Fungal X-Intrinsic Protein Aquaporin from Trichoderma atroviride: Structural and Functional Considerations

The major intrinsic protein (MIP) superfamily is a key part of the fungal transmembrane transport network. It facilitates the transport of water and low molecular weight solutes across biomembranes. The fungal uncharacterized X-Intrinsic Protein (XIP) subfamily includes the full protein diversity of MIP. Their biological functions still remain fully hypothetical. The aim of this study is still to deepen the diversity and the structure of the XIP subfamily in light of the MIP counterparts-the aquaporins (AQPs) and aquaglyceroporins (AQGPs)-and to describe for the first time their function in the development, biomass accumulation, and mycoparasitic aptitudes of the fungal bioagent Trichoderma atroviride. The fungus-XIP Glade, with one member (TriatXIP), is one of the three clades of MIPs that make up the diversity of T. atroviride MIPs, along with the AQPs (three members) and the AQGPs (three members). TriatXIP resembles those of strict aquaporins, predicting water diffusion and possibly other small polar solutes due to particularly wider ar/R constriction with a Lysine substitution at the LE2 position. The XIP loss of function in Delta TriatXIP mutants slightly delays biomass accumulation but does not impact mycoparasitic activities. Delta TriatMIP forms colonies similar to wild type; however, the hyphae are slightly thinner and colonies produce rare chlamydospores in PDA and specific media, most of which are relatively small and exhibit abnormal morphologies. To better understand the molecular causes of these deviant phenotypes, a wide-metabolic survey of the ATriatXIPs demonstrates that the delayed growth kinetic, correlated to a decrease in respiration rate, is caused by perturbations in the pentose phosphate pathway. Furthermore, the null expression of the XIP gene strongly impacts the expression of four expressed MIP-encoding genes of T. atroviride, a plausible compensating effect which safeguards the physiological integrity and life cycle of the fungus. This paper offers an overview of the fungal XIP family in the biocontrol agent T. atroviride which will be useful for further functional analysis of this particular MIP subfamily in vegetative growth and the environmental stress response in fungi. Ultimately, these findings have implications for the ecophysiology of Trichoderma spp. in natural, agronomic, and industrial systems

Functional exploration of aquaporins through structural modeling. Impact on the leaf hydraulic conductance in poplar

Les Aquaporines (AQPs) constituent une famille de protéines transmembranaires impliquées dans le transport de l’eau et d’autres petites molécules polaires. Essentielles au maintien des équilibres chimiques des cellules, elles sont conservées dans tous les embranchements du vivant et forment une famille ancienne, étendue et diverse. Cette diversité est particulièrement frappante chez les plantes qui arborent souvent plusieurs dizaines d’homologues d’AQPs dans leur génome. Malgré une base de connaissances solides sur les mécanismes moléculaires du fonctionnement et des régulations des AQPs, la conservation et l’expression d’un certain nombre de gènes de ces homologues témoignent d’une diversité fonctionnelle encore incomprise à ce jour. Chez le peuplier, plante “modèle” et sujet d’étude central de nos recherches, cette énigme s’illustre très clairement en contexte de sécheresse. Dans les feuilles en carence hydrique sévère, les gènes d’une dizaine d’homologues d’AQPs strictes qui sur la base des connaissances actuelles fonctionnent et sont régulés de manière identique, sont différentiellement exprimés dans le même référentiel de temps. De plus, la moitié d’entre eux est sur-exprimée tandis que l’autre est sous-exprimée. C’est dans l’optique de relier cette diversité génétique à une diversité fonctionnelle que nous avons décidé d’étudier le mécanisme de transport de l’eau au travers des AQPs à l’échelle atomique par modélisation moléculaire en nous focalisant sur le cas d’étude des feuilles de peuplier en carence hydrique. En s’appuyant sur des structures cristallographiques d’AQPs humaine, bactérienne et végétales, cette étude nous a permis : (i) de mettre en lumière une diversité fonctionnelle nouvelle au sein des AQPs strictes, (ii) de proposer une correction du coefficient de perméabilité osmotique (pf), indicateur communément utilisé pour caractériser les AQPs, (iii) de proposer un mécanisme moléculaire de cette diversité, (iv) de contextualiser ce mécanisme dans la diversité génétique chez le peuplier en carence hydrique (v) d’étendre ce concept à l’ensemble de la famille AQP. De manière parallèle, des résultats préliminaires autour d’un autre mécanisme de régulation des AQPs végétales faisant intervenir une boucle intra-cellulaire ainsi que l’étude de la diversité de cette famille chez Trichoderma spp. nous a également permis : (i) d’ouvrir des pistes de recherche annexes et (ii) d’unifier les travaux de l’équipe pour proposer un archétype fonctionnel d’AQP.Aquaporins (AQPs) are transmembrane proteins involved in water and other small polar solutes transport. Because of their essential role in cells homeostasis, they are conserved in all branches of life and constitute a wide and ancient protein family. The diversity of these water channels is striking in plants where it is common to find dozens of AQPs homologues in there genome. Despite a good understanding of the molecular mechanisms underlying AQPs functions and regulations, the conservation and the expression of that many genes in some species still rise questions. This diversity is well illustrated in poplar, the woody “plant model” species studied in our team, experiencing drought. In the leaves of trees undergoing severe water deficiency, 10 AQP homologues, which according to our current knowledge are supposed to function in the same way, are differentially expressed at a given time point. Moreover, half of them are up-regulated while the other half are down-regulated. In order to link this genetic diversity to a functional diversity, we studied water transport across AQPs at the atomic resolution through molecular simulations, focusing on this particular biological context of poplar leaves undergoing water deficiency. Starting from the available crystallographic structures resolved from human, bacteria and plants, we managed to : (i) highlight a new functional diversity in strict AQPs, (ii) propose a correction constant for the osmotic permeability coefficient (pf), a commonly used AQPs permeability indicator, (iii) propose the underlying molecular mechanism, (iv) contextualize this mechanism with poplar genetic diversity during drought and (v) extend the concept to the whole AQP family. Conjointly, preliminary results obtained regarding a plant AQP gating mechanism previously described as well as the study of Trichoderma spp. AQPs allowed us to : (i) open new research opportunities and (ii) unify our team’s prior works to propose an AQP functional archetype

Exploration fonctionnelle d'aquaporines par modélisation structurale. Impact sur les conductances hydriques foliaires chez le peuplier

Aquaporins (AQPs) are transmembrane proteins involved in water and other small polar solutes transport. Because of their essential role in cells homeostasis, they are conserved in all branches of life and constitute a wide and ancient protein family. The diversity of these water channels is striking in plants where it is common to find dozens of AQPs homologues in there genome. Despite a good understanding of the molecular mechanisms underlying AQPs functions and regulations, the conservation and the expression of that many genes in some species still rise questions. This diversity is well illustrated in poplar, the woody “plant model” species studied in our team, experiencing drought. In the leaves of trees undergoing severe water deficiency, 10 AQP homologues, which according to our current knowledge are supposed to function in the same way, are differentially expressed at a given time point. Moreover, half of them are up-regulated while the other half are down-regulated. In order to link this genetic diversity to a functional diversity, we studied water transport across AQPs at the atomic resolution through molecular simulations, focusing on this particular biological context of poplar leaves undergoing water deficiency. Starting from the available crystallographic structures resolved from human, bacteria and plants, we managed to : (i) highlight a new functional diversity in strict AQPs, (ii) propose a correction constant for the osmotic permeability coefficient (pf), a commonly used AQPs permeability indicator, (iii) propose the underlying molecular mechanism, (iv) contextualize this mechanism with poplar genetic diversity during drought and (v) extend the concept to the whole AQP family. Conjointly, preliminary results obtained regarding a plant AQP gating mechanism previously described as well as the study of Trichoderma spp. AQPs allowed us to : (i) open new research opportunities and (ii) unify our team’s prior works to propose an AQP functional archetype.Les Aquaporines (AQPs) constituent une famille de protéines transmembranaires impliquées dans le transport de l’eau et d’autres petites molécules polaires. Essentielles au maintien des équilibres chimiques des cellules, elles sont conservées dans tous les embranchements du vivant et forment une famille ancienne, étendue et diverse. Cette diversité est particulièrement frappante chez les plantes qui arborent souvent plusieurs dizaines d’homologues d’AQPs dans leur génome. Malgré une base de connaissances solides sur les mécanismes moléculaires du fonctionnement et des régulations des AQPs, la conservation et l’expression d’un certain nombre de gènes de ces homologues témoignent d’une diversité fonctionnelle encore incomprise à ce jour. Chez le peuplier, plante “modèle” et sujet d’étude central de nos recherches, cette énigme s’illustre très clairement en contexte de sécheresse. Dans les feuilles en carence hydrique sévère, les gènes d’une dizaine d’homologues d’AQPs strictes qui sur la base des connaissances actuelles fonctionnent et sont régulés de manière identique, sont différentiellement exprimés dans le même référentiel de temps. De plus, la moitié d’entre eux est sur-exprimée tandis que l’autre est sous-exprimée. C’est dans l’optique de relier cette diversité génétique à une diversité fonctionnelle que nous avons décidé d’étudier le mécanisme de transport de l’eau au travers des AQPs à l’échelle atomique par modélisation moléculaire en nous focalisant sur le cas d’étude des feuilles de peuplier en carence hydrique. En s’appuyant sur des structures cristallographiques d’AQPs humaine, bactérienne et végétales, cette étude nous a permis : (i) de mettre en lumière une diversité fonctionnelle nouvelle au sein des AQPs strictes, (ii) de proposer une correction du coefficient de perméabilité osmotique (pf), indicateur communément utilisé pour caractériser les AQPs, (iii) de proposer un mécanisme moléculaire de cette diversité, (iv) de contextualiser ce mécanisme dans la diversité génétique chez le peuplier en carence hydrique (v) d’étendre ce concept à l’ensemble de la famille AQP. De manière parallèle, des résultats préliminaires autour d’un autre mécanisme de régulation des AQPs végétales faisant intervenir une boucle intra-cellulaire ainsi que l’étude de la diversité de cette famille chez Trichoderma spp. nous a également permis : (i) d’ouvrir des pistes de recherche annexes et (ii) d’unifier les travaux de l’équipe pour proposer un archétype fonctionnel d’AQP

Application pratique de la dynamique moléculaire au passage de l'eau dans une aquaporine.

Application pratique de la dynamique moléculaire au passage de l'eau dans une aquaporine.. Conférence de découverte sur la dynamique moléculair

Propriétés hydrauliques du xylème de mutants d'Arabidospsis thaliana

MasterLa résistance à la cavitation est considérée comme un trait fonctionnel et écologique des plus significatifs, et il est corrélé à la tolérance à la sécheresse des espèces ligneuses. Cependant, les bases génétiques de cette résistance à la cavitation restent méconnues. Dans cette étude, nous avons voulu tester le rôle de gènes récemment mis en évidence dans la formation des ponctuations, des structures clés dans la résistance à la cavitation. Pour cela, nous avons utilisé des lignées d’insertion d’ADN-T d’Arabidopsis thaliana, une espèce récemment validée comme modèle d’étude des propriétés hydrauliques du xylème. Nous avons génotypé 10 lignées d’insertion d’ADN-T pour ces gènes et nous avons caractérisé leurs propriétés hydrauliques et structurelles du xylème. Une lignée mutée pour ROPGAP3, une petite GTPase de régulation, montre une vulnérabilité à la cavitation significativement supérieure à celle de la lignée témoin. Une autre, mutée pour MAP70-1, une protéine associée aux microtobules, montre une conductivité spécifique significativement diminuée. Nous avons également développé une méthode de mesure directe de la cavitation par microtomographie chez Arabidopsis, permettant de confirmer l’existence de phénomène de cavitation chez cette espèce herbacée, et ouvrant des perspectives dans l’analyse des lignées mutantes

Cortisol Interaction with Aquaporin-2 Modulates Its Water Permeability: Perspectives for Non-Genomic Effects of Corticosteroids

Aquaporins (AQPs) are water channels widely distributed in living organisms and involved in many pathophysiologies as well as in cell volume regulations (CVR). In the present study, based on the structural homology existing between mineralocorticoid receptors (MRs), glucocorticoid receptors (GRs), cholesterol consensus motif (CCM) and the extra-cellular vestibules of AQPs, we investigated the binding of corticosteroids on the AQP family through in silico molecular dynamics simulations of AQP2 interactions with cortisol. We propose, for the first time, a putative AQPs corticosteroid binding site (ACBS) and discussed its conservation through structural alignment. Corticosteroids can mediate non-genomic effects; nonetheless, the transduction pathways involved are still misunderstood. Moreover, a growing body of evidence is pointing toward the existence of a novel membrane receptor mediating part of these rapid corticosteroids’ effects. Our results suggest that the naturally produced glucocorticoid cortisol inhibits channel water permeability. Based on these results, we propose a detailed description of a putative underlying molecular mechanism. In this process, we also bring new insights on the regulatory function of AQPs extra-cellular loops and on the role of ions in tuning the water permeability. Altogether, this work brings new insights into the non-genomic effects of corticosteroids through the proposition of AQPs as the membrane receptor of this family of regulatory molecules. This original result is the starting point for future investigations to define more in-depth and in vivo the validity of this functional model

Cortisol Interaction with Aquaporin-2 Modulates Its Water Permeability: Perspectives for Non-Genomic Effects of Corticosteroids

International audienceAquaporins (AQPs) are water channels widely distributed in living organisms and involved in many pathophysiologies as well as in cell volume regulations (CVR). In the present study, based on the structural homology existing between mineralocorticoid receptors (MRs), glucocorticoid receptors (GRs), cholesterol consensus motif (CCM) and the extra-cellular vestibules of AQPs, we investigated the binding of corticosteroids on the AQP family through in silico molecular dynamics simulations of AQP2 interactions with cortisol. We propose, for the first time, a putative AQPs corticosteroid binding site (ACBS) and discussed its conservation through structural alignment. Corticosteroids can mediate non-genomic effects; nonetheless, the transduction pathways involved are still misunderstood. Moreover, a growing body of evidence is pointing toward the existence of a novel membrane receptor mediating part of these rapid corticosteroids' effects. Our results suggest that the naturally produced glucocorticoid cortisol inhibits channel water permeability. Based on these results, we propose a detailed description of a putative underlying molecular mechanism. In this process, we also bring new insights on the regulatory function of AQPs extra-cellular loops and on the role of ions in tuning the water permeability. Altogether, this work brings new insights into the non-genomic effects of corticosteroids through the proposition of AQPs as the membrane receptor of this family of regulatory molecules. This original result is the starting point for future investigations to define more in-depth and in vivo the validity of this functional model

A Perspective for Ménière’s Disease: In Silico Investigations of Dexamethasone as a Direct Modulator of AQP2

International audienceMénière’s disease is a chronic illness characterized by intermittent episodes of vertigo associated with fluctuating sensorineural hearing loss, tinnitus and aural pressure. This pathology strongly correlates with a dilatation of the fluid compartment of the endolymph, so-called hydrops. Dexamethasone is one of the therapeutic approaches recommended when conventional antivertigo treatments have failed. Several mechanisms of actions have been hypothesized for the mode of action of dexamethasone, such as the anti-inflammatory effect or as a regulator of inner ear water homeostasis. However, none of them have been experimentally confirmed so far. Aquaporins (AQPs) are transmembrane water channels and are hence central in the regulation of transcellular water fluxes. In the present study, we investigated the hypothesis that dexamethasone could impact water fluxes in the inner ear by targeting AQP2. We addressed this question through molecular dynamics simulations approaches and managed to demonstrate a direct interaction between AQP2 and dexamethasone and its significant impact on the channel water permeability. Through compartmentalization of sodium and potassium ions, a significant effect of Na+ upon AQP2 water permeability was highlighted as well. The molecular mechanisms involved in dexamethasone binding and in its regulatory action upon AQP2 function are described

Plant Aquaporin Gating Is Reversed by Phosphorylation on Intracellular Loop D—Evidence from Molecular Dynamics Simulations

Aquaporins (AQPs) constitute a wide and ancient protein family of transmembrane channels dedicated to the regulation of water exchange across biological membranes. In plants, higher numbers of AQP homologues have been conserved compared to other kingdoms of life such as in animals or in bacteria. As an illustration of this plant-specific functional diversity, plasma membrane intrinsic proteins (PIPs, i.e., a subfamily of plant AQPs) possess a long intracellular loop D, which can gate the channel by changing conformation as a function of the cellular environment. However, even though the closure of the AQP by loop D conformational changes is well described, the opening of the channel, on the other hand, is still misunderstood. Several studies have pointed to phosphorylation events as the trigger for the transition from closed- to open-channel states. Nonetheless, no clear answer has been obtained yet. Hence, in order to gain a more complete grasp of plant AQP regulation through this intracellular loop D gating, we investigated the opening of the channel in silico through molecular dynamics simulations of the crystallographic structure of Spinacia oleracea PIP2;1 (SoPIP2;1). Through this technique, we addressed the mechanistic details of these conformational changes, which eventually allowed us to propose a molecular mechanism for PIP functional regulation by loop D phosphorylation. More precisely, our results highlight the phosphorylation of loop D serine 188 as a trigger of SoPIP2;1 water channel opening. Finally, we discuss the significance of this result for the study of plant AQP functional diversity

Deciphering Molecular Mechanisms Involved in the Modulation of Human Aquaporins’ Water Permeability by Zinc Cations: A Molecular Dynamics Approach

Aquaporins (AQPs) constitute a wide family of water channels implicated in all kind of physiological processes. Zinc is the second most abundant trace element in the human body and a few studies have highlighted regulation of AQP0 and AQP4 by zinc. In the present work, we addressed the putative regulation of AQPs by zinc cations in silico through molecular dynamics simulations of human AQP0, AQP2, AQP4, and AQP5. Our results align with other scales of study and several in vitro techniques, hence strengthening the reliability of this regulation by zinc. We also described two distinct putative molecular mechanisms associated with the increase or decrease in AQPs’ water permeability after zinc binding. In association with other studies, our work will help deciphering the interaction networks existing between zinc and channel proteins