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.

Cambium dynamics of Pinus sylvestris and Betula spp. in the northern boreal forest in Finland

Abstract

Dinâmica da dormência e brotação de gemas de vimeiro no Planalto Sul Catarinense

O objetivo deste trabalho foi avaliar a dinâmica e a heterogeneidade de dormência de gemas, e avaliar o efeito de diferentes épocas de colheita de ramos na brotação do vimeiro, no Planalto Sul Catarinense. Ramos de ano foram colhidos nos meses de abril a agosto de 2005, nos municípios catarinenses de Lages, Bocaina do Sul e Urupema. A dormência de gemas foi avaliada em estacas retiradas da base (0 a 12 cm), centro (12 a 24 cm) e ápice (24 a 36 cm) de ramos com comprimento superior a 150 cm. Foram utilizadas estacas com e sem redução para uma única gema. As brotações das plantas em campo também foram avaliadas. A dormência mais intensa de gemas de ramos de ano ocorreu em maio, quando a intensidade de dormência e a freqüência de gemas dormentes decresceram do ápice para a base do segmento basal dos ramos. A colheita do vime em abril induziu a brotação antes do inverno e prejudicou a brotação primaveril.

Ancient dispersal of the human fungal pathogen Cryptococcus gattii from the Amazon rainforest

Over the past two decades, several fungal outbreaks have occurred, including the high-profile 'Vancouver Island' and 'Pacific Northwest' outbreaks, caused by Cryptococcus gattii, which has affected hundreds of otherwise healthy humans and animals. Over the same time period, C. gattii was the cause of several additional case clusters at localities outside of the tropical and subtropical climate zones where the species normally occurs. In every case, the causative agent belongs to a previously rare genotype of C. gattii called AFLP6/VGII, but the origin of the outbreak clades remains enigmatic. Here we used phylogenetic and recombination analyses, based on AFLP and multiple MLST datasets, and coalescence gene genealogy to demonstrate that these outbreaks have arisen from a highly-recombining C. gattii population in the native rainforest of Northern Brazil. Thus the modern virulent C. gattii AFLP6/VGII outbreak lineages derived from mating events in South America and then dispersed to temperate regions where they cause serious infections in humans and animals.

An open source browser-based software tool for graph drawing and visualisation

In this research work we searched for open source libraries which supports graph drawing and visualisation and can run in a browser. Subsequent these libraries were evaluated to find out which one is the best for this task. The result was the d3.js is that library which has the greatest functionality, flexibility and customisability. Afterwards we developed an open source software tool where d3.js was included and which was written in JavaScript so that it can run browser-based.

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Impacts of the application of swine wastewater in percolate and in soil cultivated with soybean

The aim of this study was to evaluate the possible impacts caused in the soil and in the percolate in lysimeters of drainage with application of different rates of swine wastewater (SW) during the cycle of soybean culture and to assess the productivity of it. The experiment was conducted at the Agricultural Engineering Experimental Center of UNIOESTE. The soil was classified as typical Distroferric Red Latosol. There were twenty-four drainage lysimeters in the area in which the soybean was cultivated, cultivar CD 214. Four SW depths (0; 100; 200 and 300 m³ ha-1) were applied to the soil seven days before the sowing in a single application combined with two mineral fertilizations in the sowing (with and without recommended fertilization during sowing), and three repetitions per treatment. It was realized three collections of percolate in each experimental portion, the first was conducted 40 days after sowing (DAS); the second at 72 DAS, and the third at the end of crop cycle (117 DAS). It was evaluated in the percolate the pH, calcium, magnesium, potassium, phosphorus, and total nitrogen. Based on the results, it was possible to observe that the level of K, P and N in the soil increased according tothe increase of SW rates. The levels of K and P in the percolate were higher for higher rates of SW. The productivity was not influenced by the application of SW or by fertilization.

Estudo da associação de água residuária de suinocultura e adubação mineral na cultura do milho e no solo

RESUMOO uso de água residuária em solo agrícola pode-se caracterizar como uma fonte alternativa de adubação, além de melhorar as condições edáficas do solo devido à matéria orgânica adicionada. Neste contexto, este trabalho teve por objetivo avaliar os efeitos na cultura do milho e em Latossolo Vermelho distroférrico típico, devido à aplicação de água residuária de suinocultura associada com adubação química. No experimento, utilizou-se de cinco doses de ARS (0; 112,5; 225; 337,5 e 450 m3 ha-1) durante o ciclo do milho, combinadas com duas adubações químicas (50 e 75% da dose recomendada), para compor os respectivos tratamentos, com três repetições. Os parâmetros da cultura do milho avaliados foram: produtividade (PROD), altura de plantas (AP), teor de macronutrientes (N, P, K e Mg) e de micronutrientes (Fe, Zn, Cu e B). Os parâmetros de solo avaliados foram: conteúdo de matéria orgânica (MO), soma e saturação por bases (SB e V), pH e teores disponíveis de N, P, K, Cu, Zn, Fe e Mn. A partir dos resultados obtidos, concluiu-se que: na cultura do milho, a aplicação de água residuária da suinocultura é fator predominante no aumento da produtividade e na nutrição mineral em K, Mg, Fe, Cu e B, e insuficiente para N e P; no solo, de modo geral, a água residuária da suinocultura propiciou aumentos positivos nos parâmetros de P, Zn, Cu, pH e SB, negativos nos teores de K e Fe, e nenhum efeito nos parâmetros N, V, MO e Mn; os parâmetros P, Cu e Zn aumentaram significativamente os teores no solo ao longo do experimento, indicando a importância do fator tempo nesse tipo de estudo.

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