Distribution of events of positive selection and population differentiation in a metabolic pathway: the case of asparagine N-glycosylation

Background: Asparagine N-Glycosylation is one of the most important forms of protein post-translational modification in eukaryotes. This metabolic pathway can be subdivided into two parts: an upstream sub-pathway required for achieving proper folding for most of the proteins synthesized in the secretory pathway, and a downstream sub-pathway required to give variability to trans-membrane proteins, and involved in adaptation to the environment andinnate immunity. Here we analyze the nucleotide variability of the genes of this pathway in human populations, identifying which genes show greater population differentiation and which genes show signatures of recent positive selection. We also compare how these signals are distributed between the upstream and the downstream parts of the pathway, with the aim of exploring how forces of population differentiation and positive selection vary among genes involved in the same metabolic pathway but subject to different functional constraints. Results:Our results show that genes in the downstream part of the pathway are more likely to show a signature of population differentiation, while events of positive selection are equally distributed among the two parts of the pathway. Moreover, events of positive selection arefrequent on genes that are known to be at bifurcation points, and that are identified as beingin key position by a network-level analysis such as MGAT3 and GCS1.Conclusions: These findings indicate that the upstream part of the Asparagine N-Glycosylation pathway has lower diversity among populations, while the downstream part is freer to tolerate diversity among populations. Moreover, the distribution of signatures of population differentiation and positive selection can change between parts of a pathway, especially between parts that are exposed to different functional constraints. Our results support the hypothesis that genes involved in constitutive processes can be expected to show lower population differentiation, while genes involved in traits related to the environment should show higher variability. Taken together, this work broadens our knowledge on how events of population differentiation and of positive selection are distributed among different parts of a metabolic pathway.

Lifespan extension by calorie restriction relies on the Sty1 MAP kinase stress pathway

Either calorie restriction, loss of function of the nutrient-dependent PKA or TOR/SCH9 pathways, or activation of stress defences improves longevity in different eukaryotes. However, the molecular links between glucose depletion, nutrient-dependent pathways and stress responses are unknown. Here we show that either calorie restriction or inactivation of nutrient-dependent pathways induces life-span extension in fission yeast, and that such effect is dependent on the activation of the stress-dependent Sty1 MAP kinase. During transition to stationary phase in glucose-limiting conditions, Sty1 becomes activated and triggers a transcriptional stress program, whereas such activation does not occur under glucose-rich conditions. Deletion of the genes coding for the SCH9-homologue Sck2 or the Pka1 kinases, or mutations leading to constitutive activation of the Sty1 stress pathway increase life span under glucose-rich conditions, and importantly such beneficial effects depend ultimately on Sty1. Furthermore, cells lacking Pka1 display enhanced oxygen consumption and Sty1 activation under glucose-rich conditions. We conclude that calorie restriction favours oxidative metabolism, reactive oxygen species production and Sty1 MAP kinase activation, and this stress pathway favours life-span extension.

FGF signaling controls caudal hindbrain specification through Ras-ERK1/2 pathway

Background: During early steps of embryonic development the hindbrain undergoes a regionalization process along the anterior-posterior (AP) axis that leads to a metameric organization in a series of rhombomeres (r). Refinement of the AP identities within the hindbrain requires the establishment of local signaling centers, which emit signals that pattern territories in their vicinity. Previous results demonstrated that the transcription factor vHnf1 confers caudal identity to the hindbrain inducing Krox20 in r5 and MafB/Kreisler in r5 and r6, through FGF signaling [1].Results: We show that in the chick hindbrain, Fgf3 is transcriptionally activated as early as 30 min after mvHnf1 electroporation, suggesting that it is a direct target of this transcription factor. We also analyzed the expression profiles of FGF activity readouts, such as MKP3 and Pea3, and showed that both are expressed within the hindbrain at early stages of embryonic development. In addition, MKP3 is induced upon overexpression of mFgf3 or mvHnf1 in the hindbrain, confirming vHnf1 is upstream FGF signaling. Finally, we addressed the question of which of the FGF-responding intracellular pathways were active and involved in the regulation of Krox20 and MafB in the hindbrain. While Ras-ERK1/2 activity is necessary for MKP3, Krox20 and MafB induction, PI3K-Akt is not involved in that process.Conclusion: Based on these observations we propose that vHnf1 acts directly through FGF3, and promotes caudal hindbrain identity by activating MafB and Krox20 via the Ras-ERK1/2 intracellular pathway.

Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule.

Cell death is achieved by two fundamentally different mechanisms: apoptosis and necrosis. Apoptosis is dependent on caspase activation, whereas the caspase-independent necrotic signaling pathway remains largely uncharacterized. We show here that Fas kills activated primary T cells efficiently in the absence of active caspases, which results in necrotic morphological changes and late mitochondrial damage but no cytochrome c release. This Fas ligand-induced caspase-independent death is absent in T cells that are deficient in either Fas-associated death domain (FADD) or receptor-interacting protein (RIP). RIP is also required for necrotic death induced by tumor necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL). In contrast to its role in nuclear factor kappa B activation, RIP requires its own kinase activity for death signaling. Thus, Fas, TRAIL and TNF receptors can initiate cell death by two alternative pathways, one relying on caspase-8 and the other dependent on the kinase RIP.

Regulation of the NaCI cotransporter by the SGK1-Nedd4-2 pathway

SummaryRegulation of renal Na+ transport is essential for controlling blood pressure, as well as Na+ and K+ homeostasis. Aldosterone stimulates Na+ reabsorption in the aldosterone-sensitive distal nephron (ASDN), via the Na+-CI" cotransporter (NCC) in the distal convoluted tubule (DCT), and the epithelial Na+ channel (ENaC) in the late DCT, connecting tubule and collecting duct. Importantly, aldosterone increases NCC protein expression by an unknown post-translational mechanism. The ubiquitin-protein ligase Nedd4-2 is expressed along the ASDN and regulates ENaC: under aldosterone induction, the serum/glucocorticoid-regulated kinase SGK1 phosphorylates Nedd4-2 on S328, thus preventing the Nedd4-2/ENaC interaction, ubiquitylation and degradation of the channel. Here, we present evidence that Nedd4-2 regulates NCC. In transfected HEK293 cells, Nedd4-2 co-immunoprecipitates with NCC and stimulates NCC ubiquitylation at the cell surface. In Xenopus laevis oocytes, co- expression of NCC with wild-type Nedd4-2, but not its catalytically inactive mutant, strongly decreases NCC activity and surface expression. This inhibition is prevented by SGK1 in a kinase-dependent manner. Moreover, we show that NCC expression is up-regulated in inducible renal tubule-specific Nedd4-2 knockout mice and in mDCT15 cells silenced for Nedd4-2. On the other hand, in inducible renal tubule-specific SGK1 knockout mice, NCC expression is down-regulated.Interestingly, in contrast to ENaC, Nedd4-2-mediated NCC inhibition is independent of a PY motif in NCC. Moreover, whereas single mutations of Nedd4-2 S328 or S222 to alanine do not interfere with SGK1 action, the double mutation enhances Nedd4-2 activity and abolishes SGK1-dependent inhibition. These results indicate that NCC expression and activity is controlled by a regulatory pathway involving SGK1 and Nedd4-2, and provides an explanation for the well-known aldosterone-induced increase in NCC protein expression.RésuméLa régulation du transport de sodium est cruciale dans le maintien de la pression artérielle. L'aldostérone stimule la réabsorption de Na+ dans la partie du néphron sensible à l'aldostérone (ASDN), via le co-transporteur Na+-CI" (NCC) au niveau du tubule contourné distale et via le canal à sodium (Epithelial Na+ Channel ; ENaC) dans la deuxième partie du tubule contourné distale, dans le tube connecteur et le tube collecteur. L'aldostérone augmente l'expression de NCC au niveau protéique par un mécanisme non élucidé. La protéine ubiquitine ligase Nedd4-2 est exprimée tout le long du néphron sensible à l'aldostérone. ENaC est connu pour être régulé par Nedd4-2. Suite à une stimulation par l'aldostérone, la kinase Ser/Thr SGK1 phosphoryle Nedd4-2, ce qui empêche l'interaction entre Nedd4-2 et ENaC. Dans des cellules HEK293 transfectées, nous avons montré que Nedd4-2 interagit avec le co-transporteur NCC et stimule l'ubiquitylation de NCC à la surface. Nous avons montré dans les oocytes de Xenopus laevis que l'expression de NCC avec Nedd4-2 diminue l'activité du co-transporteur. Cette diminution n'est pas observée lorsqu'on exprime NCC avec le mutant inactif de Nedd4-2. Cette inhibition de NCC est contrée par SGK1. L'effet de SGK1 sur NCC dépend de son activité kinase. Nous avons montré dans des souris knock-out pour Nedd4-2, dans le néphron et de manière inductible, que l'expression de NCC est augmentée. Nous avons également montré que la suppression de la protéine Nedd4-2 dans les cellules mDCT15 provoque l'augmentation de NCC. Au contraire dans les souris knock-out pour la kinase SGK1, dans le néphron et de manière inductible, nous observons une diminution de la protéine NCC. Contrairement à ce qui a été montré pour le canal ENaC l'inhibition de NCC par Nedd4-2 est indépendante des motifs PY. De plus, La mutation des sérines 328 ou 222 sur Nedd4-2 en alanine n'interfère pas avec l'action de SGK1 pour prévenir l'inhibition. Par contre, la double mutation, les sérines 222 et 328 mutées en alanine, augmente l'action de Nedd4-2 sur l'activité de NCC et prévient l'effet de SGK1. Ces résultats montrent que l'expression et l'activité de NCC sont contrôlées par une voie de régulation impliquant Nedd4-2-SGK1 et nous fournissent une explication pour l'augmentation de NCC observé après une induction avec l'aldostérone.Résumé large publicOn estime que des millions de personnes seraient hypertendues. L'hypertension artérielle est responsable d'environ 8 millions de décès par ans dans le monde. L'hypertension est responsable de la moitié environs des accidents cardiaques, mais aussi des accidents vasculaires cérébraux. Il est très important de comprendre les mécanismes qui se trouvent derrière cette pathologie.Le co-transporteur NCC joue un grand rôle dans le maintien de la balance sodique. Il a été montré que des perturbations dans l'expression de NCC pouvaient engendrer de l'hypertension.Le co-transporteur NCC est exprimé dans la partie distale du néphron, l'unité fonctionnelle du rein. Plusieurs études ont montrées que NCC était sous le contrôle de l'hormone aldostérone.Le travail de cette thèse consiste à étudier les mécanismes impliqués dans la régulation de NCC. On a ainsi pu montrer que NCC interagit avec la protéine ubiquitine ligase Nedd4-2. La protéine Nedd4-2 diminue l'expression de NCC à la surface cellulaire et aussi son activité Nous avons également montré que la kinase SGK1 pouvait prévenir l'interaction entre Nedd4-2 et NCC par phosphorylation de Nedd4-2. Nous avons montré dans des souris deletée pour Nedd4-2, dans le néphron, que l'expression de NCC est augmentée. Nous avons également montré que la suppression de la protéine Nedd4-2 dans les cellules mDCT15 provoque l'augmentation de NCC. Au contraire, dans les souris deletée pour la kinase SGK1, dans le néphron, nous observons une diminution de la protéine NCC. La connaissance des processus impliqués dans la régulation du co-transporteur NCC pourrait amener au développement de nouveau médicaments pour soigner l'hypertension.

Novel Agents Targeting the IGF-1R/PI3K Pathway Impair Cell Proliferation and Survival in Subsets of Medulloblastoma and Neuroblastoma.

The receptor tyrosine kinase (RTK)/phosphoinositide 3-kinase (PI3K) pathway is fundamental for cancer cell proliferation and is known to be frequently altered and activated in neoplasia, including embryonal tumors. Based on the high frequency of alterations, targeting components of the PI3K signaling pathway is considered to be a promising therapeutic approach for cancer treatment. Here, we have investigated the potential of targeting the axis of the insulin-like growth factor-1 receptor (IGF-1R) and PI3K signaling in two common cancers of childhood: neuroblastoma, the most common extracranial tumor in children and medulloblastoma, the most frequent malignant childhood brain tumor. By treating neuroblastoma and medulloblastoma cells with R1507, a specific humanized monoclonal antibody against the IGF-1R, we could observe cell line-specific responses and in some cases a strong decrease in cell proliferation. In contrast, targeting the PI3K p110α with the specific inhibitor PIK75 resulted in broad anti-proliferative effects in a panel of neuro- and medulloblastoma cell lines. Additionally, sensitization to commonly used chemotherapeutic agents occurred in neuroblastoma cells upon treatment with R1507 or PIK75. Furthermore, by studying the expression and phosphorylation state of IGF-1R/PI3K downstream signaling targets we found down-regulated signaling pathway activation. In addition, apoptosis occurred in embryonal tumor cells after treatment with PIK75 or R1507. Together, our studies demonstrate the potential of targeting the IGF-1R/PI3K signaling axis in embryonal tumors. Hopefully, this knowledge will contribute to the development of urgently required new targeted therapies for embryonal tumors.

Therapeutic PD-1 pathway blockade augments with other modalities of immunotherapy T-cell function to prevent immune decline in ovarian cancer.

The tumor microenvironment mediates induction of the immunosuppressive programmed cell death-1 (PD-1) pathway, and targeted interventions against this pathway can help restore antitumor immunity. To gain insight into these responses, we studied the interaction between PD-1 expressed on T cells and its ligands (PD-1:PD-L1, PD-1:PD-L2, and PD-L1:B7.1), expressed on other cells in the tumor microenvironment, using a syngeneic orthotopic mouse model of epithelial ovarian cancer (ID8). Exhaustion of tumor-infiltrating lymphocytes (TIL) correlated with expression of PD-1 ligands by tumor cells and tumor-derived myeloid cells, including tumor-associated macrophages (TAM), dendritic cells, and myeloid-derived suppressor cells (MDSC). When combined with GVAX or FVAX vaccination (consisting of irradiated ID8 cells expressing granulocyte macrophage colony-stimulating factor or FLT3 ligand) and costimulation by agonistic α-4-1BB or TLR 9 ligand, antibody-mediated blockade of PD-1 or PD-L1 triggered rejection of ID8 tumors in 75% of tumor-bearing mice. This therapeutic effect was associated with increased proliferation and function of tumor antigen-specific effector CD8(+) T cells, inhibition of suppressive regulatory T cells (Treg) and MDSC, upregulation of effector T-cell signaling molecules, and generation of T memory precursor cells. Overall, PD-1/PD-L1 blockade enhanced the amplitude of tumor immunity by reprogramming suppressive and stimulatory signals that yielded more powerful cancer control.

Degradation of ZAP-70 following antigenic stimulation in human T lymphocytes: role of calpain proteolytic pathway.

T cell activation by the specific Ag results in dramatic changes of the T cell phenotype that include a rapid and profound down-regulation and degradation of triggered TCRs. In this work, we investigated the fate of the TCR-associated ZAP-70 kinase in Ag-stimulated T cells. T cells stimulated by peptide-pulsed APCs undergo an Ag dose-dependent decrease of the total cellular content of ZAP-70, as detected by FACS analysis and confocal microscopy on fixed and permeabilized T cell-APC conjugates and by Western blot on total cell lysates. The time course of ZAP-70 consumption overlaps with that of zeta-chain degradation, indicating that ZAP-70 is degraded in parallel with TCR internalization and degradation. Pharmacological activation of protein kinase C (PKC) does not induce ZAP-70 degradation, which, on the contrary, requires activation of protein tyrosine kinases. Two lines of evidence indicate that the Ca2+-dependent cysteine protease calpain plays a major role in initiating ZAP-70 degradation: 1) treatment of T cells with cell-permeating inhibitors of calpain markedly reduces ZAP-70 degradation; 2) ZAP-70 is cleaved in vitro by calpain. Our results show that, in the course of T cell-APC cognate interaction, ZAP-70 is rapidly degraded via a calpain-dependent mechanism.

Anorectal Malformations: Finding the Pathway out of the Labyrinth.

Anorectal malformations (ARMs) are a complex group of congenital anomalies involving the distal anus and rectum, as well as the urinary and genital tracts in a significant number of cases. Most ARMs result from abnormal development of the urorectal septum in early fetal life. In most cases, the anus is not perforated and the distal enteric component ends blindly (atresia) or as a fistula into the urinary tract, genital tract, or perineum. ARMs are also present in a great number of syndromes and associations of congenital anomalies. The classification of ARMs is mainly based on the position of the rectal pouch relative to the puborectal sling, the presence or absence of fistulas, and the types and locations of the fistulas. All of this information is crucial in determining the most appropriate surgical approach for each case. Imaging studies play a key role in evaluation and classification of ARMs. In neonates, clinical and radiologic examinations in the first 3 days of life help determine the type of ARM and the need for early colostomy. In older children, preoperative pelvic magnetic resonance imaging is the most efficient diagnostic method for evaluating the size, morphology, and grade of development of the sphincteric musculature.

LDLs induce fibroblast spreading independently of the LDL receptor via activation of the p38 MAPK pathway.

Because adventitial fibroblasts play an important role in the repair of blood vessels, we assessed whether elevation in LDL concentrations would affect fibroblast function and whether this depended on activation of intracellular signaling pathways. We show here that in primary human fibroblasts, LDLs induced transient activation of the p38 mitogen-activated protein kinase (MAPK) pathway, but not the c-Jun N-terminal kinase MAPK pathway. This activation did not require the recruitment of the LDL receptor (LDLR), because LDLs efficiently stimulated the p38 MAPK pathway in human and mouse fibroblasts lacking functional LDLR, and because receptor-associated protein, an LDLR family antagonist, did not block the LDL-induced p38 activation. LDL particles also induced lamellipodia formation and cell spreading. These effects were blocked by SB203580, a specific p38 inhibitor. Our data demonstrate that LDLs can regulate the shape of fibroblasts in a p38 MAPK-dependent manner, a mechanism that may participate in wound healing or vessel remodeling as in atherosclerosis.

PlGF-1 and VEGFR-1 pathway regulation of the external epithelial hemato-ocular barrier. A model for retinal edema.

VEGF is considered as an important factor in the pathogenesis of macular edema. VEGF induces the rupture of the blood retinal barrier and may also influence the retinal pigment epithelial (RPE) outer retinal barrier. The aim of this work was to analyze the influence of the VEGF receptor pathways in the modulation of the RPE barrier breakdown in vitro and in vivo. The ARPE19 human junctions in culture are modulated by VEGF through VEGFR-1 but not through VEGFR-2. PlGF-1, that is a pure agonist of VEGFR-1, is produced in ARPE-19 cells under hypoxic conditions and mimics VEGF effects on the external retinal barrier as measured by TER and inulin flux. In vivo, the intravitreous injection of PlGF-1 induces a rupture of the external retinal barrier together with a retinal edema. This effect is reversible within 4 days. VEGF-E, that is a pure agonist of VEGFR-2, does not induce any acute effect on the RPE barrier. These results demonstrate that PlGF-1 can reproduce alterations of the RPE barrier occurring during diabetic retinopathy.

n-3 PUFAs modulate T-cell activation via protein kinase C-alpha and -epsilon and the NF-kappaB signaling pathway.

We elucidated the mechanisms of action of two n-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in Jurkat T-cells. Both DHA and EPA were principally incorporated into phospholipids in the following order: phosphatidylcholine < phosphatidylethanolamine < phosphatidylinositol/phosphatidylserine. Furthermore, two isoforms of phospholipase A(2) (i.e., calcium-dependent and calcium-independent) were implicated in the release of DHA and EPA, respectively, during activation of these cells. The two fatty acids inhibited the phorbol 12-myristate 13-acetate (PMA)-induced plasma membrane translocation of protein kinase C (PKC)-alpha and -epsilon. The two n-3 PUFAs also inhibited the nuclear translocation of nuclear factor kappaB (NF-kappaB) and the transcription of the interleukin-2 (IL-2) gene in PMA-activated Jurkat T-cells. Together, these results demonstrate that DHA and EPA, being released by two isoforms of phospholipase A(2), modulate IL-2 gene expression by exerting their action on two PKC isoforms and NF-kappaB in Jurkat T-cells.

The inositol Inpp5k 5-phosphatase affects osmoregulation through the vasopressin-aquaporin 2 pathway in the collecting system.

Inositol Inpp5k (or Pps, SKIP) is a member of the inositol polyphosphate 5-phosphatases family with a poorly characterized function in vivo. In this study, we explored the function of this inositol 5-phosphatase in mice and cells overexpressing the 42-kDa mouse Inpp5k protein. Inpp5k transgenic mice present defects in water metabolism characterized by a reduced plasma osmolality at baseline, a delayed urinary water excretion following a water load, and an increased acute response to vasopressin. These defects are associated with the expression of the Inpp5k transgene in renal collecting ducts and with alterations in the arginine vasopressin/aquaporin-2 signalling pathway in this tubular segment. Analysis in a mouse collecting duct mCCD cell line revealed that Inpp5k overexpression leads to increased expression of the arginine vasopressin receptor type 2 and increased cAMP response to arginine vasopressin, providing a basis for increased aquaporin-2 expression and plasma membrane localization with increased osmotically induced water transport. Altogether, our results indicate that Inpp5k 5-phosphatase is important for the control of the arginine vasopressin/aquaporin-2 signalling pathway and water transport in kidney collecting ducts.

Structural and Resting State Functional Connectivity of the Subthalamic Nucleus: Identification of Motor STN Parts and the Hyperdirect Pathway.

Deep brain stimulation (DBS) for Parkinson's disease often alleviates the motor symptoms, but causes cognitive and emotional side effects in a substantial number of cases. Identification of the motor part of the subthalamic nucleus (STN) as part of the presurgical workup could minimize these adverse effects. In this study, we assessed the STN's connectivity to motor, associative, and limbic brain areas, based on structural and functional connectivity analysis of volunteer data. For the structural connectivity, we used streamline counts derived from HARDI fiber tracking. The resulting tracks supported the existence of the so-called "hyperdirect" pathway in humans. Furthermore, we determined the connectivity of each STN voxel with the motor cortical areas. Functional connectivity was calculated based on functional MRI, as the correlation of the signal within a given brain voxel with the signal in the STN. Also, the signal per STN voxel was explained in terms of the correlation with motor or limbic brain seed ROI areas. Both right and left STN ROIs appeared to be structurally and functionally connected to brain areas that are part of the motor, associative, and limbic circuit. Furthermore, this study enabled us to assess the level of segregation of the STN motor part, which is relevant for the planning of STN DBS procedures.