proangiogenic proteins and its negative regulation of potent inhibitors of angiogenesis, we propose that the oncoprotein Mdm2 is the angiogenic switch. These findings refine our understanding of p53 interactions and activation, specifically for p53-p73 induced cell death and p53-VHL inhibition of angiogenesis. / 2020-08-05

La polyglobulie (PG) a un arrière plan clinique et physiopathologique varié, et dont la connaissance a beaucoup évolué depuis 5 ans. Elle a pour caractéristique nécessaire et commune une augmentation du volume globulaire total qui peut résulter de mécanismes moléculaires différents. L'érythrocytose est une forme de la PG ou` seule la lignée érythroïde semble être augmentée. Dans ce contexte, nous nous sommes intéressés à la mutation JAK2 (Val617Phe) et aux gênes EPOR, VHL, PHD2 chez des patients présentant une érythrocytose d'origine inconnue (98 familles). Nous avons trouvé 3 nouvelles mutations délétionnelles et d'autres mutations faux-sens et silencieuses sur EPOR. Les cellules FDCP-1 et 32D transfectées, qui expriment les récepteurs tronqués ont montré une hypersensibilité à l'EPO à concentration basse. Les analyses EPO-dose-réponse pour une mutation faux-sens étaient semblables à celles du type sauvage. La mutation JAK2(Val617Phe) était présente avec une faible fréquence dans la série de patients étudiés. La recherche des mutations dans le gêne PHD2 nous a permis de trouver 3 nouvelles mutations frame-shift et non-sens, les seules connues à ce jour: Elles entraînent la perte d'une partie ou de l'ensemble du site catalytique de PHD2 dans l'hypothèse où elles seraient synthétisées. Ainsi, ces mutations et les deux autres mutations faux-sens décrites démontrent l'importance de PHD2 dans la régulation de la voie de HIF-a, et qu'une perturbation de cette régulation pourrait conduire à une PG. La majorité des patients reste sans défaut moléculaire identifié, et des recherches complémentaires sont nécessaires. Nous avons également étudié deux Hbs augmentant l'affinité pour l'oxygène: les Hbs Nantes et La Coruña. Enfin, nous avons cherché des mutations dans les 3 exons de DPGM chez 4 patients avec un taux de 2,3-DPG bas. Une seule mutation a été trouvée dans la région 5`, dans l'exon 1 non traduit, elle nécessite des études complémentaires. / Polycythemia has a varied clinical and physiopathological background, of which our knowledge has greatly evaluated since 5 years. It is characterised by augmentation of the red cell mass which can be resulted from different molecular mechanisms. Erythrocytosis is a form of polycythemia where only the erythrocytes are augmented. In this context, we looked for the mutation JAK2 (Val617Phe), and other mutations in the genes of EPOR, VHL, PHD2 in a series of patients with erythrocytosis of unknown origin (98 families). We found 3 new frame-shift mutations and other missense and silent mutations in the EPOR. Transfected FDCP-1 and 32D cell lines expressing a truncated EpoR showed increased sensitivity at low concentration of Epo. However, in the case of a missense EPOR mutation, the Epo-dose response assays were similar to that of the wild type. The mutation JAK2 (Val617Phe) was found at a low frequency in the studied patients. Looking for mutations in the PHD2 gene allowed us to find 3 new frame-shift and nonsense mutations, the only reported to date. The encoded PHD2, if synthesized, would lose its active site or a part of it. These mutations with the other two missense mutations reported provide the evidence of the importance of PHD2 in the regulation of HIF-a pathway, and that disturbing the oxygen-sensing pathway might be a cause of polycythemia. The molecular basis of polycythemia in the majority of patients is still to be identified, and complementary studies are necessary. We studied then two examples of Hbs with a high oxygen affinity: Hb Nantes et La Coruña. Finally, we looked for mutations in the 3 exons of DPGM in a series of 4 patients. One mutation in exon 1 in the 5`region was found, and needs further studies.

La polyglobulie (PG) a un arrière plan clinique et physiopathologique varié, et dont la connaissance a beaucoup évolué depuis 5 ans. Elle a pour caractéristique nécessaire et commune une augmentation du volume globulaire total qui peut résulter de mécanismes moléculaires différents. L'érythrocytose est une forme de la PG ou` seule la lignée érythroïde semble être augmentée. Dans ce contexte, nous nous sommes intéressés à la mutation JAK2 (Val617Phe) et aux gènes EPOR, VHL, PHD2 chez des patients présentant une érythrocytose d'origine inconnue (98 familles). Nous avons trouvé 3 nouvelles mutations délétionnelles et d'autres mutations faux-sens et silencieuses sur EPOR. Les cellules FDCP-1 et 32D transfectées, qui expriment les récepteurs tronqués ont montré une hypersensibilité à l'Epo à concentration basse. Les analyses Epo-dose-réponse pour une mutation faux-sens étaient semblables à celles du type sauvage. La mutation JAK2(Val617Phe) était présente avec une faible fréquence dans la série de patients étudiés. La recherche des mutations dans le gène PHD2 nous a permis de trouver 3 nouvelles mutations frame-shift et non-sens, les seules connues à ce jour: Elles entraînent la perte d'une partie ou de l'ensemble du site catalytique de PHD2 dans l'hypothèse où elles seraient synthétisées. Ainsi, ces mutations et les deux autres mutations faux-sens décrites démontrent l'importance de PHD2 dans la régulation de la voie de HIF-α, et qu'une perturbation de cette régulation pourrait conduire à une PG. La majorité des patients reste sans défaut moléculaire identifié, et des recherches complémentaires sont nécessaires. Nous avons également étudié deux Hbs augmentant l'affinité pour l'oxygène: les Hbs Nantes et La Coruña. Enfin, nous avons cherché des mutations dans les 3 exons de DPGM chez 4 patients avec un taux de 2,3-DPG bas. Une seule mutation a été trouvée dans la région 5`, dans l'exon 1 non traduit, elle nécessite des études complémentaires.

Marking proteins for degradation by the proteasome is a classical function of ubiquitination. This process of covalent attachment of a chain of ubiquitin molecules to target proteins is governed by the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2) and the ubiquitin ligase (E3). The von Hippel-Lindau (VHL) tumour suppressor protein forms an E3 ubiquitin ligase, ECV (Elongins BC/Cul2/VHL), which targets the alpha subunit of hypoxia-inducible factor (HIF) for ubiquitin-mediated destruction under normal oxygen tension. Tumour hypoxia promotes accumulation of HIFalpha, whose expression is associated with cancer progression, poor prognosis and resistance to conventional therapies, thus establishing HIF as a therapeutic target. Notably, VHL is functionally inactivated in VHL disease, a hereditary cancer syndrome characterized by the formation of tumours in multiple organs, as well as in the majority of sporadic clear-cell renal cell carcinomas (CCRCC) and haemangioblastomas. Recently, certain VHL mutations have been shown to cause the congenital disorder Chuvash polycythemia. Work contained in this thesis describes the temporally coordinated activation of the ECV, whereby oxygen-dependent recognition of HIFalpha by VHL triggers Cul2 modification by the ubiquitin-like molecule NEDD8, which enhances ECV ubiquitin ligase activity by recruiting the E2. In addition, the feasibility of ‘bio-tailored’ enzymes in the treatment of cancer is introduced by creating a bioengineered VHL capable of targeting HIFalpha for degradation irrespective of oxygen tension, which leads to the dramatic inhibition of CCRCC tumour growth and angiogenesis in a xenograft model. Furthermore, a ubiquitin ligase composed of two F-box proteins, VHL and suppressor of cytokine signalling 1 (SOCS1), was identified and shown to be paramount for the negative regulation of erythropoiesis by targeting phosphorylated Janus kinase 2 (JAK2) for ubiquitin-mediated destruction. The malfunction of this ubiquitin ligase explains the excessive erythrocytosis observed in Chuvash polycythemia patients and reveals a novel genetic link between the seemingly distinct genes VHL and JAK2 in the development of polycythemia.

Marking proteins for degradation by the proteasome is a classical function of ubiquitination. This process of covalent attachment of a chain of ubiquitin molecules to target proteins is governed by the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2) and the ubiquitin ligase (E3). The von Hippel-Lindau (VHL) tumour suppressor protein forms an E3 ubiquitin ligase, ECV (Elongins BC/Cul2/VHL), which targets the alpha subunit of hypoxia-inducible factor (HIF) for ubiquitin-mediated destruction under normal oxygen tension. Tumour hypoxia promotes accumulation of HIFalpha, whose expression is associated with cancer progression, poor prognosis and resistance to conventional therapies, thus establishing HIF as a therapeutic target. Notably, VHL is functionally inactivated in VHL disease, a hereditary cancer syndrome characterized by the formation of tumours in multiple organs, as well as in the majority of sporadic clear-cell renal cell carcinomas (CCRCC) and haemangioblastomas. Recently, certain VHL mutations have been shown to cause the congenital disorder Chuvash polycythemia. Work contained in this thesis describes the temporally coordinated activation of the ECV, whereby oxygen-dependent recognition of HIFalpha by VHL triggers Cul2 modification by the ubiquitin-like molecule NEDD8, which enhances ECV ubiquitin ligase activity by recruiting the E2. In addition, the feasibility of ‘bio-tailored’ enzymes in the treatment of cancer is introduced by creating a bioengineered VHL capable of targeting HIFalpha for degradation irrespective of oxygen tension, which leads to the dramatic inhibition of CCRCC tumour growth and angiogenesis in a xenograft model. Furthermore, a ubiquitin ligase composed of two F-box proteins, VHL and suppressor of cytokine signalling 1 (SOCS1), was identified and shown to be paramount for the negative regulation of erythropoiesis by targeting phosphorylated Janus kinase 2 (JAK2) for ubiquitin-mediated destruction. The malfunction of this ubiquitin ligase explains the excessive erythrocytosis observed in Chuvash polycythemia patients and reveals a novel genetic link between the seemingly distinct genes VHL and JAK2 in the development of polycythemia.