N-linked glycosylation supports cross-talk between receptor tyrosine kinases and androgen receptor

Prostate cancer is the second most common cause of cancer-associated deaths in men and signalling via a transcription factor called androgen receptor (AR) is an important driver of the disease. Androgen treatment is known to affect the expression and activity of other oncogenes including receptor tyrosine kinases (RTKs). In this study we report that AR-positive prostate cancer cell-lines express 50% higher levels of enzymes in the hexosamine biosynthesis pathway (HBP) than AR-negative prostate cell-lines. HBP produces hexosamines that are used by endoplasmic reticulum and golgi enzymes to glycosylate proteins targeted to plasma-membrane and secretion. Inhibition of O-linked glycosylation by ST045849 or N-linked glycosylation with tunicamycin decreased cell viability by 20%. In addition, tunicamycin inhibited the androgen-induced expression of AR target genes KLK3 and CaMKK2 by 50%. RTKs have been shown to enhance AR activity and we used an antibody array to identify changes in the phosphorylation status of RTKs in response to androgen stimulation. Hormone treatment increased the activity of Insulin like Growth Factor 1-Receptor (IGF-1R) ten-fold and this was associated with a concomitant increase in the N-linked glycosylation of the receptor, analyzed by lectin enrichment experiments. Glycosylation is known to be important for the processing and stability of RTKs. Inhibition of N-linked glycosylation resulted in accumulation of IGF-1R pro-receptor with altered mobility as shown by immunoprecipitation. Confocal imaging revealed that androgen induced plasma-membrane localization of IGF-1R was blocked by tunicamycin. In conclusion we have established that the glycosylation of IGF-1R is necessary for the full activation of the receptor in response to androgen treatment and that perturbing this process can break the feedback loop between AR and IGF-1R activation in prostate cells. Achieving similar results selectively in a clinical setting will be an important challenge in the future.

Clathrin adaptor epsinR is required for retrograde sorting on early endosomal membranes

Retrograde transport links early/recycling endosomes to the trans-Golgi network (TGN), thereby connecting the endocytic and the biosynthetic/secretory pathways. To determine how internalized molecules are targeted to the retrograde route, we have interfered with the function of clathrin and that of two proteins that interact with it, AP1 and epsinR. We found that the glycosphingolipid binding bacterial Shiga toxin entered cells efficiently when clathrin expression was inhibited. However, retrograde transport of Shiga toxin to the TGN was strongly inhibited. This allowed us to show that for Shiga toxin, retrograde sorting on early/recycling endosomes depends on clathrin and epsinR, but not AP1. EpsinR was also involved in retrograde transport of two endogenous proteins, TGN38/46 and mannose 6-phosphate receptor. In conclusion, our work reveals the existence of clathrin-independent and -dependent transport steps in the retrograde route, and establishes a function for clathrin and epsinR at the endosome-TGN interface.

Localization of polymorphic N-acetyltransferase (NAT2) in tissues of inbred mice

Like humans, mice exhibit polymorphism in the N-acetylation of aromatic amines, many of which are toxic and/or carcinogenic. Mice have three N-acetyltransferase (Nat) genes, Nat1, Nat2 and Nat3, and Nat2 is known to be polymorphic. There is a dramatic difference in the acetylation of NAT2 substrates by blood from fast (C57BL/6J) compared with slow acetylator (A/J) mice. However, the acetylation of these substrates by liver cytosols from the two strains is very similar. In order to determine whether the expression of the NAT2 protein corresponded with the activities measured, a polyclonal antipeptide antisera was raised against the C-terminal decapeptide of NAT2 and characterized using recombinant murine NAT2 antigen. Enzyme-linked immunosorbent assays (ELISAs) demonstrated that the anti-NAT2 antiserum bound in a concentration-dependent fashion to recombinant NAT2. Immunochemical analysis of mouse liver cytosols from C57BL/6J or A/J livers indicated that the level of NAT2 protein expressed in the two strains was similar. Immunohistochemical staining of C57BL/6J liver with anti-NAT2 antiserum showed that NAT2 was expressed in hepatocytes throughout the liver although the intensity of staining in the perivenous (centrilobular) region was higher than that in the periportal region. NAT2 was also detected in epithelial cells in the lung, kidney, bladder, small intestine and skin as well as in erythrocytes and lymphocytes in the spleen and hair follicles and sebaceous glands in the skin. Characterization of the distribution of NAT2 will be of value in elucidating the role of polymorphic N-acetylation in protecting the organism from environmental insults as well as in endogenous metabolism.

The dual EGFR/HER2 inhibitor lapatinib synergistically enhances the antitumor activity of the histone deacetylase inhibitor panobinostat in colorectal cancer models

As key molecules that drive progression and chemoresistance in gastrointestinal cancers, epidermal growth factor receptor (EGFR) and HER2 have become efficacious drug targets in this setting. Lapatinib is an EGFR/HER2 kinase inhibitor suppressing signaling through the RAS/RAF/MEK (MAP/ERK kinase)/MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase)/AKT pathways. Histone deacetylase inhibitors (HDACi) are a novel class of agents that induce cell cycle arrest and apoptosis following the acetylation of histone and nonhistone proteins modulating gene expression and disrupting HSP90 function inducing the degradation of EGFR-pathway client proteins. This study sought to evaluate the therapeutic potential of combining lapatinib with the HDACi panobinostat in colorectal cancer (CRC) cell lines with varying EGFR/HER2 expression and KRAS/BRAF/PIK3CA mutations. Lapatinib and panobinostat exerted concentration-dependent antiproliferative effects in vitro (panobinostat range 7.2-30 nmol/L; lapatinib range 7.6-25.8 μmol/L). Combined lapatinib and panobinostat treatment interacted synergistically to inhibit the proliferation and colony formation in all CRC cell lines tested. Combination treatment resulted in rapid induction of apoptosis that coincided with increased DNA double-strand breaks, caspase-8 activation, and PARP cleavage. This was paralleled by decreased signaling through both the PI3K and MAPK pathways and increased downregulation of transcriptional targets including NF-κB1, IRAK1, and CCND1. Panobinostat treatment induced downregulation of EGFR, HER2, and HER3 mRNA and protein through transcriptional and posttranslational mechanisms. In the LoVo KRAS mutant CRC xenograft model, the combination showed greater antitumor activity than either agent alone, with no apparent increase in toxicity. Our results offer preclinical rationale warranting further clinical investigation combining HDACi with EGFR and HER2-targeted therapies for CRC treatment.

The dual EGFR/HER-2 tyrosine kinase inhibitor lapatinib sensitizes colon and gastric cancer cells to the irinotecan active metabolite SN-38

Members of the human epidermal receptor (HER) family are frequently associated with aggressive disease and poor prognosis in multiple malignancies. Lapatinib is a dual tyrosine kinase inhibitor targeting the epidermal growth factor receptor (EGFR) and HER-2. This study evaluated the therapeutic potential of lapatinib, alone and in combination with SN-38, the active metabolite of irinotecan (CPT-11), in colon and gastric cancer cell lines. Concentration-dependent antiproliferative effects of both lapatinib and SN-38 were observed in all colon and gastric cancer cell lines tested but varied significantly between individual cell lines (lapatinib range 0.08-11.7 muM; SN-38 range 3.6-256 nM). Lapatinib potently inhibited the growth of a HER-2 overexpressing gastric cancer cell line and demonstrated moderate activity in gastric and colon cancer cells with detectable HER-2 expression. The combination of lapatinib and SN-38 interacted synergistically to inhibit cell proliferation in all colon and gastric cancer cell lines tested. Cotreatment with lapatinib and SN-38 also resulted in enhanced cell cycle arrest and the induction of apoptosis with subsequent cellular pharmacokinetic analysis demonstrating that lapatinib promoted the increased intracellular accumulation and retention of SN-38 when compared to SN-38 treatment alone. Finally, the combination of lapatinib and CPT-11 demonstrated synergistic antitumor efficacy in the LoVo colon cancer mouse xenograft model with no apparent increase in toxicity compared to CPT-11 monotherapy. These results provide compelling preclinical rationale indicating lapatinib to be a potentially efficacious chemotherapeutic combination partner for irinotecan in the treatment of gastrointestinal carcinomas.

Study of influence of regulatory polymorphisms of expression in development of breast cancer

The human genome has millions of genetics variants that can affect gene expression. These variants are known as cis-regulatory variants and are responsible for intra-species phenotypic differences and individual susceptibility to disease. One of the diseases affected by cis-regulatory variants is breast cancer. Breast cancer is one of the most common cancers, with approximately 4500 new cases each year in Portugal. Breast cancer has many genes mutated and TP53 has been shown to be relevant for this disease. TP53 is one of the most commonly mutated genes in human cancer and it is involved in cell cycle regulation and apoptosis. Previous work by Maia et al has shown that TP53 has differential allelic expression (DAE), which suggests that this gene may be under the influence of cis-regulatory variants. Also, its DAE pattern is totally altered in breast tumours with normal copy number. We hypothesized that cis-regulatory variants affecting TP53 may have a role in breast cancer development and treatment. The present work aims to identify the cis-regulatory variants playing a role in TP53 expression, using in silico, in vitro and in vivo approaches. By bioinformatic tools we have identified candidate cis-regulatory variants and predicted the possible transcription factor binding sites that they affect. By EMSA we studied DNA-protein interactions in this region of TP53. The in silico analysis allowed us to identified three candidate cis-regulatory SNPs which may affect the binding of seven transcription factors. However, the EMSA experiments have not been conclusive and we have not yet confirmed whether any of the identified SNPs are associated with gene expression control of TP53. We will carry out further experiments to validate our findings.

Molecular structure and functional analysis of runx3 in zebrafish

Tese de doutoramento, Ciências Biomédicas, Universidade do Algarve, Departamento de Ciências Biomédicas e Medicina, 2014

Control of endosomal Toll-like receptor (TLR) signaling by nanoparticles and applications in cancer therapy

Thesis (Ph.D.)--University of Washington, 2014

Exploring the modulatory role of A2A receptors in oligodendrogenesis derived from neural stem/progenitor cells of the subventricular zone

Tese de mestrado, Neurociências, Faculdade de Medicina, Universidade de Lisboa, 2015

The role of Di-iron proteins in pathogen resistance

Dissertation presented to obtain the Ph.D. degree in Biochemistry

The role of α2,3- and α2,6-sialylation

RESUMO: Os glicoconjugados que decoram a superfície celular e os lípidos e proteínas secretados ocupam o ponto de encontro onde normalmente ocorrem interacções críticas homólogas (hospedeiro-hospedeiro) e heterólogas (hospedeiro-patogénio). Apesar de ser largamente aceite que os glicanos são parte integrante do processo de imunidade, continua a não ser claro qual o papel que os glicanos, em toda a sua diversidade, tomam no quadro geral da imunidade. Os glicanos, que são frequentemente terminados por resíduos de ácido siálico, podem ser alterados por factores externos, tais como patogénios, ou por acontecimentos fisiológicos celulares específicos. Normalmente em posição terminal, as glico-estruturas que contêm ácido siálico assumem um papel fundamental numa quantidade substancial de receptores imunes envolvidos na adesividade e tráfico celular, tal como as Selectinas e as Siglecs, das quais se sabe apresentarem uma relevante função imune. À altura do início desta tese, era sabido que os ácidos siálicos expressos à superfície das células poderiam modular mecanismos importantes nas respostas imunes adaptativas. Considerando a posição de charneira que as células dendríticas (DCs) ocupam na transição da resposta imune inata para a adaptativa, antecipámos que os ácidos siálicos poderiam também modular mecanismos relevantes nas DCs humanas. As DCs têm uma função muito relevante na verificação e captura antigénica, migração para os gânglios linfáticos e apresentação antigénica aos linfócitos, uma sequência de funções que conduz, em ultima instância, à indução da resposta inata adaptativa. Considerando estas premissas, a nossa hipótese principal foi que os ácidos siálicos podem influenciar funções relevantes das DCs, tais como captura de antigénios, maturação, migração para os gânglios linfáticos e apresentação antigénica às células Para testar esta hipótese, dividimos o trabalho em quatro partes: 1) Analisámos os glicanos sialilados de superfície, expressos durante a diferenciação de monócitos humanos em DCs (moDCs). Os nossos dados mostraram que a expressão dos glicanos com ligações em O (O-glicanos) e sialilados em α2,3, assim como glicanos com ligações em N (N-glicanos) sialilados em α2,6 e α2,3 aumentou durante o processo de diferenciação das moDCs. Contribuindo para esta nova configuração glicosídica, três sialiltransferases (STs) poderão estar envolvidas: a ST6Gal-1 correlaciona-se com a expressão aumentada de N-glicanos sialilados em α2,6; a ST3Gal-1 contribui para a sialilação em α2,3 de O-glicanos, em especial de antigénios T; e a ST3Gal-4 poderá ser responsável pelo aumento de N-glicanos sialilados em α2,3. Após estímulo e consequente maturação das moDCs, ambos os níveis de expressão génica de ST6Gal-1 e ST3Gal-4 são negativamente modificados sendo, também, que a expressão de ST3Gal-1 varia consoante o estímulo. 2) Estudámos posteriormente as consequências da modulação dos ácidos siálicos de superfície nas funções das DCs. Observámos que a remoção dos ácidos siálicos de superfície diminui significativamente a capacidade de macropinocitose e endocitose mediada por receptores nas moDCs. Em contrapartida, o tratamento com sialidase aumentou significativamente a capacidade das moDCs para fagocitar Escherichia coli. Determinou-se também que este mecanismo requer a existência de ácido siálico presente nas E. coli indicando um mecanismo de interacção hospedeiro-patogénio dependente de ácido siálico em ambas as partes envolvidas. As moDCs tratadas com sialidase também apresentam um nível superior de expressão de moléculas de MHC e moléculas co-estimulatórias, sugerindo um fenótipo celular mais maduro. Recorrendo ao modelo de ratinho, utilizaram-se DCs derivadas de células da medula (BMDCs) de ratinhos deficientes em ST3Gal-1 e ST6Gal-1. Estes ensaios revelaram que quer a endocitose quer a maturação são influenciadas por modificações 37 nos glicanos sialilados em α2,3 ou α2,6. A detecção e quantificação de proteínas Nglicosiladas e sialiladas em α2,6 apontou para um potencial envolvimento de integrinas β2 nestes mecanismos. 3) O efeito da sialilação em α2,6 na migração das DCs para os gânglios linfáticos foi também analisado. Observámos que BMDCs deficientes para ST6Gal-1 apresentam uma redução de cerca de 50% nos níveis de migração das DCs para os gânglios linfáticos, tal como aferido em ensaios de inflamação in situ e estudos de transferência adoptiva de células. Uma redução dos níveis deste tipo de migração foi também observada quando BMDCs nativas foram transferidas para ratinhos receptores deficientes em ST6Gal-1. São, contudo, necessários mais ensaios de forma a identificar as moléculas envolvidas neste processo. 4) Por último, analisámos o impacto da sialilação na estimulação antigénica das DCs às células T. Assim, concluiu-se que moDCs tratadas com sialidase apresentam um nível de expressão superior de IL-12, TNF-ɑ, IL-6 e IL-10, e activação do factor de transcrição nuclear kappa B (NF-κB). As DCs tratadas com sialidase induziram uma maior proliferação nas células T, com expressão correspondente de interferão-γ. Este dado sugere que a remoção de ácidos siálicos de superfície contribui para o desenvolvimento de uma resposta pro-inflamatória do tipo 1 por células T auxiliares (resposta Th1). Considerando estes dados no seu todo, concluímos que o ácido siálico tem um papel marcante nas funções imunes das DCs. Alterações à concentração de ácido siálico à superfície das células podem alterar a endocitose/fagocitose, maturação, migração para os tecidos e gânglios linfáticos e capacidade estimulatória para com as células T. Complementando estes dados, as ligações glicosídicas de ácidos siálicos criados por ST6Gal-1 e ST3Gal-1 são funcionalmente relevantes. A modulação programada da sialilação do glicocálice, mediada por sialidases individuais ou sialiltransferases é uma possibilidade aceitável para a melhoria da fagocitose por DCs e da sua potência imunológica. Este facto tem um significado particular para imunoterapias baseadas em DCs, podendo provar-se decisivo para a sua eficiência e aplicabilidade num futuro muito próximo.-------------------------------ABSTRACT: Glycans decorating cell surface and secreted proteins and lipids occupy the junction where critical host–host and host-pathogen interactions occur. In spite of the wide acceptance that glycans are centrally implicated in immunity, exactly how glycans and their variety and variability contribute to the overall immune response remains poorly defined. Glycans, frequently terminated by sialic acid residues, may be modified by external factors such as pathogens or upon specific physiological cellular events. The terminal, privileged positions of sialic acid-modified structures makes them key, fundamental determinants for a number of immune receptors with known involvement in cellular adhesiveness and cell trafficking, such as Selectins and Siglecs, with known relevant immune functions. At the time this thesis was initiated, it was established that sialic acids expressed at cell surface could modulate important mechanisms of the adaptive immune responses. Given the key role of dendritic cells (DCs) in the transition from innate to the adaptive immune responses, we anticipated that sialic acids could also modulate important mechanisms of human DCs. DCs have a relevant role in antigen screening and uptake, migration to lymph nodes and antigen presentation to lymphocytes, ultimately triggering the adaptive immune response. Therefore, our primary hypothesis was that sialic acids may modulate DC functions, such as antigen uptake, maturation, homing to lymph nodes and antigen presentation to T cells. To test this hypothesis, we divided our work in four parts. 1) Surface sialylated glycans expressed during differentiation from human monocytes to DCs (moDCs) were analyzed. Our data showed that α2,3-sialylated O-glycans and α2,6- and α2,3-sialylated N-glycans expression increased during moDC differentiation. Three main sialyltransferases (STs) are committed with this new glycan configuration: ST6Gal- 1 correlates with the increased expression of α2,6-sialylated N-glycans; ST3Gal-1 32 contributes for the α2,3-sialylation of O-glycans, especially T antigens; and ST3Gal-4 may contribute for the increased α2,3-sialylated N-glycans. Upon moDC maturation, ST6Gal-1 and ST3Gal-4 are downregulated and ST3Gal-1 is altered in a stimulus dependent manner. 2) We subsequently analyzed the consequences of the modulation of cell surface sialic acids in DC functions. We observed that removing surface sialic acid by sialidase significantly decreased the capacity of moDCs to micropinocytose and receptormediated endocytose. In contrast, treatment with a sialidase significantly improved the capacity of moDCs to phagocytose Escherichia coli. The improved phagocytosis mechanism required E. coli sialic acids, indicating a mechanism of host–pathogen interaction dependent on sialic acid moieties. Sialidase-treated moDCs have increased expression of MHC and co-stimulatory molecules, suggesting a more mature phenotype. Experiments using mouse bone marrow-derived DCs (BMDCs) from ST3Gal-1-/- and ST6Gal-1-/- strains indicated that endocytosis and maturation are influenced by changes in either α2,3 or α2,6-sialylated glycans. The analysis of α2,6-sialylated, N-glycosylated proteins, strongly suggested the potential involvement of β2 integrins, underlying these mechanisms. 3) The effect of α2,6-sialylation in DC homing to lymph nodes was also analyzed. We observed that BMDCs deficient for ST6Gal-1 have an almost 50% reduction in DC homing, as assayed by in situ inflammation and adoptive transfer studies. A reduction in DC homing was also observed when wild type BMDCs were transferred into ST6Gal-1-/- recipient mice. Further investigations are necessary to identify the molecules involved in this process. 4) Finally, we also analyzed the impact of sialylation on DCs ability to prime T cells. Sialidase-treated moDCs show increased gene expression of IL-12, TNF-α, IL-6 and IL- 10 cytokines, and activation of the transcription factor nuclear factor-κB. Sialidase33 treated DCs induced a higher proliferative response of T cells with concomitant higher expression of interferon-γ, suggesting that the clearance of cell surface sialic acids contributes to the development of a T helper type 1 proinflammatory response. Together, our data strongly support sialic acid’s relevance in DC immune functions. Alterations of cell surface sialic acid content can alter the endocytosis/phagocytosis, maturation, migration/homing and the ability for T cell priming in human DCs. Moreover, sialic acid linkages created by ST6Gal-1 and ST3Gal-1 are functionally relevant. The engineering of cell surface sialylation, mediated by individual sialidases or sialyltransferases is a likely possibility to fine tune DC phagocytosis and immunological potency, with particular significance to DC-based therapies.

The Role of the Fbox Protein CG6758 in Xbp1s Induced Retinal Degeneration in Drosophila

The Unfolded Protein Response (UPR) is a signaling pathway that is activated by an accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) that causes ER stress. The activation of the UPR aims to restore ER homeostasis by attenuation of ER client protein translation, increased transcription of ER chaperones and ER associated degradation (ERAD) factors. If ER stress is too long or too strong, cells may die. The main signaling branch of the UPR is mediated by the ER transmembrane protein IRE1 and the transcription factor Xbp1. The active, spliced form of Xbp1 (Xbp1spliced) acts as a transcription factor with protective function against toxic protein aggregation. However, overexpression of Xbp1spliced in the developing Drosophila eye causes degeneration of the eye (“glossy” eye phenotype).(...)

Constitutive activation of Wnt signaling favors generation of memory CD8 T cells.

T cell factor-1 (TCF-1) and lymphoid enhancer-binding factor 1, the effector transcription factors of the canonical Wnt pathway, are known to be critical for normal thymocyte development. However, it is largely unknown if it has a role in regulating mature T cell activation and T cell-mediated immune responses. In this study, we demonstrate that, like IL-7Ralpha and CD62L, TCF-1 and lymphoid enhancer-binding factor 1 exhibit dynamic expression changes during T cell responses, being highly expressed in naive T cells, downregulated in effector T cells, and upregulated again in memory T cells. Enforced expression of a p45 TCF-1 isoform limited the expansion of Ag-specific CD8 T cells in response to Listeria monocytogenes infection. However, when the p45 transgene was coupled with ectopic expression of stabilized beta-catenin, more Ag-specific memory CD8 T cells were generated, with enhanced ability to produce IL-2. Moreover, these memory CD8 T cells expanded to a larger number of secondary effectors and cleared bacteria faster when the immunized mice were rechallenged with virulent L. monocytogenes. Furthermore, in response to vaccinia virus or lymphocytic choriomeningitis virus infection, more Ag-specific memory CD8 T cells were generated in the presence of p45 and stabilized beta-catenin transgenes. Although activated Wnt signaling also resulted in larger numbers of Ag-specific memory CD4 T cells, their functional attributes and expansion after the secondary infection were not improved. Thus, constitutive activation of the canonical Wnt pathway favors memory CD8 T cell formation during initial immunization, resulting in enhanced immunity upon second encounter with the same pathogen.

Molecular mechanisms for the regulation of skin homeostasis

L'ubiquitination est une modification des protéines conservée, consistant en l'addition de résidus « ubiquitine » et régulant le destin cellulaire des protéines. La protéine « TRAF-interacting protein » TRAIP (ou TRIP) est une ligase E3 qui catalyse l'étape finale de l'ubiquitination. TRAIP est conservé dans l'évolution et est nécessaire au développement des organismes puisque l'ablation de TRAIP conduit à la mort embryonnaire aussi bien de la drosophile que de la souris. De plus, la réduction de l'expression de TRAIP dans des kératinocytes épidermiques humains réprime la prolifération cellulaire et induit un arrêt du cycle cellulaire en phase Gl, soulignant le lien étroit entre TRAIP et la prolifération cellulaire. Comme les mécanismes de régulation de la prolifération jouent un rôle majeur dans l'homéostasie de la peau, il est important de caractériser la fonction de TRAIP dans ces mécanismes. En utilisant des approches in vitro, nous avons déterminé que la protéine TRAIP est instable, modifiée par l'addition d'ubiquitine et ayant une demi-vie d'environ 4 heures. Nos analyses ont également révélé que l'expression de TRAIP est dépendante du cycle cellulaire, atteignant un pic d'expression en phase G2/M et que l'induction de son expression s'effectue principalement au cours de la transition Gl/S. Nous avons identifié le facteur de transcription E2F1 comme en étant le responsable, en régulant directement le promoteur de TRAIP. Aussi, TRAIP endogène ou surexprimée est surtout localisée au niveau du nucléole, une organelle nucléaire qui est désassemblée pendant la division cellulaire. Pour examiner la localisation subcellulaire de TRAIP pendant la mitose, nous avons imagé la protéine TRAIP fusionnée à une protéine fluorescente, à l'intérieur de cellules vivantes nommées HeLa, à l'aide d'un microscope confocal. Dans ces conditions, TRAIP est majoritairement localisée autour des chromosomes en début de mitose, puis est arrangée au niveau de l'ADN chromosomique en fin de mitose. La détection de TRAIP endogène à l'aide d'un anticorps spécifique a confirmé cette localisation. Enfin, l'inactivation de TRAIP dans les cellules HeLa par interférence ARN a inhibé leur capacité à s'arrêter en milieu de mitose. Nos résultats suggèrent que le mécanisme sous-jacent peut être lié au point de contrôle de l'assemblage du fuseau mitotique. - Ubiquitination of proteins is a post-translational modification which decides the cellular fate of the protein. The TRAF-interacting protein (TRAIP, TRIP) functions as an E3 ubiquitin ligase mediating addition of ubiquitin moieties to proteins. TRAIP interacts with the deubiquitinase CYLD, a tumor suppressor whose functional inactivation leads to skin appendage tumors. TRAIP is required for early embryonic development since removal of TRAIP either in Drosophila or mice by mutations or knock¬out is lethal due to aberrant regulation of cell proliferation and apoptosis. Furthermore, shRNA- mediated knock-down of TRAIP in human epidermal keratinocytes (HEK) repressed cell proliferation and induced a Gl/S phase block in the cell cycle. Additionally, TRAIP expression is strongly down- regulated during keratinocyte differentiation supporting the notion of a tight link between TRAIP and cell proliferation. We thus examined the biological functions of TRAIP in epithelial cell proliferation. Using an in vitro approach, we could determine that the TRAIP protein is unstable, modified by addition of ubiquitin moieties after translation and exhibits a half-life of 3.7+/-1-6 hours. Our analysis revealed that the TRAIP expression is modulated in a cell-cycle dependent manner, reaching a maximum expression level in G2/M phases. In addition, the expression of TRAIP was particularly activated during Gl/S phase transition and we could identify the transcription factor E2F1 as an activator of the TRAIP gene promoter. Both endogenous and over-expressed TRAIP mainly localized to the nucleolus, a nuclear organelle which is disassembled during cell division. To examine the subcellular localization of TRAIP during M phase, we performed confocal live-cell imaging of a functional fluorescent protein TRAIP-GFP in HeLa cells. TRAIP was distributed in the cytoplasm and accumulated around mitotic chromosomes in pro- and meta-phasic cells. TRAIP was then confined to chromosomal DNA location in anaphase and later phases of mitosis. Immune-detection of endogenous TRAIP protein confirmed its particular localization in mitosis. Finally, inactivating TRAIP expression in HeLa cells using RNA interference abrogated the cells ability to stop or delay mitosis progression. Our results suggested that TRAIP may involve the spindle assembly checkpoint.

Oxygen tension controls the expression of the monocarboxylate transporter MCT4 in cultured mouse cortical astrocytes via a hypoxia-inducible factor-1α-mediated transcriptional regulation.

The monocarboxylate transporter MCT4 is a high capacity carrier important for lactate release from highly glycolytic cells. In the central nervous system, MCT4 is predominantly expressed by astrocytes. Surprisingly, MCT4 expression in cultured astrocytes is low, suggesting that a physiological characteristic, not met in culture conditions, is necessary. Here we demonstrate that reducing oxygen concentration from 21% to either 1 or 0% restored in a concentration-dependent manner the expression of MCT4 at the mRNA and protein levels in cultured astrocytes. This effect was specific for MCT4 since the expression of MCT1, the other astrocytic monocarboxylate transporter present in vitro, was not altered in such conditions. MCT4 expression was shown to be controlled by the transcription factor hypoxia-inducible factor-1α (HIF-1α) since under low oxygen levels, transfecting astrocyte cultures with a siRNA targeting HIF-1α largely prevented MCT4 induction. Moreover, the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG) induced MCT4 expression in astrocytes cultured in presence of 21% oxygen. In parallel, glycolytic activity was enhanced by exposure to 1% oxygen as demonstrated by the increased lactate release, an effect dependent on MCT4 expression. Finally, MCT4 expression was found to be necessary for astrocyte survival when exposed for a prolonged period to 1% oxygen. These data suggest that a major determinant of astrocyte MCT4 expression in vivo is likely the oxygen tension. This could be relevant in areas of high neuronal activity and oxygen consumption, favouring astrocytic lactate supply to neurons. Moreover, it could also play an important role for neuronal recovery after an ischemic episode.