Impaired aldehyde dehydrogenase 1 subfamily member 2A-dependent retinoic acid signaling is related with a mesenchymal-like phenotype and an unfavorable prognosis of head and neck squamous cell carcinoma.

BACKGROUND: An inverse correlation between expression of the aldehyde dehydrogenase 1 subfamily A2 (ALDH1A2) and gene promoter methylation has been identified as a common feature of oropharyngeal squamous cell carcinoma (OPSCC). Moreover, low ALDH1A2 expression was associated with an unfavorable prognosis of OPSCC patients, however the causal link between reduced ALDH1A2 function and treatment failure has not been addressed so far. METHODS: Serial sections from tissue microarrays of patients with primary OPSCC (n = 101) were stained by immunohistochemistry for key regulators of retinoic acid (RA) signaling, including ALDH1A2. Survival with respect to these regulators was investigated by univariate Kaplan-Meier analysis and multivariate Cox regression proportional hazard models. The impact of ALDH1A2-RAR signaling on tumor-relevant processes was addressed in established tumor cell lines and in an orthotopic mouse xenograft model. RESULTS: Immunohistochemical analysis showed an improved prognosis of ALDH1A2(high) OPSCC only in the presence of CRABP2, an intracellular RA transporter. Moreover, an ALDH1A2(high)CRABP2(high) staining pattern served as an independent predictor for progression-free (HR: 0.395, p = 0.007) and overall survival (HR: 0.303, p = 0.002), suggesting a critical impact of RA metabolism and signaling on clinical outcome. Functionally, ALDH1A2 expression and activity in tumor cell lines were related to RA levels. While administration of retinoids inhibited clonogenic growth and proliferation, the pharmacological inhibition of ALDH1A2-RAR signaling resulted in loss of cell-cell adhesion and a mesenchymal-like phenotype. Xenograft tumors derived from FaDu cells with stable silencing of ALDH1A2 and primary tumors from OPSCC patients with low ALDH1A2 expression exhibited a mesenchymal-like phenotype characterized by vimentin expression. CONCLUSIONS: This study has unraveled a critical role of ALDH1A2-RAR signaling in the pathogenesis of head and neck cancer and our data implicate that patients with ALDH1A2(low) tumors might benefit from adjuvant treatment with retinoids.

E2F1 mediates sustained lipogenesis and contributes to hepatic steatosis.

E2F transcription factors are known regulators of the cell cycle, proliferation, apoptosis, and differentiation. Here, we reveal that E2F1 plays an essential role in liver physiopathology through the regulation of glycolysis and lipogenesis. We demonstrate that E2F1 deficiency leads to a decrease in glycolysis and de novo synthesis of fatty acids in hepatocytes. We further demonstrate that E2F1 directly binds to the promoters of key lipogenic genes, including Fasn, but does not bind directly to genes encoding glycolysis pathway components, suggesting an indirect effect. In murine models, E2F1 expression and activity increased in response to feeding and upon insulin stimulation through canonical activation of the CDK4/pRB pathway. Moreover, E2F1 expression was increased in liver biopsies from obese, glucose-intolerant humans compared with biopsies from lean subjects. Finally, E2f1 deletion completely abrogated hepatic steatosis in different murine models of nonalcoholic fatty liver disease (NAFLD). In conclusion, our data demonstrate that E2F1 regulates lipid synthesis and glycolysis and thus contributes to the development of liver pathology.

Cell cycle control in Alphaproteobacteria.

NlmCategory="UNASSIGNED">Alphaproteobacteria include many medically and environmentally important organisms. Despite the diversity of their niches and lifestyles, from free-living to host-associated, they usually rely on very similar mechanisms to control their cell cycles. Studies on Caulobacter crescentus still lay the foundation for understanding the molecular details of pathways regulating DNA replication and cell division and coordinating these two processes with other events of the cell cycle. This review highlights recent discoveries on the regulation and the mode of action of conserved global regulators and small molecules like c-di-GMP and (p)ppGpp, which play key roles in cell cycle control. It also describes several newly identified mechanisms that modulate cell cycle progression in response to stresses or environmental conditions.

Hepatic circadian clock oscillators and nuclear receptors integrate microbiome-derived signals.

The liver is a key organ of metabolic homeostasis with functions that oscillate in response to food intake. Although liver and gut microbiome crosstalk has been reported, microbiome-mediated effects on peripheral circadian clocks and their output genes are less well known. Here, we report that germ-free (GF) mice display altered daily oscillation of clock gene expression with a concomitant change in the expression of clock output regulators. Mice exposed to microbes typically exhibit characterized activities of nuclear receptors, some of which (PPARα, LXRβ) regulate specific liver gene expression networks, but these activities are profoundly changed in GF mice. These alterations in microbiome-sensitive gene expression patterns are associated with daily alterations in lipid, glucose, and xenobiotic metabolism, protein turnover, and redox balance, as revealed by hepatic metabolome analyses. Moreover, at the systemic level, daily changes in the abundance of biomarkers such as HDL cholesterol, free fatty acids, FGF21, bilirubin, and lactate depend on the microbiome. Altogether, our results indicate that the microbiome is required for integration of liver clock oscillations that tune output activators and their effectors, thereby regulating metabolic gene expression for optimal liver function.

Hepatic circadian clock oscillators and nuclear receptors integrate microbiome-derived signals.

The liver is a key organ of metabolic homeostasis with functions that oscillate in response to food intake. Although liver and gut microbiome crosstalk has been reported, microbiome-mediated effects on peripheral circadian clocks and their output genes are less well known. Here, we report that germ-free (GF) mice display altered daily oscillation of clock gene expression with a concomitant change in the expression of clock output regulators. Mice exposed to microbes typically exhibit characterized activities of nuclear receptors, some of which (PPARα, LXRβ) regulate specific liver gene expression networks, but these activities are profoundly changed in GF mice. These alterations in microbiome-sensitive gene expression patterns are associated with daily alterations in lipid, glucose, and xenobiotic metabolism, protein turnover, and redox balance, as revealed by hepatic metabolome analyses. Moreover, at the systemic level, daily changes in the abundance of biomarkers such as HDL cholesterol, free fatty acids, FGF21, bilirubin, and lactate depend on the microbiome. Altogether, our results indicate that the microbiome is required for integration of liver clock oscillations that tune output activators and their effectors, thereby regulating metabolic gene expression for optimal liver function.

Developmental and tissue-specific involvement of peroxisome proliferator-activated receptor-alpha in the control of mouse uncoupling protein-3 gene expression.

Uncoupling protein-3 (UCP3) is a member of the mitochondrial carrier family expressed preferentially in skeletal muscle and heart. It appears to be involved in metabolic handling of fatty acids in a way that minimizes excessive production of reactive oxygen species. Fatty acids are powerful regulators of UCP3 gene transcription. We have found that the role of peroxisome proliferator-activated receptor-α (PPARα) on the control of UCP3 gene expression depends on the tissue and developmental stage. In adults, UCP3 mRNA expression is unaltered in skeletal muscle from PPARα-null mice both in basal conditions and under the stimulus of starvation. In contrast, UCP3 mRNA is down-regulated in adult heart both in fed and fasted PPARα-null mice. This occurs despite the increased levels of free fatty acids caused by fasting in PPARα-null mice. In neonates, PPARα-null mice show impaired UCP3 mRNA expression in skeletal muscle in response to milk intake, and this is not a result of reduced free fatty acid levels. The murine UCP3 promoter is activated by fatty acids through either PPARα or PPARδ but not by PPARγ or retinoid X receptor alone. PPARδ-dependent activation could be a potential compensatory mechanism to ensure appropriate expression of UCP3 gene in adult skeletal muscle in the absence of PPARα. However, among transcripts from other PPARα and PPARδ target genes, only those acutely induced by milk intake in wild-type neonates were altered in muscle or heart from PPARα-null neonates. Thus, PPARα-dependent regulation is required for appropriate gene regulation of UCP3 as part of the subset of fatty-acid-responsive genes in neonatal muscle and heart.

Impact of T cell receptor affinity on the molecular mechanisms modulating CD8 T cell signalling and function against NY-ESO-1 tumour antigen

It is well established that cytotoxic T lymphocytes play a pivotal role in the protection against intracellular pathogens and tumour cells. Such protective immune responses rely on the specific T cell receptor (TCR)-mediated recognition by CD8 T cells of small antigenic peptides presented in the context of class-I Major Histocompatibility Complex molecules (pMHCs) on the surface of infected or malignant cells. The strength (affinity/avidity) of this interaction is a major correlate of protection. Although tumour-reactive CD8 T cells can be observed in cancer patients, anti-tumour immune responses are often ineffective in controlling or eradicating the disease due to the relative low TCR affinity of these cells. To overcome this limitation, tumour-specific CD8 T cells can be genetically modified to express TCRs of improved binding strength against a defined tumour antigen before adoptive cell transfer into cancer patients. We previously generated a panel of TCRs specific for the cancer-testis antigen NY-ESO-l,57.165 with progressively increased affinities for the pMHC complex, thus providing us with a unique tool to investigate the causal link between the surface expression of such TCRs and T cell activation and function. We recently demonstrated that anti-tumour CD8 T cell reactivity could only be improved within physiological affinity limits, beyond which drastic functional declines were observed, suggesting the presence of multiple regulatory mechanisms limiting T cell activation and function in a TCR affinity-dependent manner. The overarching goal of this thesis was (i) to assess the precise impact of TCR affinity on T cell activation and signalling at the molecular level and (ii) to gain further insights on the mechanisms that regulate and delimitate maximal/optimized CD8 T cell activation and signalling. Specifically, by combining several technical approaches we characterized the activation status of proximal (i.e. CD3Ç, Lek, and ZAP-70) and distal (i.e. ERK1/2) signalling molecules along the TCR affinity gradient. Moreover, we assessed the extent of TCR downmodulation, a critical step for initial T cell activation. CD8 T cells engineered with the optimal TCR affinity variants showed increased activation levels of both proximal and distal signalling molecules when compared to the wild-type T cells. Our analyses also highlighted the "paradoxical" status of tumour-reactive CD8 T cells bearing very high TCR affinities, which retained strong proximal signalling capacity and TCR downmodulation, but were unable to propagate signalling distally (i.e. pERKl/2), resulting in impaired cell-mediated functions. Importantly, these very high affinity T cells displayed maximal levels of SHP-1 and SHP-2 phosphatases, two negative regulatory molecules, and this correlated with a partial pERKl/2 signalling recovery upon pharmacological SHP-l/SHP-2 inhibition. These findings revealed the putative presence of inhibitory regulators of the TCR signalling cascade acting very rapidly following tumour-specific stimulation. Moreover, the very high affinity T cells were only able to transiently express enhanced proximal signalling molecules, suggesting the presence of an additional level of regulation that operates through the activation of negative feedback loops over time, limiting the duration of the TCR-mediated signalling. Overall, the determination of TCR-pMHC binding parameters eliciting optimal CD8 T cell activation, signalling, and effector function while guaranteeing high antigen specificity, together with the identification of critical regulatory mechanisms acting proximally in the TCR signalling cascade, will directly contribute to optimize and support the development of future TCR-based adoptive T cell strategies for the treatment of malignant diseases. -- Les lymphocytes T CD8 cytotoxiques jouent un rôle prédominant dans la protection contre les pathogènes intracellulaires et les cellules tumorales. Ces réponses immunitaires dépendent de la spécificité avec laquelle les récepteurs T (TCR) des lymphocytes CD8 reconnaissent les peptides antigéniques présentés par les molécules du complexe Majeur de Histocompatibilité de classe I (pCMH) à la surface des cellules infectées ou malignes. La force (ou affinité/avidité) de l'interaction du TCR-pCMH est un corrélat majeur de protection. Les réponses immunitaires sont cependant souvent inefficaces et ne permettent pas de contrôler ou d'éliminer les cellules tumorales chez les patients atteint du cancer, et ce à cause de la relative faible reconnaissance des TCRs exprimés par les lymphocytes T CD8 envers les antigènes tumoraux. Afin de surmonter cette limitation, les cellules T anti-tumorales peuvent être génétiquement modifiées en les dotant de TCRs préalablement optimisés afin d'augmenter leur reconnaissance ou affinité contre les antigènes tumoraux, avant leur ré¬infusion dans le patient. Nous avons récemment généré des cellules T CD8 exprimant un panel de TCRs spécifiques pour l'antigène tumoral NY-ESO-l157.16J avec des affinités croissantes, permettant ainsi d'investiguer la causalité directe entre l'affinité du TCR-pCMH et la fonction des cellules T CD8. Nous avons démontré que la réactivité anti-tumorale pouvait être améliorée en augmentant l'affinité du TCR dans une intervalle physiologique, mais au delà duquel nous observons un important déclin fonctionnel. Ces résultats suggèrent la présence de mécanismes de régulation limitant l'activation des cellules T de manière dépendante de l'affinité du TCR. Le but de cette thèse a été (i) de définir l'impact précis de l'affinité du TCR sur l'activation et la signalisation des cellules T CD8 au niveau moléculaire et (ii) d'acquérir de nouvelles connaissances sur les mécanismes qui régulent et délimitent l'activation et la signalisation maximale des cellules T CD8 optimisées. Spécifiquement, en combinant plusieurs approches technologiques, nous avons caractérisé l'état d'activation de différentes protéines de la voie de signalisation proximale (CD3Ç, Lek et ZAP-70) et distale (ERK1/2) le long du gradient d'affinité du TCR, ainsi que l'internalisation du TCR, une étape clef dans l'activation initiale des cellules T. Les lymphocytes T CD8 exprimant des TCRs d'affinité optimale ont montré des niveaux d'activation augmentés des molécules proximales et distales par rapport aux cellules de type sauvage (wild-type). Nos analyses ont également mis en évidence un paradoxe chez les cellules T CD8 équipées avec des TCRs de très haute affinité. En effet, ces cellules anti-tumorales sont capables d'activer leurs circuits biochimiques au niveau proximal et d'internaliser efficacement leur TCR, mais ne parviennent pas à propager les signaux biochimiques dépendants du TCR jusqu'au niveau distal (via phospho-ERKl/2), avec pour conséquence une limitation de leur capacité fonctionnelle. Finalement, nous avons démontré que SHP-1 et SHP-2, deux phosphatases avec des propriétés régulatrices négatives, étaient majoritairement exprimées dans les cellules T CD8 de très hautes affinités. Une récupération partielle des niveaux d'activation de ERK1/2 a pu être observée après l'inhibition pharmacologique de ces phosphatases. Ces découvertes révèlent la présence de régulateurs moléculaires qui inhibent le complexe de signalisation du TCR très rapidement après la stimulation anti-tumorale. De plus, les cellules T de très hautes affinités ne sont capables d'activer les molécules de la cascade de signalisation proximale que de manière transitoire, suggérant ainsi un second niveau de régulation via l'activation de mécanismes de rétroaction prenant place progressivement au cours du temps et limitant la durée de la signalisation dépendante du TCR. En résumé, la détermination des paramètres impliqués dans l'interaction du TCR-pCMH permettant l'activation de voies de signalisation et des fonctions effectrices optimales ainsi que l'identification des mécanismes de régulation au niveau proximal de la cascade de signalisation du TCR contribuent directement à l'optimisation et au développement de stratégies anti-tumorales basées sur l'ingénierie des TCRs pour le traitement des maladies malignes.

Neuronal activity controls the antagonistic between PGC-1α and SMRT in regulating antioxidant defences

Transcriptional coactivators and corepressors often have multiple targets and can have opposing actions on transcription and downstream physiological events. The coactivator peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α is under-expressed in Huntington's disease and is a regulator of antioxidant defenses and mitochondrial biogenesis. We show that in primary cortical neurons, expression of PGC-1α strongly promotes resistance to excitotoxic and oxidative stress in a cell autonomous manner, whereas knockdown increases sensitivity. In contrast, the transcriptional corepressor silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) specifically antagonizes PGC-1α-mediated antioxidant effects. The antagonistic balance between PGC-1α and SMRT is upset in favor of PGC-1α by synaptic activity. Synaptic activity triggers nuclear export of SMRT reliant on multiple regions of the protein. Concommitantly, synaptic activity post-translationally enhances the transactivating potential of PGC-1α in a p38-dependent manner, as well as upregulating cyclic-AMP response element binding protein-dependent PGC-1α transcription. Activity-dependent targeting of PGC-1α results in enhanced gene expression mediated by the thyroid hormone receptor, a prototypical transcription factor coactivated by PGC-1α and repressed by SMRT. As a consequence of these events, SMRT is unable to antagonize PGC-1α-mediated resistance to oxidative stress in synaptically active neurons. Thus, PGC-1α and SMRT are antagonistic regulators of neuronal vulnerability to oxidative stress. Further, this coactivatorcorepressor antagonism is regulated by the activity status of the cell, with implications for neuronal viability.

Mitotic regulation by Polo-like kinase 1 and the Chromosomal Passenger Complex

During mitosis, the duplicated genome must be accurately divided between two daughter cells. Polo-like kinase 1 (Plk1) and Aurora B kinase, together with its binding partners Incenp, Survivin and Borealin (chromosomal passenger complex, CPC), have key roles in coordinating mitotic events. The accuracy of cell division is safeguarded by a signaling cascade termed the mitotic spindle checkpoint (SC), which ensures that chromosomes are not physically separated before correct bipolar attachments have been formed between kinetochores and spindle microtubules (MT). An inhibitory “wait anaphase” signal, which delays chromosome separation (anaphase onset), is created at individual kinetochores and broadcasted throughout the cell in response to lack of kinetochore-microtubule (kMT) attachment or proper interkinetochore tension. It is believed that the fast turnover of SC molecules at kinetochores contributes to the cell’s ability to produce this signal and enables rapid responses to changing cellular conditions. Kinetochores that lack MT attachment and tension express a certain phosphoepitope called the 3F3/2 phosphoepitope, which has been linked to SC signaling. In the experimental part, we investigated the regulation of the 3F3/2 phosphoepitope, analyzed whether CPC molecules turn over at centromeres, and dissected the mitotic roles of the CPC using a microinjection technique that allowed precise temporal control over its function. We found that the kinetochore 3F3/2 phosphoepitope is created by Plk1, and that CPC proteins exhibit constant exchange at centromeres. Moreover, we found that CPC function is necessary in the regulation of chromatid movements and spindle morphology in anaphase. In summary, we identified new functions of key mitotic regulators Plk1 and CPC, and provided insighs into the coordination of mitotic events.

Negative Regulation of Receptor Tyrosine Kinases by T-cell Protein Tyrosine Phosphatase

Protein tyrosine phosphorylation controls a wide array of cellular responses such as growth, migration, proliferation, differentiation, metabolism and cytoskeletal organisation. Tyrosine phosphorylation is a dynamic process involving the competing activities of protein tyrosine kinases and protein tyrosine phosphatases. The protein tyrosine kinases are further divided into non-receptor- and receptor tyrosine kinases. The latter are transmembrane glycoproteins activated by the binding of specific ligands, mostly growth factors, to their extracellular domain, transmitting different signals to the cell. Growth factor receptors such as the epidermal growth factor receptor, vascular endothelial growth factor receptor 2 and platelet-derived growth factor receptor β, belong to the receptor tyrosine kinases, the signalling of which is often disturbed in various diseases, including cancer. This has led to the development of receptor tyrosine kinase antagonists for use as anti-cancer drugs. As the receptor tyrosine kinases, also the protein tyrosine phosphatases can be divided into receptor- and non-receptor types. The protein tyrosine phosphatases have attained much less attention than the receptor tyrosine kinases partly because they were identified later. However, accumulating evidence shows that the protein tyrosine phosphatases have important roles as specific and active regulators of tyrosine phosphorylation in cells and of physiological processes. Consequently, the protein tyrosine phosphatases are receiving arising interest as novel drug targets. The aim of this work was to elucidate the negative regulation of receptor tyrosine kinases by one non-receptor protein tyrosine phosphatase, T-cell protein tyrosine phosphatase TCPTP. The results show that TCPTP activated by cell adhesion receptor integrin α1 functions as a negative regulator of the epidermal growth factor receptor. It was also found that TCPTP affects vascular endothelial growth factor receptor 2 signalling and angiogenesis. Lastly, a High-throughput screen with 64,280 compounds was performed to identify novel TCPTP activators, resulting in identification of one small molecule compound capable of exerting similar effects on TCPTP signalling as integrin α1. This compound is shown to downregulate signalling of epidermal growth factor receptor and platelet-derived growth factor receptor β, as well as to inhibit cell proliferation and angiogenesis. Our results suggest that a suitable small-molecule TCPTP activator could be utilized in the development of novel anti-cancer drugs.

β1 integrin regulation

Integrins are heterodimeric adhesion receptors mediating adhesion to extracellular matrix proteins and to other cells. Integrins are important in embryonic development, structural integrity of connective tissue, blood thrombus formation, and immune defense system. Integrins are transmembrane proteins whose ligand binding capacity (activity) is regulated by large conformational changes. Extracellular ligand binding or intracellular effector binding to integrin cytoplasmic face regulate integrin activity. Integrins are thus able to mediate bi-directional signaling. Integrin function is also regulated by intracellular location. Integrins are constantly recycled from endocytic vesicles to plasma membrane, and this has been shown to be important for cell migration and invasion as well. Deregulation of integrin functionality can lead to deleterious illnesses, such as bleeding or inflammatory disorders. It is also evident that integrin deregulation is associated with cancer progression. In this study, a novel Beta1 integrin associating protein, Rab21, was characterized. Rab21 binding to integrin cytoplasmic tail was shown to be important for Beta1 integrin endo- and exocytosis – intracellular trafficking. It was furher shown that this interaction has an important role in cell adhesion, migration, as well as in the final step of cell division, cytokinesis. This work showed that abrogation of Rab21 function or β1 integrin endocytic traffic, can lead to defects in cell division and results in formation of multinucleated cells. Multinucleation and especially tetraploidy can be a transient pathway to aneuploidy and tumorigenesis. This work characterized chromosomal deletions in rab21 locus in ovarian and prostate cancer samples and showed that a cell line with rab21 deletion also had impairment in cell division, which could be rescued by Rab21 re-expression. The work demonstrates an important role for Rab21 and Beta1 integrin traffic regulation in cell adhesion and division, and suggests a probable associaton with tumorigenesis. In this study, Beta1 integrin activity regulation was also addressed. A novel cell array platform for genome-scale RNAi screenings was characterized here. More than 4500 genes were knocked-down in prostate cancer cells using siRNA-mediated silencing. The effects on Beta1 integrin activity were analyzed upon knock-downs. The screen identified more that 400 putative regulators of Beta1 integrin activity in prostate cancer. In conclusion, this work will help us to understand complex regulatory pathways involved in cancer cell adhesion and migration.

Rcor2 underexpression in senescent mice: a target for inflammaging?

BACKGROUND: Aging is characterized by a low-grade systemic inflammation that contributes to the pathogenesis of neurodegenerative disorders such as Alzheimer's disease (AD). However, little knowledge is currently available on the molecular processes leading to chronic neuroinflammation. In this context, recent studies have described the role of chromatin regulators in inflammation and longevity including the REST corepressor (Rcor)-2 factor, which seems to be involved in an inflammatory suppressive program. METHODS: To assess the impact of Rcor2 in age-related inflammation, gene expression levels were quantified in different tissues and ages of the spontaneous senescence-accelerated P8 mouse (P8) using the SAMR1 mouse (R1) as a control. Specific siRNA transfection in P8 and R1 astrocyte cultures was used to determine Rcor2 involvement in the modulation of neuroinflammation. The effect of lipopolysaccharide (LPS) treatment on Rcor2 levels and neuroinflammation was analyzed both in vivo and in vitro. RESULTS: P8 mice presented a dramatic decrease in Rcor2 gene expression compared with R1 controls in splenocytes, an alteration also observed in the brain cortex, hippocampus and primary astrocytes of these mice. Rcor2 reduction in astrocytes was accompanied by an increased basal expression of the interleukin (Il)-6 gene. Strikingly, intraperitoneal LPS injection in R1 mice downregulated Rcor2 in the hippocampus, with a concomitant upregulation of tumor necrosis factor (Tnf-α), Il1-β and Il6 genes. A negative correlation between Rcor2 and Il6 gene expression was also verified in LPS-treated C6 glioma cells. Knock down of Rcor2 by siRNA transfection (siRcor2) in R1 astrocytes upregulated Il6 gene expression while siRcor2 further increased Il6 expression in P8 astrocytes. Moreover, LPS activation provoked a further downregulation of Rcor2 and an amplified induction of Il6 in siRcor2-tranfected astrocytes. CONCLUSIONS: Data presented here show interplay between Rcor2 downregulation and increased inflammation and suggest that Rcor2 may be a key regulator of inflammaging

Interferentes endócrinos no ambiente

Although the hypothesis that environmental chemicals may exhibit endocrine disrupting effects is not new, the issue has been a growing level of concern due to reports of increased incidences of endocrine-related disease in humans, including declining male fertility, and more significantly, to adverse physiological effects observed in wildlife where cause and effect relationships are more evident. The list of endocrine disrupting chemicals (EDCs) includes a range of anthropogenic compounds, phytoestrogens, naturally occurring sex steroids and synthetic estrogens. Within the aquatic environment, the presence of EDCs has concerned many scientists and water quality regulators. Discharge of effluents from treatment facilities is likely to be a significant source of input of contaminants to many systems, and the potential for concentration of hydrophilic compounds and transformation products within sludges has implications for their disposal. Then, understanding the processes and the fate of EDCs on the environment, as well as the mechanisms of endocrine disruption, may facilitate controlling or limiting exposure of both humans and the environment to these compounds.

Progressive Purkinje Cell Degeneration in tambaleante Mutant Mice Is a Consequence of a Missense Mutation in HERC1 E3 Ubiquitin Ligase

The HERC gene family encodes proteins with two characteristic domains: HECT and RCC1-like. Proteins with HECT domain shave been described to function as ubiquitin ligases, and those that contain RCC1-like domains have been reported to function as GTPases regulators. These two activities are essential in a number of important cellular processes such as cell cycle, cell signaling, and membrane trafficking. Mutations affecting these domains have been found associated with retinitis pigmentosa, amyotrophic lateral sclerosis, and cancer. In humans, six HERC genes have been reported which encode two subgroups of HERC proteins: large (HERC1-2) and small (HERC3-6). The giant HERC1 protein was the first to be identified. It has been involved in membrane trafficking and cell proliferation/growth through its interactions with clathrin, M2-pyruvate kinase, and TSC2 proteins. Mutations affecting other members of the HERC family have been found to be associated with sterility and growth retardation. Here, we report the characterization of a recessive mutation named tambaleante, which causes progressive Purkinje cell degeneration leading to severe ataxia with reduced growth and lifespan in homozygous mice aged over two months. We mapped this mutation in mouse chromosome 9 and then performed positional cloning. We found a GuA transition at position 1448, causing a Gly to Glu substitution (Gly483Glu) in the highly conserved N- terminal RCC1-like domain of the HERC1 protein. Successful transgenic rescue, with either a mouse BAC containing the normal copy of Herc1 or with the human HERC1 cDNA, validated our findings. Histological and biochemical studies revealed extensive autophagy associated with an increase of the mutant protein level and a decrease of mTOR activity. Our observations concerning this first mutation in the Herc1 gene contribute to the functional annotation of the encoded E3 ubiquitin ligase and underline the crucial and unexpected role of this protein in Purkinje cell physiology.

New mechanisms regulating human Th1 and Th2 cell differentiation

Selective development of human T helper (Th) cells into functionally distinct Th1 and Th2 subtypes plays an essential role in the host immune response towards pathogens. However, abnormal function or differentiation of these cells can lead to development of various autoimmune diseases as well as asthma and allergy. Therefore, identification of key factors and the molecular mechanisms mediating Th1 and Th2 cell differentiation is important for understanding the molecular mechanisms of these diseases. The goal of this study was to identify novel factors involved in the regulation of Th1 and Th2 differentiation processes. A new method was optimized for enrichment of transiently transfected resting human primary T lymphocytes, that allowed the study of the influence of genes of interest in human Th1/Th2 cell differentiation and other primary Th cell functions. Functional characterization of PRELI, a novel activation-induced protein in human Th cells, identified it as a mitochondrial protein involved in the regulation of Th cell differentiation and apoptosis. By influencing the intracellular redox state, PRELI induces mitochondrial apoptosis pathway and downregulates STAT6 and Th2 differentiation. The data suggested that Calpain, an oxidative stress induced cysteine protease, is involved as a mediator in PRELI-induced downregulation of STAT6. PIM serine/threonine-specific kinases were identified as new regulators of human Th1 cell differentiation. PIM1 and PIM2 kinases were shown to be preferentially expressed in Th1 cells as compared to Th2 cells. RNA interference studies showed that PIM kinases enhance the production of IFN, the hallmark cytokine produced by Th1 cells. They also induce the expression of the key Th1-driving factor T-bet and the IL-12 signaling pathway during early phases of Th1 cell differentiation. Taken together, new regulators of human T helper cell differentiation were identified in this study, which provides new insights into the signaling mechanisms controlling the selective activation of human Th cell subsets.