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.

Defining the RNA polymerase III transcriptome: Genome-wide localization of the RNA polymerase III transcription machinery in human cells.

Our view of the RNA polymerase III (Pol III) transcription machinery in mammalian cells arises mostly from studies of the RN5S (5S) gene, the Ad2 VAI gene, and the RNU6 (U6) gene, as paradigms for genes with type 1, 2, and 3 promoters. Recruitment of Pol III onto these genes requires prior binding of well-characterized transcription factors. Technical limitations in dealing with repeated genomic units, typically found at mammalian Pol III genes, have so far hampered genome-wide studies of the Pol III transcription machinery and transcriptome. We have localized, genome-wide, Pol III and some of its transcription factors. Our results reveal broad usage of the known Pol III transcription machinery and define a minimal Pol III transcriptome in dividing IMR90hTert fibroblasts. This transcriptome consists of some 500 actively transcribed genes including a few dozen candidate novel genes, of which we confirmed nine as Pol III transcription units by additional methods. It does not contain any of the microRNA genes previously described as transcribed by Pol III, but reveals two other microRNA genes, MIR886 (hsa-mir-886) and MIR1975 (RNY5, hY5, hsa-mir-1975), which are genuine Pol III transcription units.

Beta-lactams against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) have developed resistance to virtually all non-experimental antibiotics. They are intrinsically resistant to beta-lactams by virtue of newly acquired low-affinity penicillin-binding protein 2A (PBP2A). Because PBP2A can build the wall when other PBPs are blocked by beta-lactams, designing beta-lactams capable of blocking this additional target should help solve the issue. Older molecules including penicillin G, amoxicillin and ampicillin had relatively good PBP2A affinities, and successfully treated experimental endocarditis caused by MRSA, provided that the bacterial penicillinase could be inhibited. Newer anti-PBP2A beta-lactams with over 10-fold greater PBP2A affinities and low minimal inhibitory concentrations were developed, primarily in the cephem and carbapenem classes. They are also very resistant to penicillinase. Most have demonstrated anti-MRSA activity in animal models of infection, and two--the carbapenem CS-023 and the cephalosporin ceftopibrole medocaril--have proceeded to Phase II and Phase III clinical evaluation. Thus, clinically useful anti-MRSA beta-lactams are imminent.

Local Ca(2+) detection and modulation of synaptic release by astrocytes.

Astrocytes communicate with synapses by means of intracellular calcium ([Ca(2+)](i)) elevations, but local calcium dynamics in astrocytic processes have never been thoroughly investigated. By taking advantage of high-resolution two-photon microscopy, we identify the characteristics of local astrocyte calcium activity in the adult mouse hippocampus. Astrocytic processes showed intense activity, triggered by physiological transmission at neighboring synapses. They encoded synchronous synaptic events generated by sparse action potentials into robust regional (∼12 μm) [Ca(2+)](i) elevations. Unexpectedly, they also sensed spontaneous synaptic events, producing highly confined (∼4 μm), fast (millisecond-scale) miniature Ca(2+) responses. This Ca(2+) activity in astrocytic processes is generated through GTP- and inositol-1,4,5-trisphosphate-dependent signaling and is relevant for basal synaptic function. Thus, buffering astrocyte [Ca(2+)](i) or blocking a receptor mediating local astrocyte Ca(2+) signals decreased synaptic transmission reliability in minimal stimulation experiments. These data provide direct evidence that astrocytes are integrated in local synaptic functioning in adult brain.

Final antigenic Melan-A peptides produced directly by the proteasomes are preferentially selected for presentation by HLA-A*0201 in melanoma cells.

The melanoma-associated protein Melan-A contains the immunodominant CTL epitope Melan-A(26/27-35)/HLA-A*0201 against which a high frequency of T lymphocytes has been detected in many melanoma patients. In this study we show that the in vitro degradation of a polypeptide encompassing Melan-A(26/27-35) by proteasomes produces both the final antigenic peptide and N-terminally extended intermediates. When human melanoma cells expressing the corresponding fragments were exposed to specific CTL, those expressing the minimal antigenic sequence were recognized more efficiently than those expressing the N-terminally extended intermediates. Using a tumor-reactive CTL clone, we confirmed that the recognition of melanoma cells expressing an N-terminally extended intermediate of Melan-A is inefficient. We demonstrated that the inefficient cytosolic trimming of N-terminally extended intermediates could offer a selective advantage for the preferred presentation of Melan-A peptides directly produced by the proteasomes. These results imply that both the proteasomes and postproteasomal peptidases limit the availability of antigenic peptides and that the efficiency of presentation may be affected by conditions that alter the ratio between fully and partially processed proteasomal products.

Rapid Biodiversity Assessment and Monitoring Method for Highly Diverse Benthic Communities: A Case Study of Mediterranean Coralligenous Outcrops

Increasing anthropogenic pressures urge enhanced knowledge and understanding of the current state of marine biodiversity. This baseline information is pivotal to explore present trends, detect future modifications and propose adequate management actions for marine ecosystems. Coralligenous outcrops are a highly diverse and structurally complex deep-water habitat faced with major threats in the Mediterranean Sea. Despite its ecological, aesthetic and economic value, coralligenous biodiversity patterns are still poorly understood. There is currently no single sampling method that has been demonstrated to be sufficiently representative to ensure adequate community assessment and monitoring in this habitat. Therefore, we propose a rapid non-destructive protocol for biodiversity assessment and monitoring of coralligenous outcrops providing good estimates of its structure and species composition, based on photographic sampling and the determination of presence/absence of macrobenthic species. We used an extensive photographic survey, covering several spatial scales (100s of m to 100s of km) within the NW Mediterranean and including 2 different coralligenous assemblages: Paramuricea clavata (PCA) and Corallium rubrum assemblage (CRA). This approach allowed us to determine the minimal sampling area for each assemblage (5000 cm² for PCA and 2500 cm²for CRA). In addition, we conclude that 3 replicates provide an optimal sampling effort in order to maximize the species number and to assess the main biodiversity patterns of studied assemblages in variability studies requiring replicates. We contend that the proposed sampling approach provides a valuable tool for management and conservation planning, monitoring and research programs focused on coralligenous outcrops, potentially also applicable in other benthic ecosystems

What can we learn from international comparisons of costs by DRG ?

OBJECTIVES: The objective of this study was to compare costs data by diagnosis related group (DRG) between Belgium and Switzerland. Our hypotheses were that differences between countries can probably be explained by methodological differences in cost calculations, by differences in medical practices and by differences in cost structures within the two countries. METHODS: Classifications of DRG used in the two countries differ (AP-DRGs version 1.7 in Switzerland and APR-DRGs version 15.0 in Belgium). The first step of this study was to transform Belgian summaries into Swiss AP-DRGs. Belgian and Swiss data were calculated with a clinical costing methodology (full costing). Belgian and Swiss costs were converted into US$ PPP (purchasing power parity) in order to neutralize differences in purchasing power between countries. RESULTS: The results of this study showed higher costs in Switzerland despite standardization of cost data according to PPP. The difference is not explained by the case-mix index because this was similar for inliers between the two countries. The length of stay (LOS) was also quite similar for inliers between the two countries. The case-mix index was, however, higher for high outliers in Belgium, as reflected in a higher LOS for these patients. Higher costs in Switzerland are thus probably explained mainly by the higher number of agency staff by service in this country or because of differences in medical practices. CONCLUSIONS: It is possible to make international comparisons but only if there is standardization of the case-mix between countries and only if comparable accountancy methodologies are used. Harmonization of DRGs groups, nomenclature and accountancy is thus required.

In vitro and in vivo evaluation of sunitinib eluting beads

Background: Chemoembolization is used to treat liver malignancies. However recurrence occurs frequently, possibly because of neoangiogenesis triggered by ischemia caused by the embolic agent. In this context, the combination of an embolic agent with an anti-angiogenic drug seems appealing. This study characterizes the in vitro loading and release profile of sunitinib eluting beads of different sizes and their pharmacokinetic profile in a rabbit model. Methods: 70-150 μm and 100-300 μm drug eluting beads (DC Bead, Biocompatibles UK) were loaded by incubation in a sunitinib hydrochloride solution. Drug was quantified by spectrophotometry at 430 nm. Drug release was measured over one-week periods and normalized using an internal standard in 30% ethanol in NaCl 0.9%. New-Zealand white rabbits were used. Eight animals received 0.2 ml of 100-300 μm DC Bead loaded with 6 mg of sunitinib in the hepatic artery (group 1) and 4 animals received 6 mg of sunitinib p.o. (group 2). Half of the animals were sacrificed after 6 hours and half after24 hours. Liver enzymes were measured at 0, 6 and 24 hours in both groups. Plasmatic sunitinib concentration was determined by tandem mass spectroscopy (LC MS/MS) at 0, 1, 2, 3, 4, 5, 6 and 24 hours. At sacrifice, the livers were harvested and sunitinib concentration in liver tissue was assessed by LC MS/MS. Results: High drug loading was obtained for both microsphere bead sizes. Particle shrinking was observed with adsorption of sunitinib. Almost complete release of sunitinib was detected under physiological conditions, with very similar release for 70-150 μm and 100-300 μm (t50%=1.2 h) DC Bead. Conclusions: Sunitinib eluting beads are well tolerated by rabbits when administered in the hepatic artery. No unexpected toxicity was observed. Very high drug concentration can be obtained at the site of embolization with minimal systemic passage.

Harvest site influences the growth properties of adipose derived stem cells.

The therapeutic potential of adult stem cells may become a relevant option in clinical care in the future. In hand and plastic surgery, cell therapy might be used to enhance nerve regeneration and help surgeons and clinicians to repair debilitating nerve injuries. Adipose-derived stem cells (ASCs) are found in abundant quantities and can be harvested with a low morbidity. In order to define the optimal fat harvest location and detect any potential differences in ASC proliferation properties, we compared biopsies from different anatomical sites (inguinal, flank, pericardiac, omentum, neck) in Sprague-Dawley rats. ASCs were expanded from each biopsy and a proliferation assay using different mitogenic factors, basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) was performed. Our results show that when compared with the pericardiac region, cells isolated from the inguinal, flank, omental and neck regions grow significantly better in growth medium alone. bFGF significantly enhanced the growth rate of ASCs isolated from all regions except the omentum. PDGF had minimal effect on ASC proliferation rate but increases the growth of ASCs from the neck region. Analysis of all the data suggests that ASCs from the neck region may be the ideal stem cell sources for tissue engineering approaches for the regeneration of nervous tissue.

Scoring system for renal pathology in Fabry disease: report of the international study group of fabry nephropathy (ISGFN).

BACKGROUND: In Fabry nephropathy, alpha-galactosidase deficiency leads to accumulation of glycosphingolipids in all kidney cell types, proteinuria and progressive loss of kidney function. METHODS: An international working group of nephrologists from 11 Fabry centres identified adult Fabry patients, and pathologists scored histologic changes on renal biopsies. A standardized scoring system was developed with a modified Delphi technique assessing 59 Fabry nephropathy cases. Each case was scored independently of clinical information by at least three pathologists with an average final score reported. RESULTS: We assessed 35 males (mean age 36.4 years) and 24 females (43.9 years) who mostly had clinically mild Fabry nephropathy. The average serum creatinine was 1.3 mg/dl (114.9 micromol/l); estimated glomerular filtration rate was 81.7 ml/min/1.73 m(2) and urine protein to creatinine ratio was 1.08 g/g (122.0 mg/mmol). Males had greater podocyte vacuolization on light microscopy (mean score) and glycosphingolipid inclusions on semi-thin sections than females. Males also had significantly more proximal tubule, peritubular capillary and vascular intimal inclusions. Arteriolar hyalinosis was similar, but females had significantly more arterial hyalinosis. Chronic kidney disease stage correlated with arterial and glomerular sclerosis scores. Significant changes, including segmental and global sclerosis, and interstitial fibrosis were seen even in patients with stage 1-2 chronic kidney disease with minimal proteinuria. CONCLUSIONS: The development of a standardized scoring system of both disease-specific lesions, i.e. lipid deposition related, and general lesions of progression, i.e. fibrosis and sclerosis, showed a spectrum of histologic appearances even in early clinical stage of Fabry nephropathy. These findings support the role of kidney biopsy in the baseline evaluation of Fabry nephropathy, even with mild clinical disease. The scoring system will be useful for longitudinal assessment of prognosis and responses to therapy for Fabry nephropathy.

Integrating species distribution models (SDMs) and phylogeography for two species of Alpine Primula.

The major intention of the present study was to investigate whether an approach combining the use of niche-based palaeodistribution modeling and phylo-geography would support or modify hypotheses about the Quaternary distributional history derived from phylogeographic methods alone. Our study system comprised two closely related species of Alpine Primula. We used species distribution models based on the extant distribution of the species and last glacial maximum (LGM) climate models to predict the distribution of the two species during the LGM. Phylogeographic data were generated using amplified fragment length polymorphisms (AFLPs). In Primula hirsuta, models of past distribution and phylogeographic data are partly congruent and support the hypothesis of widespread nunatak survival in the Central Alps. Species distribution models (SDMs) allowed us to differentiate between alpine regions that harbor potential nunatak areas and regions that have been colonized from other areas. SDMs revealed that diversity is a good indicator for nunataks, while rarity is a good indicator for peripheral relict populations that were not source for the recolonization of the inner Alps. In P. daonensis, palaeo-distribution models and phylogeographic data are incongruent. Besides the uncertainty inherent to this type of modeling approach (e.g., relatively coarse 1-km grain size), disagreement of models and data may partly be caused by shifts of ecological niche in both species. Nevertheless, we demonstrate that the combination of palaeo-distribution modeling with phylogeographical approaches provides a more differentiated picture of the distributional history of species and partly supports (P. hirsuta) and partly modifies (P. daonensis and P. hirsuta) hypotheses of Quaternary distributional history. Some of the refugial area indicated by palaeodistribution models could not have been identified with phylogeographic data.

Cutting edge: cell autonomous rather than environmental factors control bacterial superantigen-induced T cell anergy in vivo.

Anergic T cells display a marked decrease in their ability to produce IL-2 and to proliferate in the presence of an appropriate antigenic signal. Two nonmutually exclusive classes of models have been proposed to explain the persistence of T cell anergy in vivo. While some reports indicate that anergic T cells have intrinsic defects in signaling pathways or transcriptional activities, other studies suggest that anergy is maintained by environmental "suppressor" factors such as cytokines or Abs. To distinguish between these conflicting hypotheses, we employed the well-characterized bacterial superantigen model system to evaluate in vivo the ability of a trace population of adoptively transferred naive or anergized T cells to proliferate in a naive vs anergic environment upon subsequent challenge. Our data clearly demonstrate that bacterial superantigen-induced T cell anergy is cell autonomous and independent of environmental factors.

A statistical procedure for testing social reciprocity at group, dyadic and individual levels

This paper examines statistical analysis of social reciprocity, that is, the balance between addressing and receiving behaviour in social interactions. Specifically, it focuses on the measurement of social reciprocity by means of directionality and skew-symmetry statistics at different levels. Two statistics have been used as overall measures of social reciprocity at group level: the directional consistency and the skew-symmetry statistics. Furthermore, the skew-symmetry statistic allows social researchers to obtain complementary information at dyadic and individual levels. However, having computed these measures, social researchers may be interested in testing statistical hypotheses regarding social reciprocity. For this reason, it has been developed a statistical procedure, based on Monte Carlo sampling, in order to allow social researchers to describe groups and make statistical decisions.

Mutations in the human UBR1 gene and the associated phenotypic spectrum.

Johanson-Blizzard syndrome (JBS) is a rare, autosomal recessive disorder characterized by exocrine pancreatic insufficiency, typical facial features, dental anomalies, hypothyroidism, sensorineural hearing loss, scalp defects, urogenital and anorectal anomalies, short stature, and cognitive impairment of variable degree. This syndrome is caused by a defect of the E3 ubiquitin ligase UBR1, which is part of the proteolytic N-end rule pathway. Herein, we review previously reported (n = 29) and a total of 31 novel UBR1 mutations in relation to the associated phenotype in patients from 50 unrelated families. Mutation types include nonsense, frameshift, splice site, missense, and small in-frame deletions consistent with the hypothesis that loss of UBR1 protein function is the molecular basis of JBS. There is an association of missense mutations and small in-frame deletions with milder physical abnormalities and a normal intellectual capacity, thus suggesting that at least some of these may represent hypomorphic UBR1 alleles. The review of clinical data of a large number of molecularly confirmed JBS cases allows us to define minimal clinical criteria for the diagnosis of JBS. For all previously reported and novel UBR1 mutations together with their clinical data, a mutation database has been established at LOVD.