The zinc transporter SLC39A13/ZIP13 is required for connective tissue development; its involvement in BMP/TGF-beta signaling pathways.

BACKGROUND: Zinc (Zn) is an essential trace element and it is abundant in connective tissues, however biological roles of Zn and its transporters in those tissues and cells remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that mice deficient in Zn transporter Slc39a13/Zip13 show changes in bone, teeth and connective tissue reminiscent of the clinical spectrum of human Ehlers-Danlos syndrome (EDS). The Slc39a13 knockout (Slc39a13-KO) mice show defects in the maturation of osteoblasts, chondrocytes, odontoblasts, and fibroblasts. In the corresponding tissues and cells, impairment in bone morphogenic protein (BMP) and TGF-beta signaling were observed. Homozygosity for a SLC39A13 loss of function mutation was detected in sibs affected by a unique variant of EDS that recapitulates the phenotype observed in Slc39a13-KO mice. CONCLUSIONS/SIGNIFICANCE: Hence, our results reveal a crucial role of SLC39A13/ZIP13 in connective tissue development at least in part due to its involvement in the BMP/TGF-beta signaling pathways. The Slc39a13-KO mouse represents a novel animal model linking zinc metabolism, BMP/TGF-beta signaling and connective tissue dysfunction.

The ArgR regulatory protein, a helper to the anaerobic regulator ANR during transcriptional activation of the arcD promoter in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, when deprived of oxygen, generates ATP from arginine catabolism by enzymes of the arginine deiminase pathway, encoded by the arcDABC operon. Under conditions of low oxygen tension, the transcriptional activator ANR binds to a site centered 41.5 bp upstream of the arcD transcriptional start. ANR-mediated anaerobic induction was enhanced two- to threefold by extracellular arginine. This arginine effect depended, in trans, on the transcriptional regulator ArgR and, in cis, on an ArgR binding site centered at -73.5 bp in the arcD promoter. Binding of purified ArgR protein to this site was demonstrated by electrophoretic mobility shift assays and DNase I footprinting. This ArgR recognition site contained a sequence, 5'-TGACGC-3', which deviated in only 1 base from the common sequence motif 5'-TGTCGC-3' found in other ArgR binding sites of P. aeruginosa. Furthermore, an alignment of all known ArgR binding sites confirmed that they consist of two directly repeated half-sites. In the absence of ANR, arginine did not induce the arc operon, suggesting that ArgR alone does not activate the arcD promoter. According to a model proposed, ArgR makes physical contact with ANR and thereby facilitates initiation of arc transcription.

Mécanismes moléculaires de l'homodimérisation de la protéine Scaffold IB1 et son implication dans la sécrétion d'insuline

RÉSUMÉ Les kinases activées par des mitogènes (MAPKs) constituent une importante famille d'enzymes conservée dans l'évolution. Elles forment un réseau de signalisation qui permet à la cellule de réguler spécifiquement divers processus impliqués dans la différenciation, la survie ou l'apoptose. Les kinases formant le module MAPK sont typiquement disposées en cascades de trois partenaires qui s'activent séquentiellement par phosphorylation. Le module minimum est constitué d'une MAPK kinase kinase (MAPKKK), d'une MAPK kinase (MAPKK) et d'une MAPK. Une fois activée, la MAPK phosphoryle différents substrats tels que des facteurs de transcription ou d'autres protéines. Chez les mammifères, trois groupes principaux de MAPKs ont été identifiés. Il s'agit du groupe des kinases régulées par des signaux extracellulaires du type «mitogènes » (ERK), ainsi que des groupes p38 et cJun NH2-terminal kinase (JNK), ou SAPK pour stress activated protein kinase, plutôt activées par des stimuli de type «stress ». De nombreuses études ont impliqué JNK dans la régulation de différents processus physiologiques et pathologiques, comme le diabète, les arthrites rhumatoïdes, l'athérosclérose, l'attaque cérébrale, les maladies de Parkinson et d'Alzheimer. JNK, en particulier joue un rôle dans la mort des cellules sécrétrices d'insuline induite par l'interleukine (IL)-1 β, lors du développement du diabète de type 1. IB1 est une protéine scaffold (échafaud) qui participe à l'organisation du module de JNK. IB1 est fortement exprimée dans les neurones et les cellules β du pancréas. Elle a été impliquée dans la survie des cellules, la régulation de l'expression du transporteur du glucose de type 2 (Glut-2) et dans le processus de sécrétion d'insuline glucose-dépendante. IBl est caractérisée par plusieurs domaines d'interaction protéine-protéine : un domaine de liaison à JNK (JBD), un domaine homologue au domaine 3 de Src (SH3) et un domaine d'interaction avec des tyrosines phosphorylées (PID). Des partenaires d'IB1, incluant les membres de la familles des kinases de lignée mélangée (MLKs), la MAPKK MKK7, la phosphatase 7 des MAPKs (MKP-7) ainsi que la chaîne légère de la kinésine, ont été isolés. Tous ces facteurs, sauf les MLKs et MKK7 interagissent avec le domaine PID ou l'extrême partie C-terminale d'IBl (la chaîne légère de la kinésine). Comme d'autres protéines scaffolds déjà décrites, IBl et un autre membre de la famille, IB2, sont capables d'homo- et d'hétérodimériser. L'interaction a lieu par l'intermédiaire de leur région C-terminale, contenant les domaines SH3 et PID. Mais ni le mécanisme moléculaire, ni la fonction de la dimérisation n'ont été caractérisés. Le domaine SH3 joue un rôle central lors de l'assemblage de complexes de macromolécules impliquées dans la signalisation intracellulaire. Il reconnaît de préférence des ligands contenant un motif riche en proline de type PxxP et s'y lie. Jusqu'à maintenant, tous les ligands isolés se liant à un domaine SH3 sont linéaires. Bien que le domaine SH3 soit un domaine important de la transmission des signaux, aucun partenaire interagissant spécifiquement avec le domaine SH3 d'IB1 n'a été identifié. Nous avons démontré qu'IBl homodimérisait par un nouveau set unique d'interaction domaine SH3 - domaine SH3. Les études de cristallisation ont démontré que l'interface recouvrait une région généralement impliquée dans la reconnaissance classique d'un motif riche en proline de type PxxP, bien que le domaine SH3 d'IB1 ne contienne aucun motif PxxP. L'homodimère d'IB1 semble extrêmement stable. Il peut cependant être déstabilisé par trois mutations ponctuelles dirigées contre des résidus clés impliqués dans la dimérisation. Chaque mutation réduit l'activation basale de JNK dépendante d'IB 1 dans des cellules 293T. La déstabilisation de la dimérisation induite par la sur-expression du domaine SH3, provoque une diminution de la sécrétion d'insuline glucose dépendant. SUMMARY Mitogen activated kinases (MAPK) are an important and conserved enzyme family. They form a signaling network required to specifically regulate process involved in cell differentiation, proliferation or death. A MAPK module is typically organized in a threekinase cascade which are activated by sequential phosphorylation. The MAPK kinase kinase (MAPKKK), the MAPK kinase (MAPKK) and the MAPK constitute the minimal module. Once activated, the MAPK phosphorylates its targets like transcription factors or other proteins. In mammals, three major groups of MAPKs have been identified : the group of extra-cellular regulated kinase (ERK) which is activated by mitogens and the group of p38 and cJun NH2-terminal kinase (JNK) or SAPK for stress activated protein kinase, which are activated by stresses. Many studies implicated JNK in many physiological or pathological process regulations, like diabetes, rheumatoid arthritis, arteriosclerosis, strokes or Parkinson and Alzheimer disease. In particular, JNK plays a crucial role in pancreatic β cell death induced by Interleukin (IL)-1 β in type 1 diabetes. Islet-brain 1 (IB 1) is a scaffold protein that interacts with components of the JNK signal-transduction pathway. IB 1 is expressed at high levels in neurons and in pancreatic β-cells, where it has been implicated in cell survival, in regulating expression of the glucose transporter type 2 (Glut-2) and in glucose-induced insulin secretion. It contains several protein-protein interaction domains, including a JNK-binding domain (JBD), a Src homology 3 domain (SH3) and a phosphotyrosine interaction domain (PID). Proteins that have been shown to associate with IB 1 include members of the Mixed lineage kinase family (MLKs), the MAPKK MKK7, the MAPK phosphatase-7 MKP7, as well as several other ligands including kinesin light chain, LDL receptor related family members and the amyloid precursor protein APP. All these factors, except MLK3 and MKK7 have been shown to interact with the PID domain or the extreme C-terminal part (Kinesin light chain) of IB 1. As some scaffold already described, IB 1 and another member of the family, IB2, have previously been shown to engage in oligomerization through their respective C-terminal regions that include the SH3 and PID domains. But neither the molecular mechanisms nor the function of dimerization have yet been characterized. SH3 domains are central in the assembly of macromolecular complexes involved in many intracellular signaling pathways. SH3 domains are usually characterized by their preferred recognition of and association with canonical PxxP motif. In all these cases, a single linear sequence is sufficient for binding to the SH3 domain. However, although SH3 domains are important elements of signal transduction, no protein that interacts specifically with the SH3 domain of IB 1 has been identified so far. Here, we show that IB 1 homodimerizes through a navel and unique set of SH3-SH3 interactions. X-ray crystallography studies indicate that the dieter interface covers a region usually engaged in PxxP-mediated ligand recognition, even though the IB 1 SH3 domain lacks this motif. The highly stable IB 1 homodimer can be significantly destabilized in vitro by individual point-mutations directed against key residues involved in dimerization. Each mutation reduces IB 1-dependent basal JNK activity in 293T cells. Impaired dimerization induced by over-expression of the SH3 domain also results in a significant reduction in glucose-dependent insulin secretion in pancreatic β-cells.

Creatine deficiency syndromes and the importance of creatine synthesis in the brain.

Creatine deficiency syndromes, due to deficiencies in AGAT, GAMT (creatine synthesis pathway) or SLC6A8 (creatine transporter), lead to complete absence or very strong decrease of creatine in CNS as measured by magnetic resonance spectroscopy. Brain is the main organ affected in creatine-deficient patients, who show severe neurodevelopmental delay and present neurological symptoms in early infancy. AGAT- and GAMT-deficient patients can be treated by oral creatine supplementation which improves their neurological status, while this treatment is inefficient on SLC6A8-deficient patients. While it has long been thought that most, if not all, of brain creatine was of peripheral origin, the past years have brought evidence that creatine can cross blood-brain barrier, however, only with poor efficiency, and that CNS must ensure parts of its creatine needs by its own endogenous synthesis. Moreover, we showed very recently that in many brain structures, including cortex and basal ganglia, AGAT and GAMT, while found in every brain cell types, are not co-expressed but are rather expressed in a dissociated way. This suggests that to allow creatine synthesis in these structures, guanidinoacetate must be transported from AGAT- to GAMT-expressing cells, most probably through SLC6A8. This new understanding of creatine metabolism and transport in CNS will not only allow a better comprehension of brain consequences of creatine deficiency syndromes, but will also contribute to better decipher creatine roles in CNS, not only in energy as ATP regeneration and buffering, but also in its recently suggested functions as neurotransmitter or osmolyte.

Adipose reduction in the activity of the repressor ICER is responsible for insulin resistance elicited by the transcriptional factors CREB in obesity

BACKGROUND AND AIMS: Sustained adipose activation of the transcriptional activators cAMP response binding proteins (CREB) in obesity leads to impaired expression of the glucose transporter GLUT4 and adiponectin (adipoq) in mice model of obesity. Diminution of GLUT4 and adipoq caused by CREB is indirect and relies on the increased repressive activity of the CREB target gene activating transcription factor 3 (ATF3). Specific inactivation of CREB in adipocytes decreases ATF3 production and improves whole-body insulin sensitivity of mice in the context of diet-induced obesity. Thus, elevation of CREB activity is a key mechanism responsible for adipocyte dysfunction and systemic insulin resistance. The inducible cAMP early repressor (ICER) is a negative regulator of the CREB activity. In fact, ICER antagonizes the CREB factor by competing for the regulation of similar target genes. The goal of the study was to investigate whether loss of ICER expression in adipocytes could be responsible for increased CREB activity in obesity. MATERIALS AND METHODS: Mice C57bl6 were fed with a high fat diet (HFD) for 12 weeks to increase body weight and generate insulin resistance. Biopsies of visceral adipose tissues (VAT) were prepared from human lean (BMI=24}0.5 Kg/m2) or obese subjects (BMI>35 Kg/m2). Total RNA and protein were prepared from white adipose tissues (WAT) of chow- or HFD-fed mice and VAT of lean and obese subjects. Activities of CREBs and ICER were monitored by electromobility shift assays (EMSA). The role of ICER on CREB activity was confirmed in 3T3-L1 adipocytes cells. Briefly after differentiation, the cells were electroporated with the plasmid coding for ICER cDNA. Gene expression was quantified by quantitative real-time PCR and western Blotting experiments. RESULTS: The expression of ICER is reduced in WAT of HFD-induced obese mice when compared to chow mice as measured by real-time PCR and EMSA. Similar result was found in human tissues. Reduction in ICER expression was associated with increased ATF3 expression and decreased adipoq and GLUT4 contents. Diminution in ICER levels was observed in adipocytes fraction whereas its expression was unchanged in stroma vascular fraction of WAT. Overexpression of ICER in 3T3-L1 adipocytes silenced the expression of ATF3, confirming the regulation of the factor by ICER. The expression of ICER is regulated by histone deacetylases activity (HDAC). Inhibition of HDACs in 3T3-L1 adipocytes cells using trichostatin inhibited the production of ICER. The whole activity of HDAC was reduced in WAT and VAT of obese mice and human obese subjects. CONCLUSION: Impaired adipose expression of ICER is responsible of increased CREB activity in adipocytes in obesity. This mechanism relies on reduction of the HDAC activity.

The neuronal monocarboxylate transporter MCT2: its regulation by BDNF and its role in learning and memory

Glucose has been considered the major, if not the exclusive, energy substrate for the brain. But under certain physiological and pathological conditions other substrates, namely monocarboxylates (lactate, pyruvate and ketone bodies), can contribute significantly to satisfy brain energy demands. These monocarboxylates need to be transported across the blood-brain barrier or out of astrocytes into the extracellular space and taken up into neurons. It has been shown that monocarboxylates are transported by a family of proton-linked transporters called monocarboxylate transporters (MCTs). In the central nervous system, MCT2 is the predominant neuronal isoform and little is known about the regulation of its expression. Noradrenaline (NA), insulin and IGF-1 were previously shown to enhance the expression of MCT2 in cultured cortical neurons via a translational mechanism. Here we demonstrate that the well known brain neurotrophic factor BDNF enhances MCT2 protein expression in cultured cortical neurons and in synaptoneurosome preparations in a time- and concentrationdependent manner without affecting MCT2 mRNA levels. We observed that BDNF induced MCT2 expression by activation of MAPK as well as PI3K/Akt/mTOR signaling pathways. Furthermore, we investigated the possible post-transcriptional regulation of MCT2 expression by a neuronal miRNA. Then, we demonstrated that BDNF enhanced MCT2 expression in the hippocampus in vivo, in parallel with some post-synaptic proteins such as PSD95 and AMPA receptor GluR2/3 subunits, and two immediate early genes Arc and Zif268 known to be expressed in conditions related to synaptic plasticity. In the last part, we demonstrated in vivo that a downregulation of hippocampal MCT2 via silencing with an appropriate lentiviral vector in mice caused an impairment of working memory without reference memory deficit. In conclusion, these results suggest that regulation of neuronal monocarboxylate transporter MCT2 expression could be a key event in the context of synaptic plasticity, allowing an adequate energy substrate supply in situations of altered synaptic efficacy. - Le glucose représente le substrat énergétique majeur pour le cerveau. Cependant, dans certaines conditions physiologiques ou pathologiques, le cerveau a la capacité d'utiliser des substrats énergéiques appartenant à la classe des monocarboxylates (lactate, pyruvate et corps cétoniques) afin de satisfaire ses besoins énergétiques. Ces monocarboxylates doivent être transportés à travers la barrière hématoencéphalique mais aussi hors des astrocytes vers l'espace extracellulaire puis re-captés par les neurones. Leur transport est assuré par une famillle de transporteurs aux monocarboxylates (MCTs). Dans le système nerveux central, les neurones expriment principalement l'isoforme MCT2 mais peu d'informations sont disponibles concernant la régulation de son expression. Il a été montré que la noradrénaline, l'insuline et l'IGF-1 induisent l'expression de MCT2 dans des cultures de neurones corticaux par un mécanisme traductionnel. Dans cette étude nous démontrons dans un premier temps que le facteur neurotrophique BDNF augmente l'expression de MCT2 à la fois dans des cultures de neurones corticaux et dans les préparations synaptoneurosomales selon un décours temporel et une gamme de concentrations propre. Aucun changement n'a été observé concernant les niveaux d'ARNm de MCT2. Nous avons observé que le BDNF induisait l'expression de MCT2 par l'activation simultanée des voies de signalisation MAPK et PI3K/Akt/mTOR. De plus, nous nous sommes intéressés à une potentielle régulation par les micro-ARNs de la synthèse de MCT2. Ensuite, nous avons démontré que le BDNF induit aussi l'expression de MCT2 dans l'hippocampe de la souris en parallèle avec d'autres protéines post-synaptiques telles que PSD95 et GluR2/3 et avec deux « immediate early genes » tels que Arc et Zif268 connus pour être exprimés dans des conditions de plasticité synaptique. Dans un dernier temps, nous avons démontré qu'une diminution d'expression de MCT2 induite par le biais d'un siRNA exprimé via un vecteur lentiviral dans l'hippocampe de souris générait des déficits de mémoire de travail sans affecter la mémoire de référence. En conclusion, ces résultats nous suggèrent que le transporteur aux monocarboxylates neuronal MCT2 serait essentiel pour l'apport énergétique du lactate pour les neurones dans des conditions de haute activité neuronale comme c'est le cas pendant les processus de plasticité synaptique.

Revisiting G3BP1 as a RasGAP binding protein: sensitization of tumor cells to chemotherapy by the RasGAP 317-326 sequence does not involve G3BP1.

RasGAP is a multifunctional protein that controls Ras activity and that is found in chromosomal passenger complexes. It also negatively or positively regulates apoptosis depending on the extent of its cleavage by caspase-3. RasGAP has been reported to bind to G3BP1 (RasGAP SH3-domain-binding protein 1), a protein regulating mRNA stability and stress granule formation. The region of RasGAP (amino acids 317-326) thought to bind to G3BP1 corresponds exactly to the sequence within fragment N2, a caspase-3-generated fragment of RasGAP, that mediates sensitization of tumor cells to genotoxins. While assessing the contribution of G3BP1 in the anti-cancer function of a cell-permeable peptide containing the 317-326 sequence of RasGAP (TAT-RasGAP₃₁₇₋₃₂₆), we found that, in conditions where G3BP1 and RasGAP bind to known partners, no interaction between G3BP1 and RasGAP could be detected. TAT-RasGAP₃₁₇₋₃₂₆ did not modulate binding of G3BP1 to USP10, stress granule formation or c-myc mRNA levels. Finally, TAT-RasGAP₃₁₇₋₃₂₆ was able to sensitize G3BP1 knock-out cells to cisplatin-induced apoptosis. Collectively these results indicate that G3BP1 and its putative RasGAP binding region have no functional influence on each other. Importantly, our data provide arguments against G3BP1 being a genuine RasGAP-binding partner. Hence, G3BP1-mediated signaling may not involve RasGAP.

Human-to-bovine jump of Staphylococcus aureus CC8 is associated with the loss of a β-hemolysin converting prophage and the acquisition of a new staphylococcal cassette chromosome.

Staphylococcus aureus can colonize and infect both humans and animals, but isolates from both hosts tend to belong to different lineages. Our recent finding of bovine-adapted S. aureus showing close genetic relationship to the human S. aureus clonal complex 8 (CC8) allowed us to examine the genetic basis of host adaptation in this particular CC. Using total chromosome microarrays, we compared the genetic makeup of 14 CC8 isolates obtained from cows suffering subclinical mastitis, with nine CC8 isolates from colonized or infected human patients, and nine S. aureus isolates belonging to typical bovine CCs. CC8 isolates were found to segregate in a unique group, different from the typical bovine CCs. Within this CC8 group, human and bovine isolates further segregated into three subgroups, among which two contained a mix of human and bovine isolates, and one contained only bovine isolates. This distribution into specific clusters and subclusters reflected major differences in the S. aureus content of mobile genetic elements (MGEs). Indeed, while the mixed human-bovine clusters carried commonly human-associated β-hemolysin converting prophages, the bovine-only isolates were devoid of such prophages but harbored an additional new non-mec staphylococcal cassette chromosome (SCC) unique to bovine CC8 isolates. This composite cassette carried a gene coding for a new LPXTG-surface protein sharing homologies with a protein found in the environmental bacterium Geobacillus thermoglucosidans. Thus, in contrast to human CC8 isolates, the bovine-only CC8 group was associated with the combined loss of β-hemolysin converting prophages and gain of a new SCC probably acquired in the animal environment. Remaining questions are whether the new LPXTG-protein plays a role in bovine colonization or infection, and whether the new SCC could further acquire antibiotic-resistance genes and carry them back to human.

GLUT2 in pancreatic and extra-pancreatic gluco-detection (review).

Detection of variations in blood glucose concentrations by pancreatic beta-cells and a subsequent appropriate secretion of insulin are key events in the control of glucose homeostasis. Because a decreased capability to sense glycemic changes is a hallmark of type 2 diabetes, the glucose signalling pathway leading to insulin secretion in pancreatic beta-cells has been extensively studied. This signalling mechanism depends on glucose metabolism and requires the presence of specific molecules such as GLUT2, glucokinase and the K(ATP) channel subunits Kir6.2 and SUR1. Other cells are also able to sense variations in glycemia or in local glucose concentrations and to modulate different physiological functions participating in the general control of glucose and energy homeostasis. These include cells forming the hepatoportal vein glucose sensor, which controls glucose storage in the liver, counterregulation, food intake and glucose utilization by peripheral tissues and neurons in the hypothalamus and brainstem whose firing rates are modulated by local variations in glucose concentrations or, when not protected by a blood-brain barrier, directly by changes in blood glucose levels. These glucose-sensing neurons are involved in the control of insulin and glucagon secretion, food intake and energy expenditure. Here, recent physiological studies performed with GLUT2-/- mice will be described, which indicate that this transporter is essential for glucose sensing by pancreatic beta-cells, by the hepatoportal sensor and by sensors, probably located centrally, which control activity of the autonomic nervous system and stimulate glucagon secretion. These studies may pave the way to a fine dissection of the molecular and cellular components of extra-pancreatic glucose sensors involved in the control of glucose and energy homeostasis.