HIV-1 Tat protein inhibits neurosecretion by binding to phosphatidylinositol 4,5-bisphosphate.

HIV-1 transcriptional activator (Tat) enables viral transcription and is also actively released by infected cells. Extracellular Tat can enter uninfected cells and affect some cellular functions. Here, we examine the effects of Tat protein on the secretory activity of neuroendocrine cells. When added to the culture medium of chromaffin and PC12 cells, Tat was actively internalized and strongly impaired exocytosis as measured by carbon fiber amperometry and growth hormone release assay. Expression of Tat mutants that do not bind to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] did not affect secretion, and overexpression of phosphatidylinositol 4-phosphate 5-kinase (PIP5K), the major PtdIns(4,5)P2 synthesizing enzyme, significantly rescued the Tat-induced inhibition of neurosecretion. This suggests that the inhibition of exocytosis may be the consequence of PtdIns(4,5)P2 sequestration. Accordingly, expression of Tat in PC12 cells interfered with the secretagogue-dependent recruitment of annexin A2 to the plasma membrane, a PtdIns(4,5)P2-binding protein that promotes the formation of lipid microdomains that are required for exocytosis. In addition Tat significantly prevented the reorganization of the actin cytoskeleton necessary for the movement of secretory vesicles towards plasma membrane fusion sites. Thus, the capacity of extracellular Tat to enter neuroendocrine cells and sequester plasma membrane PtdIns(4,5)P2 perturbs several PtdIns(4,5)P2-dependent players of the exocytotic machinery, thereby affecting neurosecretion. We propose that Tat-induced inhibition of exocytosis is involved in the neuronal disorders associated with HIV-1 infection.journal articleresearch support, non-u.s. gov't2013 Jan 152012 11 23importe

Metabolisme des acides gras et dynamique des phospholipides dans l'erythrocyte infecte par le parasite du paludisme

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Mode d'entrée de la protéine Tat de HIV-1 dans les lymphocytes T CD4+

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Mécanismes de sécrétion non conventionnelle et d'inhibition de la phagocytose par la protéine Tat du VIH-1

La protéine Tat du VIH-1 est essentielle à la réplication virale. Sécrétée de manière non conventionnelle par les cellules infectées, elle entre dans les cellules non infectées et perturbe leur fonctionnement. La Tat extracellulaire agit donc comme une toxine virale et constitue un facteur déterminant de l'évolution vers le SIDA. Nous avons étudié le mécanisme de sécrétion de Tat par le lymphocyte T CD4+ (T4). Nous montrons que les T4 infectés exportent la majorité de leur Tat, et que le partenaire de Tat responsable de son recrutement est le PI(4,5)P2 membranaire. La fixation de Tat sur le PIP2 est cruciale pour sa sécrétion. Son affinité est telle que Tat peut déplacer les protéines cellulaires du PIP2. Nous avons cherché à déterminer si cette fixation de Tat sur le PIP2 était responsable de l'inhibition de la phagocytose observée chez les monocytes et les macrophages des patients infectés. Nos résultats indiquent que Tat pourrait effectivement être impliquée dans ce défaut de phagocytose. En effet, la Tat extracellulaire inhibe fortement la phagocytose par les macrophages. Pour cela, Tat se localise au niveau de la coupe phagocytaire, zone fortement enrichie en PIP2 où Tat bloque le recrutement de protéines liant le cytosquelette à la membrane, empêchant ainsi l'internalisation des particules opsonisées. Cette action de la Tat extracellulaire était inconnue jusqu'alors et pourrait participer au développement des infections opportunistes lors du SIDAHIV-1 Tat protein is essential for viral replication and transcription. Although devoid of a signal sequence, Tat is secreted by infected cells using an unconventional secretion mechanism. Circulating Tat can then enter and affect uninfected cells, inducing T-cell or neuron death for instance. Extracellular Tat thus behaves like a viral toxin and is a key virulence factor for progression to AIDS. We characterized Tat secretion by one of its main producer cell, the T CD4+ lymphocyte (T4). We show that T4 export most of their Tat, in agreement with Tat accumulation at their plasma membrane. Tat recruitment at this level is mediated by a tight binding to phosphatidylinositol-4,5-bisphosphate (PIP2), a lipid that exclusively localizes to the inner leaflet of the plasma membrane. Tat-PIP2 interaction is crucial for Tat secretion. The molecular bases of this interaction are original and the affinity is so high that Tat can displace cellular proteins from PIP2. We examined whether Tat fixation on PIP2 could be responsible for the phagocytosis defect observed in phagocytic cells from infected patients. Our results indicate that Tat could actually be involved in this phagocytosis inhibition. Indeed, extracellular Tat strongly inhibits phagocytosis by macrophages. For this, Tat localizes to the phagocytic cup, a region that is strongly enriched in PIP2 where Tat interfere with the recruitment of proteins such as ezrin that connects the cytoskeleton to the plasma membrane, thereby preventing phagocytosis of opsonized particles. This is an original extracellular Tat activity that might contribute to the development of opportunistic infections during AIDSMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Correlation of the efficiency of fatty acid derivatives in suppressing Plasmodium falciparum growth in culture with their inhibitory effect on acyl-CoA synthetase activity

International audienceThe intraerythrocytic malaria parasite depends on the surrounding medium for a supply of phospholipid precursors. Efficient inhibition (IC50 7–90 μM) of Plasmodium falciparum growth in vitro was achieved using modified fatty acids. The fatty acid analogues most effective in suppressing P. falciparum growth in culture were also the most active inhibitors of acyl-CoA synthetase from the monkey parasite P. knowlesi

Phosphatidylinositol (4,5)-bisphosphate-mediated pathophysiological effect of HIV-1 Tat protein

International audienceHuman immunodeficiency virus (HIV)-infected cells actively release the transcriptional activator (Tat) viral protein that is required for efficient HIV gene transcription. Extracellular Tat is able to enter uninfected cells. We recently reported that internalized Tat escapes endosomes to reach the cytosol and is then recruited to the plasma membrane by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). As a consequence, Tat strongly impairs different critical cellular functions in several cell types. Here we will review recent evidences showing that Tat, by affecting the interaction of key cellular effectors with PtdIns(4,5)P2, blocks exocytosis from neuroendocrine cells, perturbs the synaptic vesicle exo-endocytosis cycle, prevents efficient phagocytosis by macrophages, and alters potassium channel activity in cardiac cells. Potential mechanistic aspects of Tat effects on these cellular processes will be discussed

Mécanismes d'interaction de la protéine Tat du VIH-1 avec les membranes plasmique et endosomale

La protéine transactivatrice Tat joue un rôle crucial dans la multiplication du VIH-1. Elle a un rôle établi dans la transcription virale. Elle possède de plus la propriété d'être sécrétée par les cellules infectées. Elle peut ensuite pénétrer par endocytose dans les cellules non-infectées, où elle va provoquer divers dysfonctionnements. Dans ce travail, nous avons mis en évidence que Tat est sécrétée directement à travers la membrane plasmique en utilisant un mécanisme de sécrétion non conventionnel. Nous avons montré que le phosphatidylinositol-4,5-bisphosphate, PI(4,5)P2, est responsable du recrutement de Tat sur le feuillet interne de la membrane plasmique. En effet, Tat se fixe au PI(4,5)P2 avec une grande spécificité et une forte affinité. Cette interaction de stochiométrie 1:1 implique un triplet de résidus basiques de Tat (résidus 49-51). Elle est stabilisée par l'insertion membranaire de la chaîne latérale du Trp11. Ce mécanisme de fixation original restreint la fixation de Tat au PI(4,5)P2 membranaire mais permet aussi une très haute affinité. Celle-ci va permettre à Tat d'être sécrétée mais aussi de déplacer des protéines cellulaires du PI(4,5)P2. D'autre part, nous avons élucidé le mécanisme moléculaire permettant l'insertion de Tat dans la membrane endosomale. Elle est induite par le pH acide (pH~5.3) de ces compartiments. Ce pH acide est détecté par un système "sensor" impliquant Tat-Glu2, résidu acide qui est relié par un pont salin avec un triplet d'Arg (55-57). La protonation de Glu2 rompt les ponts salins, permettant l'exposition du Trp11 et l'insertion membranaire de Tat, préalable à sa translocation vers le cytosolThe HIV-1 transactivating factor Tat, plays a critical role in HIV-1 pathogenesis. It is strictly required for viral transcription. It can also be secreted by infected cells. Once in the bloodstream, it can enter the cytosol of uninfected cells by endocytosis and dramatically affect their biological activity. Here, we provide evidence that Tat is directly secreted through the plasma membrane using an unconventional secretion pathway. We found that phosphatidylinositol-4,5-bisphosphate, PI(4,5)P2, is responsible for Tat recruitment to the inner leaflet of the plasma membrane, thereby allowing its membrane insertion and translocation. We observed, using a variety of biophysical techniques, that Tat binds specifically and with a very high affinity to a single molecule of PI(4,5)P2. This interaction is mediated by a triplet of basic residues (residues 49-51), and is stabilized by the concomitant membrane insertion of the side chain of Tat-Trp11. This original mechanism restricts Tat binding to membrane embedded PI(4,5)P2, but also enables very tight binding. This high affinity allows Tat to be secreted and to displace cell proteins?n?nfrom the plasma membrane. We also elucidate the molecular mechanism responsible for Tat insertion into the endosome membrane. This insertion is triggered by the low pH (pH ~5.3) present within the endosome lumen. Acidic pH is detected by a sensor made of Glu2 and an Arg triplet (55-57) that are connected by a salt bridge. Glu2 protonation at low pH will destabilize the salt bridge, thereby allowing Trp11 exposure and enabling membrane penetration. This is the first step of the membrane translocation process that will allow Tat delivery to cytosolMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Total cholesterol, fatty acids, and plasmalogens can be reliably quantitated by analysis on chromarods after the methylation step required for fatty acid analysis by gas-liquid chromatography

International audienceA new method of simultaneous quantitation of total fatty acids, plasmalogens, and cholesterol using thin-layer chromatography with flame-ionization detection (TLC-FID) is presented. This Method only requires methanolysis of the crude lipid extracts, an operation usually performed for fatty acid analysis by gas-liquid chromatography (GLC), and both analyses can thus be carried out in one step. Because of its resolution power and sensitivity, the TLC-FID technique can be used to analyze microsamples routinely assayed in capillary GLC (less than 15 μg of total fatty acids). The data obtained by capillary GLC and those provided by TLC-FID after careful individual calibration of the rods are almost identical. Reproducibility of the TLC-FID measurements is lower for fatty acid methyl esters and dimethylacetals, but equivalent for cholesterol quantitation, if the samples are analyzed twice on chromarods

Autophagy restricts HIV-1 infection

International audienc