Caractérisation des périodes de sécheresse sur le domaine de l'Afrique simulée par le Modèle Régional Canadien du Climat (MRCC5)

Les conséquences des changements climatiques sur la fréquence ainsi que sur l'intensité des précipitations auront un impact direct sur les périodes de sécheresse et par conséquent sur différents secteurs économiques tels que le secteur de l'agriculture. Ainsi, dans cette étude, l'habilité du Modèle Régional Canadien du Climat (MRCC5) à simuler les différentes caractéristiques des périodes de sécheresse est évaluée pour 4 seuils de précipitation soit 0.5 mm, 1 mm, 2 mm et 3 mm. Ces caractéristiques incluent le nombre de jours secs, le nombre de périodes de sécheresse ainsi que le maximum de jours consécutifs sans précipitation associé à une récurrence de 5 ans. Les résultats sont présentés pour des moyennes annuelles et saisonnières. L'erreur de performance est évaluée en comparant le MRCC5 piloté par ERA-Interim aux données d'analyses du GPCP pour le climat présent (1997-2008). L'erreur due aux conditions aux frontières c'est-à-dire les erreurs de pilotage du MRCC5, soit par CanESM2 et par ERA-Interim ainsi que l'évaluation de la valeur ajoutée du MRCC5 face au CanESM2 sont également analysées. L'analyse de ces caractéristiques est également faite dans un contexte de climat changeant pour deux périodes futures, soit 2041-2070 et 2071-2100 à l'aide du MRCC5 piloté par le modèle de circulation générale CanESM2 de même que par le modèle CanESM2 sous le scénario RCP 4.5. Les résultats suggèrent que le MRCC5 piloté par ERA-Interim a tendance à surestimer la moyenne annuelle du nombre de jours secs ainsi que le maximum de jours consécutifs sans précipitation associé à une récurrence de 5 ans dans la plupart des régions de l'Afrique et une tendance à sous-estimer le nombre de périodes de sécheresse. En général, l'erreur de performance est plus importante que l'erreur due aux conditions aux frontières pour les différentes caractéristiques de périodes de sécheresse. Pour les régions équatoriales, les changements appréhendés par le MRCC5 piloté par CanESM2 pour les différentes caractéristiques de périodes de sécheresse et pour deux périodes futures (2041-2070 et 2071-2100), suggèrent une augmentation significatives du nombre de jours secs ainsi que du maximum de jours consécutifs sans précipitation associé à une récurrence de 5 ans. Une diminution significative du nombre de périodes de sécheresse est aussi prévue.\ud ______________________________________________________________________________ \ud MOTS-CLÉS DE L’AUTEUR : Modèle Régional du Climat, Changement climatique, Jours secs, Nombre de périodes de sécheresse, Événement de faible récurrence, Afriqu

Insights into the oligomerization process of the C-terminal domain of human plasma membrane Ca2+-ATPase

Plasma membrane calcium pumps (PMCAs) sustain a primary transport system for the specific removal of cytosolic calcium ions from eukaryotic cells. PMCAs are characterized by the presence of a C-terminal domain referred to as a regulatory domain. This domain is target of several regulatory mechanisms: activation by Ca(2+)-calmodulin complex and acidic phospholipids, phosphorylation by kinase A and C, proteolysis by calpain and oligomerization. As far as oligomerization is concerned, the C-terminal domain seems to be crucial for this process. We have cloned the C-terminal domain of the human PMCA isoform 1b, and characterized its properties in solution. The expressed protein maintains its tendency to oligomerize in aqueous solutions, but it is dissociated by amphipathic molecules such as diacylglycerol and sodium dodecyl sulphate. The presence of sodium dodecyl sulphate stabilizes the domain as a compact structure in monomeric form retaining the secondary structure elements, as shown by small angle neutron scattering and circular dichroism measurements. The importance of oligomerization for the regulation of PMCA activity and intracellular calcium concentration is discussed

Quaternary Structure Heterogeneity of Oligomeric Proteins: A SAXS and SANS Study of the Dissociation Products of Octopus vulgaris Hemocyanin

Octopus vulgaris hemocyanin shows a particular self-assembling pattern, characterized by a hierarchical organization of monomers. The highest molecular weight aggregate is a decamer, the stability of which in solution depends on several parameters. Different pH values, buffer compositions, H\u2082O/D\u2082O ratios and Hofmeister's salts result in modifications of the aggregation state of Octopus vulgaris hemocyanin. The new QUAFIT method, recently applied to derive the structure of the decameric and the monomeric assembly from small-angle scattering data, is used here to model the polydisperse system that results from changing the solution conditions. A dataset of small-angle X-rays and neutron scattering curves is analysed by QUAFIT to derive structure, composition and concentration of different assemblies present in solution. According to the hierarchy of the association/dissociation processes and the possible number of different aggregation products in solution, each sample has been considered as a heterogeneous mixture composed of the entire decamer, the dissociated "loose" monomer and all the intermediate dissociation products. Scattering curves corresponding to given experimental conditions are well fitted by using a linear combination of single particle form factors. QUAFIT has proved to be a method of general validity to describe solutions of proteins that, even after purification processes, result to be intrinsically heterogeneous

Solution structures of 2x6-meric and 4x6-meric hemocyanins of crustaceans Carcinus aestuarii, Squilla mantis and Upogebia pusilla.

Arthropod hemocyanins (Hcs) are a family of large, high molecular mass, extracellular oxygen transport proteins. They form oligomeric quaternary structures based on different arrangements of a basic 6 x 75 kDa hexameric unit. Their complex quaternary structures present binding sites for allosteric effectors and regulate the oxygen binding process in a cooperative manner. In order to describe the functional regulation of arthropod Hcs, a detailed description of their quaternary structure is necessary. We have utilized small angle X-ray scattering to characterize the structure of three arthropod Hcs in unperturbed conditions. Two different levels of complexity are evaluated: for the 2 x 6-meric case, we analyzed the Hcs of the portunid crab Carcinus aestuarii and stomatopod Squilla mantis, while in the case of 4 x 6-meric structures, we studied the Hc of the thalassinid shrimp Upogebia pusilla. While C aestuarii Hc presented a structure comparable to other 2 x 6-meric crustacean Hcs, S. mantis Hc shows a peculiar and quite unique arrangement of its building blocks, resembling a substructure of giant Hcs found among cheliceratans. For U. pusilla, the arrangement of its subunits is described as tetrahedral, in contrast to the more common square planar 4 x 6-meric structure found in other arthropod Hcs

Looking for putative phenoloxidases of compound ascidians: haemocyanin-like proteins in Polyandrocarpa misakiensis and Botryllus schlosseri

Phenoloxidases (POs) constitute a family of copper-containing enzymes widely distributed among invertebrates. They exert a pivotal role in immune defences as they induce cytotoxicity through the conversion of phenols to quinones and the production of reactive oxygen species. In ascidians, PO activity has been described and studied in both solitary and colonial species and the enzyme is involved in inflammatory and cytotoxic reactions against foreign cells or molecules, and in the formation of the cytotoxic foci which characterise the nonfusion reaction of botryllids. Expressed genes for two putative POs (CiPO1 and CiPO2) have been recently identified in C. intestinalis. In the present study, we determined the cDNA sequences of the POs from two colonial ascidians: Botryllus schlosseri from Mediterranean Sea and Polyandrocarpa misakiensis from Japan. Multiple sequence alignments evidenced the similarity between ascidian POs and crustacean proPOs whereas the analysis of the three-dimensional structure reveals high similarity with arthropod haemocyanins which share common precursors with arthropod proPOs. Ascidian POs and arthropod proPOs grouped in the same cluster and share the full conservation of the six histidines at the two copper-binding sites as well as of other motifs, also found in arthropod haemocyanin subunits, involved in the regulation of enzyme activity. Cytoenzymatic studies and in situ hybridisation (ISH) indicated that the genes are transcribed inside morula cells (MCs), a characteristic haemocyte type in ascidians, at the beginning of their differentiation. Sequence analysis allowed a better understanding of previous biochemical data and suggest some hypotheses for the regulation of enzyme activity

Looking for putative phenoloxidases of compound ascidians: haemocyanin-like proteins in Polyandrocarpa misakiensis and Botryllus schlosseri

none6siPhenoloxidases (POs) constitute a family of copper-containing enzymes widely distributed among invertebrates. They exert a pivotal role in immune defences as they induce cytotoxicity through the conversion of phenols to quinones and the production of reactive oxygen species. In ascidians, PO activity has been described and studied in both solitary and colonial species and the enzyme is involved in inflammatory and cytotoxic reactions against foreign cells or molecules, and in the formation of the cytotoxic foci which characterise the nonfusion reaction of botryllids. Expressed genes for two putative POs (CiPO1 and CiPO2) have been recently identified in C. intestinalis. In the present study, we determined the cDNA sequences of the POs from two colonial ascidians: Botryllus schlosseri from Mediterranean Sea and Polyandrocarpa misakiensis from Japan. Multiple sequence alignments evidenced the similarity between ascidian POs and crustacean proPOs whereas the analysis of the three-dimensional structure reveals high similarity with arthropod haemocyanins which share common precursors with arthropod proPOs. Ascidian POs and arthropod proPOs grouped in the same cluster and share the full conservation of the six histidines at the two copper-binding sites as well as of other motifs, also found in arthropod haemocyanin subunits, involved in the regulation of enzyme activity. Cytoenzymatic studies and in situ hybridisation (ISH) indicated that the genes are transcribed inside morula cells (MCs), a characteristic haemocyte type in ascidians, at the beginning of their differentiation. Sequence analysis allowed a better understanding of previous biochemical data and suggest some hypotheses for the regulation of enzyme activity.mixedLoriano Ballarin; Nicola Franchi; Filippo Schiavon; Silvio C.E. Tosatto; Ivan Micetic; Kazuo KawamuraBallarin, Loriano; Franchi, Nicola; Schiavon, Filippo; Tosatto, Silvio C. E.; Micetic, Ivan; Kawamura, Kazu

Biochemical Characterization of Highly Purified Leucine-Rich Repeat Kinases 1 and 2 Demonstrates Formation of Homodimers

Leucine-rich repeat kinase 1 and 2 (LRRK1 and LRRK2) are large multidomain proteins containing kinase, GTPase and multiple protein-protein interaction domains, but only mutations in LRRK2 are linked to familial Parkinson's disease (PD). Independent studies suggest that LRRK2 exists in the cell as a complex compatible with the size of a dimer. However, whether this complex is truly a homodimer or a heterologous complex formed by monomeric LRRK2 with other proteins has not been definitively proven due to the limitations in obtaining highly pure proteins suitable for structural characterization. Here, we used stable expression of LRRK1 and LRRK2 in HEK293T cell lines to produce recombinant LRRK1 and LRRK2 proteins of greater than 90% purity. Both purified LRRKs are folded, with a predominantly alpha-helical secondary structure and are capable of binding GTP with similar affinity. Furthermore, recombinant LRRK2 exhibits robust autophosphorylation activity, phosphorylation of model peptides in vitro and ATP binding. In contrast, LRRK1 does not display significant autophosphorylation activity and fails to phosphorylate LRRK2 model substrates, although it does bind ATP. Using these biochemically validated proteins, we show that LRRK1 and LRRK2 are capable of forming homodimers as shown by single-particle transmission electron microscopy and immunogold labeling. These LRRK dimers display an elongated conformation with a mean particle size of 145 angstrom and 175 angstrom respectively, which is disrupted by addition of 6M guanidinium chloride. Immunogold staining revealed double-labeled particles also in the pathological LRRK2 mutant G2019S and artificial mutants disrupting GTPase and kinase activities, suggesting that point mutations do not hinder the dimeric conformation. Overall, our findings indicate for the first time that purified and active LRRK1 and LRRK2 can form dimers in their full-lengt