Adipokinetic hormones (AKHs) are neuropeptides from the arthropod AKH/RPCH (adipokinetic hormone/ red pigment concentrating hormone) family. The typical AKH is an octa-, nona- or decamer that is synthesized, stored and released by the neurosecretory cells of the corpora cardiaca (CC) connected to the brain and primarily involved in the mobilization of energy reserves from the fat body in insects. In addition to its well established role in energy metabolism, AKH has also been implicated to be involved in stress responses specifically to oxidative stress. Oxidative stress induced elevation of AKH levels as well as a modulation of biomarkers of oxidative damage following exogenous application of AKH have been demonstrated. However, the discrete steps involved in the mode of action of AKH in triggering an anti-oxidative response is far from clear. Given the role of AKH as a neuroendocrine factor that mediates a response to oxidative stress, the mode of action of AKH at the sub-cellular level was investigated. Using isolated central nervous system (brain) as an in vitro model, we establish that AKH can potentiate an anti-oxidative response to oxidative stress. Further, we also demonstrate that AKH uses a conserved signal transduction mechanism involving both protein kinase C (PKC) and cyclic adenosine monophosphate (cAMP) and by mobilizing both intra as well as extra-cellular Ca2+ stores to elaborate its anti-oxidative response. Finally, using the genetically tractable fruit fly Drosophila melanogaster, we demonstrate through RNAi mediated knockdown of AKH synthesis as well as overexpression of AKH using the GAL4/UAS system, that the fork-head box transcription factor (dFoXO) might function downstream of AKH signaling in its stress responsive role. These results implicate AKH as a stress hormone while offering possibilities to further identify specific regulatory mechanisms and downstream effector molecules. Since stress signaling pathways are conserved, insights obtained from such studies on insects will offer some unique avenues for understanding stress responses and related pathologies in vertebrates including humans.

Schizosaccharomyces pombe est devenu un important système modèle pour étudier les processus physiologiques, biochimiques et génétiques chez l'homme. Ces travaux appartiennent à un projet sur la façon dont la fonction altérée de l'appareil de Golgi contribue à des maladies comme le cancer ou cause des anormalités génétiques. La protéine HID-1 de C. elegans et des humains est une protéine de l'appareil de Golgi qui appartient à la superfamille de protéines DYMECLIN. Les animaux ont à la fois un gène HID1 et un gène DYM. Chez les humains, une expression réduite de HID1 est impliquée dans la prolifération de tumeurs. La perte de DYM chez les humains mène à une déformation squelettique. S. pombe a trois gènes orthologues HID-1, mais pas de DYM. Par contraste, de nombreux eucaryotes unicellulaires et pluricellulaires n'ont qu'un gène DYM. Les mutants de S. pombe sans Hid1 et Hid3 étaient sensibles au stress oxydatif et la croissance de hid3Δ a été stoppée dans des milieux de culture minimum standard. L'insensibilité de hid3Δ au brefeldin A, mais sa sensibilité au golgicide A démontrent que Hid3 fonctionne dans le transport antérograde à travers l'appareil de Golgi. Afin d'explorer des rapports indiquant que le renouvellement des protéines pourrait être modifié dans hid3Δ, j'ai entrepris une étude de protéomique à la quantification label-free. La régulation positive de la voie de signalisation MAPK de tension a démontré que les cellules étaient dans un état de tension dans des conditions normales de croissance. De plus, des composants de protéine dans plusieurs voies de signalisation étaient modifiés, pouvant affecter une large gamme de processus cellulaires. / Schizosaccharomyces pombe has become an important model system to study physiological, biochemical and genetic processes in humans. This work is part of a continuing project to study how altered Golgi function contributes to diseases, such as cancer, or causes of genetic abnormalities. The HID-1 protein of C. elegans and humans are peripheral membrane proteins of the Golgi apparatus and are part of the DYMECLIN superfamily of proteins. Animals have both a HID1 and a DYM gene. In C. elegans, HID-1 maintains normal cellular growth and in humans reduced expression of HID1 has been implicated in tumour proliferation. Loss of DYM in humans leads to skeletal deformation and potentially mental retardation. S. pombe has three HID-1 orthologues, but no DYM. In contrast, many unicellular and multicellular eukaryotes have only DYM. S. pombe mutants lacking Hid1 and Hid3 were sensitive to oxidative stress and growth of hid3Δ was stopped in standard minimal media. Insensitivity of hid3Δ to brefeldin A but sensitivity to golgicide A demonstrated that Hid3 operates in anterograde protein transport through the Golgi. In order to investigate reports that protein turnover might be altered in hid3Δ, I undertook a proteomics study using label-free protein quantification. Up-regulation of the MAPK stress signalling pathway demonstrated that cells were under a state of stress under standard growth conditions. In addition, protein components of Ras signalling, microtubule dynamics and chromatin remodelling were altered potentially affecting a wide variety of processes from cell cycle regulation to metabolism.

Plant natriuretic peptides (PNPs) comprise a novel class of hormones that share some sequence similarity in the active site with their animal analogues that function as regulators of salt and water balance. A PNP present in Arabidopsis thaliana (AtPNP-A) has been assigned a role in abiotic and biotic stress responses, and the recombinant protein has been demonstrated to elicit cyclic guanosine monophosphate (cGMP)-dependent stomatal guard cell opening, regulate ion movements, and induce osmoticum-dependent water uptake. Although the importance of the hormone in maintaining ion and fluid homeostasis has been established, key components of the AtPNP-A-dependent signal transduction pathway remain unknown. Since identification of the binding partners of AtPNP-A, including its receptor(s), is fundamental to understanding the mode of its action at the molecular level, comprehensive protein-protein interaction studies, involving yeast two-hybrid screening, affinity-based assays, protein cross-linking and co-immunoprecipitation followed by mass spectrometric (MS) analyses have been performed. Several candidate binding partners of AtPNP-A identified with at least two independent methods were subsequently expressed as recombinant proteins, purified, and the specificity of their interactions with the recombinant AtPNP-A was verified using surface plasmon resonance. Several specific binary interactants of AtPNP-A were subjected to functional assays aimed at unraveling the consequences of the interactions in planta. These experiments have revealed that reactive oxygen species (ROS) are novel secondary messengers involved in the transduction of AtPNP-A signal in suspension-cultured cells of A. thaliana (Col-0). Further insight into the AtPNP-A dependent signalling events occurring in suspension-cultured cells in ROS-dependent or ROS-independent manner have been obtained from the large-scale proteomics study employing tandem mass tag (TMT) labelling followed by MS analysis to identify and relatively quantify proteins that are differentially expressed upon the treatment with nano- and picomolar concentrations of the biologically active AtPNP-A peptide at different time-points post-treatment. Characterization of both the AtPNP-A interactome and AtPNP-A dependent proteome afforded novel insights into the signal transduction pathways altered by PNPs and shed new light on the mechanisms by which these candidate interactants operate. Taken together, indications are that PNP dependent mechanisms can be harnessed for possible biotechnological applications.

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Les plantes ne pouvant pas se déplacer sont continuellement soumises aux stress environnementaux. La blessure, l’un des stress les plus fréquents auxquels la plante est soumise, peut causer d’important dégâts et faciliter l’entrée de pathogène dans les tissus de la plante. Pour répondre efficacement à la blessure, la plante a développé des mécanismes lui permettant de guérir ses tissus endommagés et d’empêcher l’infection pathogène. Les stress environnementaux sont perçus grâce à la présence de récepteurs spécifiques activant des voies de signalisation qui, à terme, conduisent à la mise en place de réponses de défense. Les modules de MAPK, composés de 3 kinases (MAP3K, MAP2K et MAPK) activées en cascades, représentent d’importantes voies de signalisation impliquées en réponse à divers stress biotiques et abiotiques. Grâce aux approches de tests de phosphorylation in vitro très maîtrisées dans le groupe « Stress signaling », j’ai pu identifier un module MAPK impliquant la MAP2K MKK3 et les MAPKs du groupe C (MPK1, 2, 7 et 14) activé par la blessure. Les MAP3Ks du sous-clade III (MAP3K13 à 20) sont transcriptionnellement induites par divers stress ce qui semble être un mécanisme assez conservé. Certains membres du sous-clade III sont induits par la blessure et parmi eux la MAP3K14 semble avoir un rôle majeur en amont du module MKK3/MPK1-2-7-14. Enfin, j’ai pu montrer que l’acide jasmonique (JA), une phytohormone importante produite en réponse à la blessure, tient un rôle important en amont du module. Ce-dernier est également activé en réponse à l’insecte herbivore Spodoptera littoralis et au champignon nécrotrophe Botrytis cinerea. Dans le contexte de blessure par l’insecte herbivore, MKK3 semble réguler la production de deux phytohormones, le JA et l’Acide Salicylique (SA). / Plants are sessile organisms. They have to cope continuously with environmental stresses. Injury, one of the most frequent stress conditions that plants must face may cause harsh damages to the plant tissues and facilitate the entry of pathogens. Therefore, plants have evolved mechanisms to respond efficiently to wounding by healing damaged tissues and preventing further pathogen infection. Wounding is a complex stress which is perceived by specific receptors which activate signaling pathways leading to those responses. Mitogen-Activated Protein Kinases modules are composed of 3 kinases (MAP3K, MAP2K and MAPK) activated in cascade and represent important signaling pathways involved in response to various biotic and abiotic stresses as well as in developmental processes. During my Ph.D I identified a MAPK module activated 30 minutes after wounding and involving the MAP2K MKK3 acting upstream of C-group MAPKs MPK1-2-7-14. In the past, the laboratory has shown that this module is dependent on the transcriptional regulation of sub-clade III MAP3Ks (MAP3K13 to 20). Some were found induced by wounding and among them MAP3K14 seems to have an important role upstream MKK3/C-group MAPKs. Finally I was able to show that Jasmonic Acid (JA), a major phytohormone produced upon wounding and involved in the mediation of defense responses, was shown to have an important role upstream the MKK3/C-group MAPKs module. The module is also activated by the herbivore Spodoptera littoralis and the necrotrophic fungus Botrytis cinerea. Upon insect feeding, MKK3 negatively regulates JA and SA levels. My work helped to better understand stress signaling events occurring upon wounding.

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