La thématique du laboratoire de l’équipe d’Adam Telerman porte sur la réversion tumorale, un processus rare au cours duquel les cellules cancéreuses perdent leur phénotype malin, et deviennent des cellules dites révertantes. Plusieurs protéines clefs impliquées dans cette transformation ont été mises en évidence, dont TCTP (Translationally Controlled Tumor Protein). La protéine TCTP est également impliquée dans la régulation de l’apoptose en interagissant et en renforçant l’activité anti-apoptotique de Mcl-1 et de Bcl-xl, deux protéines appartenant à la famille des Bcl-2. Ce projet s’attache à comprendre en termes moléculaires le mode d’action de TCTP au cours de l’apoptose. / Adam Telerman’s team research focuses on tumor reversion, a rare process in which cancer cells lose their malignant phenotype, and therefore become revertant. Many key proteins were showed to be involved in this transformation, including TCTP (translationally Controlled Tumor Protein). TCTP protein is also involved in apoptosis regulation by interacting and strengthening the anti-apoptotic activity of Mcl-1 and Bcl-xl, two proteins from Bcl-2 family.

La protéine Bcl-xL fait partie de la famille des protéines anti-apoptotiques Bcl-2. Il a été montré que cette protéine avait un rôle majeur dans la formation des plaquettes chez la souris (mégacaryopoïèse). Une dérégulation de cette protéine pourrait aboutir à une altération de la mégacaryopoïèse et donner des pathologies humaines comme des thrombopénies chroniques. Une des causes de thrombopénies chroniques est le purpura thrombopénique immunologique (ou PTI), qui associe deux mécanismes physiopathologiques : une destruction auto-immune des plaquettes et une insuffisance de leur production par la moelle osseuse. Le PTI est un diagnostic d’exclusion par élimination de toutes les causes connues de thrombopénie. Au sein de notre cohorte de patients suivis en médecine interne pour cette maladie, nous avons identifié certains patients qui présentaient un profil non-immunologique, c’est-à-dire l’absence d’auto-immunité et une non réponse à tous les traitements immunomodulateurs, ou pas d’indication à un traitement compte tenu d’un taux de plaquettes suffisant. Nous montrons dans ce travail de thèse que Bcl-xL est nécessaire pour la survie du mégacaryocyte humain pendant toute la mégacaryopoïèse, à la différence de la souris. Par ailleurs, certains patients ont une altération intrinsèque de la formation des proplaquettes, et certains d’entre eux ont également une diminution de l’ARN messager et de la protéine Bcl-xL dans leurs plaquettes. Ces observations nouvelles suggèrent l’implication de Bcl-xL dans la physiopathologie de leur maladie et ouvrent la voie à l’identification d’une potentielle nouvelle cause de thrombopénie chronique. / The Bcl-xL protein is a member of Bcl-2 anti-apoptotic proteins. It has been shown in mouse that this protein had a major role in platelet production (megakaryopoiesis). Bcl-xL deregulation could lead to megakaryopoiesis impairement and explain some human diseases such as chronic thrombocytopenias. One cause of chronic thrombocytopenia is immune thrombocytopenia (ITP) that associates 2 pathophysiological mechanisms: an immune-mediated platelet destruction and an insufficient production from the bone marrow cells. ITP is a diagnosis of exclusion when all known causes of thrombocytopenia have been ruled out by diagnosis work-up. In ITP cohort of patients followed in our internal medicine department, we have identified some patients with a haematological profile of their disease, ie absence of overt features of auto-immunity, and absence of response to immunomudulatory treatments, or no indication to such treatment because of sufficient platelet count. We demonstrate in this study that Bcl-xL is necessary for megakaryocyte survival during all megakaryopoiesis, contrary to what was found in mouse. Moreover, some patients have an intrinsically impaired proplatelet formation, and some of them also have a decrease of Bcl-xL mRNA and protein in their platelets. These novel observations suggest that a deregulation of Bcl-xL is a possible cause of their disease and lead the way to the identification of a potentially new cause of chronic thrombocytopenia in human

TCTP est une protéine de 20 kDa que l’on retrouve souvent sous forme de dimère. Elle est fortement conservée dans la phylogénie et on la trouve dans les levures, les plantes, les invertébrés et les mammifères. Elle est localisée dans tous les compartiments de la cellule : noyau, cytoplasme, et membranes. Il s’agit d’une protéine très abondante dans des cellules souches ainsi que des cellules en croissance exponentielle, y compris les cellules tumorales. Sa fonction principale est celle d’une « protéine de survie ». TCTP a été décrite comme interagissant avec de multiples protéines dont p53, MDM2, Bcl-xL et TSAP6. Le but de mon travail est de permettre de mieux caractériser ces fonctions de TCTP et pour cela, nous avons étudié ses interactions in vitro et surtout, in vivo, dans différents modèles génétiques chez la souris. / TCTP is a 20 kDa protein frequently encountered as a dimer. It is highly conserved through phylogeny and is present inn yeast, plants, invertebrates and mammals. It is localized in all compartments of the cell: nucleus, cytoplasm, membranes. This protein is highly abundant in stem cells and during the exponential growth, including in cancer cells. It mainly functions as a survivor factor. TCTP has been described as interacting with multiple proteins, including p53, MDM2, Bcl-xL and TSAP6. The purpose of my work is to better characterize these functions of TCTP; we therefore studied its interactions in vitro, but mostly in vivo, using different murine genetic models.

With previous indication of the Group VIA phospholipase A2 (iPLA2β) enzyme regulating ER-stress induced apoptosis in β-cells by regulating the anti-apoptotic protein Bcl-xL via alternative splicing, our lab postulated iPLA2β to be utilizing a similar mechanism to regulate apoptosis in mice lung. Our previous lab work has shown implications of lung function compromise in iPLA2β-/- mice, and we speculated the cause to be due altered lung architecture stemming from the attenuation of apoptosis. The western blot analysis in this study suggested that iPLA2β is involved in the regulation of Bcl-xL, but the mRNA ratios of the splice variants suggested that alternative splicing is not the mechanism iPLA2β is utilizing for the regulation in our animal models. Additionally, the observation and assessment of the lung morphology of the iPLA2β-/- and wild type mice suggested that iPLA2β does not play an integral role in lung morphology.

XIAP and Bcl-xL are critical anti-apoptotic molecules that directly inhibit caspases and block mitochondrial membrane permeabilization, respectively. In addition to preventing apoptosis, both XIAP and Bcl-xL can be generated by cap-independent translation via the utilization of an IRES in the 5'-UTR of their mRNAs. In recent years it has been shown that activation of S6K2 induces the translational upregulation of these two apoptotic regulators. Here I have determined that activation of S6K2 enhances IRES-mediated translation of XIAP and Bcl-xL by inducing the degradation of PDCD4, which I have identified as a novel regulator of XIAP and Bcl-xL IRES elements. Furthermore, I have shown that PDCD4 is a positive modulator of the Apaf-1 IRES element. The concurrent regulation of XIAP, Bcl-xL and Apaf-1 by PDCD4 suggests a model in which the level of PDCD4 expression alters the apoptotic threshold by specifically impacting IRES-mediated translation of the XIAP, Bcl-xL and Apaf-1 mRNAs.

The Bcl-2 family of proteins contains both anti-apoptotic (e.g.Bcl-2, Bcl-xL) and pro-apoptotic (e.g.Bad, Bcl-xS) proteins. The Bcl-xL and Bcl-xS are splice variants, but have different functions during apoptosis. The pro-survival kinase Akt can phosphorylate certain Bcl-2-related proteins, specifically on serine residues, to regulate their function and localization. This is an extension of the work from our laboratorys finding that haloperidol induces PC12 cell death by inducing Bcl-xS which then translocates from cytosol to mitochondria where it facilitates the release of cytochrome c. The toxicity induced by Bcl-xS is reversed by expression of constitutively active Akt. I hypothesized that Akt-mediated post-translational modification may be important for regulating the function of Bcl-xS and Bcl-xL.<p> Three specific serine residues were ultimately chosen for the characterization of Bcl-xS/xL function: Ser62 (inactivation mutant), Ser106 (putative Akt phosphorylation motif), and Ser165 in Bcl-xS (and the corresponding Ser228 in Bcl-xL) (immediately upstream of hydrophobic tail). The individual substitution of all three Serines with Alanines (which precludes phosphorylation at that site) in Bcl-xS did not affect the expression of the protein, but they did induce varying degrees of cytotoxicity in both PC12 and HEK cultures. I focused on the Ser106 substitution mutant given my hypothesis that Akt targeted this site. Overexpression of Bcl-xS(S106A) was toxic in both PC12 and HEK cultures, as expected, and this coincided with the appearance of the Bcl-xS(S106A) protein in the mitochondrial fraction. The release of cytochrome c from PC12 cell mitochondria coincided with the co-immunoprecipitation of the Bcl-xS protein with VDAC (voltage-dependent anion channel), a channel-forming protein that is known to mediate cytochrome c release, and with the initiation of caspase-dependent events. This was not the case in HEK cells, where the mitochondrial VDAC seemed to be diminished and the toxicity was cytochrome c-independent as well as caspase-independent. In addition, I was able to demonstrate that the S106A substituted protein was not able to co-immunoprecipitate with Akt, supporting Ser106 as a potential target for the Akt protein. I then studied the effects of the homologous substitutions in Bcl-xL on cell function. I chose to use treatment with the potent inducer of apoptosis, staurosporine, as a model of cytotoxicity. Again, substituted proteins exerted toxicity, but they did not potentiate the effects of staurosporine, at least not on MTT conversion. I did notice, however, that there was a clear morphological change with certain concentrations of staurosporine, and subsequently demonstrated that the Bcl-xL(S106A) protein potentiated PC12 cell differentiation induced by staurosporine. This protein also co-immunoprecipitated better with Akt, which was unexpected given my results with the Bcl-xS(S106A) protein described above. Perhaps the extra amino acids in Bcl-xL account for this.<p> It is clear that the phosphorylation of Bcl-xS and Bcl-xL proteins is an important means of regulating their function and localization within the cell. These data support the S106 residues in both Bcl-xS and Bcl-xL as novel targets for the pro-survival Akt kinase, and indicate a role for this/these residue(s) in cellular functions as diverse as apoptosis and differentiation.

XIAP and Bcl-xL are critical anti-apoptotic molecules that directly inhibit caspases and block mitochondrial membrane permeabilization, respectively. In addition to preventing apoptosis, both XIAP and Bcl-xL can be generated by cap-independent translation via the utilization of an IRES in the 5'-UTR of their mRNAs. In recent years it has been shown that activation of S6K2 induces the translational upregulation of these two apoptotic regulators. Here I have determined that activation of S6K2 enhances IRES-mediated translation of XIAP and Bcl-xL by inducing the degradation of PDCD4, which I have identified as a novel regulator of XIAP and Bcl-xL IRES elements. Furthermore, I have shown that PDCD4 is a positive modulator of the Apaf-1 IRES element. The concurrent regulation of XIAP, Bcl-xL and Apaf-1 by PDCD4 suggests a model in which the level of PDCD4 expression alters the apoptotic threshold by specifically impacting IRES-mediated translation of the XIAP, Bcl-xL and Apaf-1 mRNAs.

The Bcl-2 family of proteins contains both anti-apoptotic (e.g.Bcl-2, Bcl-xL) and pro-apoptotic (e.g.Bad, Bcl-xS) proteins. The Bcl-xL and Bcl-xS are splice variants, but have different functions during apoptosis. The pro-survival kinase Akt can phosphorylate certain Bcl-2-related proteins, specifically on serine residues, to regulate their function and localization. This is an extension of the work from our laboratorys finding that haloperidol induces PC12 cell death by inducing Bcl-xS which then translocates from cytosol to mitochondria where it facilitates the release of cytochrome c. The toxicity induced by Bcl-xS is reversed by expression of constitutively active Akt. I hypothesized that Akt-mediated post-translational modification may be important for regulating the function of Bcl-xS and Bcl-xL.<p> Three specific serine residues were ultimately chosen for the characterization of Bcl-xS/xL function: Ser62 (inactivation mutant), Ser106 (putative Akt phosphorylation motif), and Ser165 in Bcl-xS (and the corresponding Ser228 in Bcl-xL) (immediately upstream of hydrophobic tail). The individual substitution of all three Serines with Alanines (which precludes phosphorylation at that site) in Bcl-xS did not affect the expression of the protein, but they did induce varying degrees of cytotoxicity in both PC12 and HEK cultures. I focused on the Ser106 substitution mutant given my hypothesis that Akt targeted this site. Overexpression of Bcl-xS(S106A) was toxic in both PC12 and HEK cultures, as expected, and this coincided with the appearance of the Bcl-xS(S106A) protein in the mitochondrial fraction. The release of cytochrome c from PC12 cell mitochondria coincided with the co-immunoprecipitation of the Bcl-xS protein with VDAC (voltage-dependent anion channel), a channel-forming protein that is known to mediate cytochrome c release, and with the initiation of caspase-dependent events. This was not the case in HEK cells, where the mitochondrial VDAC seemed to be diminished and the toxicity was cytochrome c-independent as well as caspase-independent. In addition, I was able to demonstrate that the S106A substituted protein was not able to co-immunoprecipitate with Akt, supporting Ser106 as a potential target for the Akt protein. I then studied the effects of the homologous substitutions in Bcl-xL on cell function. I chose to use treatment with the potent inducer of apoptosis, staurosporine, as a model of cytotoxicity. Again, substituted proteins exerted toxicity, but they did not potentiate the effects of staurosporine, at least not on MTT conversion. I did notice, however, that there was a clear morphological change with certain concentrations of staurosporine, and subsequently demonstrated that the Bcl-xL(S106A) protein potentiated PC12 cell differentiation induced by staurosporine. This protein also co-immunoprecipitated better with Akt, which was unexpected given my results with the Bcl-xS(S106A) protein described above. Perhaps the extra amino acids in Bcl-xL account for this.<p> It is clear that the phosphorylation of Bcl-xS and Bcl-xL proteins is an important means of regulating their function and localization within the cell. These data support the S106 residues in both Bcl-xS and Bcl-xL as novel targets for the pro-survival Akt kinase, and indicate a role for this/these residue(s) in cellular functions as diverse as apoptosis and differentiation.

XIAP and Bcl-xL are critical anti-apoptotic molecules that directly inhibit caspases and block mitochondrial membrane permeabilization, respectively. In addition to preventing apoptosis, both XIAP and Bcl-xL can be generated by cap-independent translation via the utilization of an IRES in the 5'-UTR of their mRNAs. In recent years it has been shown that activation of S6K2 induces the translational upregulation of these two apoptotic regulators. Here I have determined that activation of S6K2 enhances IRES-mediated translation of XIAP and Bcl-xL by inducing the degradation of PDCD4, which I have identified as a novel regulator of XIAP and Bcl-xL IRES elements. Furthermore, I have shown that PDCD4 is a positive modulator of the Apaf-1 IRES element. The concurrent regulation of XIAP, Bcl-xL and Apaf-1 by PDCD4 suggests a model in which the level of PDCD4 expression alters the apoptotic threshold by specifically impacting IRES-mediated translation of the XIAP, Bcl-xL and Apaf-1 mRNAs.

The RNA-binding protein HuR controls key cellular processes by binding target mRNAs and regulating them at various post-transcriptional levels. HuR can function as an Internal Ribosome Entry Site (IRES) trans-acting factor that regulates the IRES-mediated translation of XIAP. Since XIAP and Bcl-xL expression was reported to be co-regulated, we investigated whether HuR is also a regulat or of Bcl-xL expression. We found that HuR binds the 3’end of the Bcl-xL 5’UTR in-vitro. In U2OS cells, we showed that loss of HuR by siRNA significantly increased Bcl-xL protein expression while Bcl-2 and Mcl-1 levels remained unchanged. We found that the HuR-dependent Bcl-xL increase was through translation, shown by polysome profiling. Possible transcriptional, stability and splicing changes were eliminated. At the physiological level HuR levels did not impact cell survival but altered mitochondrial morphology, partially through Bcl-xL. Thus, HuR may be involved in maintaining proper mitochondrial function by controlling Bcl-xL expression.