Functional Networks Involved in Cell Wall Biosynthesis and the Isoprenoid Pathway in the Yeast Saccharomyces cerevisiae

Ce travail porte sur la réponse de la levure à des composés chimiques, qui de manière similaire aux antifongiques, altèrent la structure pariétale et/ou la biosynthèse de l'ergostérol. Ces deux voies sont essentielles pour la survie des champignons, la paroi les protège de leur environnement, alors que l'ergostérol garantit un fonctionnement correct de la membrane plasmique. Pour comprendre ces mécanismes et leurs interactions, nous avons choisi un organisme modèle, la levure Saccharomyces cerevisiae, et étudié sa réponse à des agents perturbant la synthèse de la paroi (populacandine B, rouge Congo, caféine et Calcofluor White), ainsi qu'à des inhibiteurs de la biosynthèse de l'ergostérol (lovastatine et acide zaragozic). Nous avons découvert que la caféine inhibe Tor1p et provoque une baisse rapide et transitoire du niveau intracellulaire en AMPc. Nous avons ainsi montré que Rom2p, un élément de la voie du maintien de l’intégrité pariétale, agit sous l'effet de la caféine comme une protéine "pivot" en transmettant le signal à la kinase Mpk1, mais également à la voie de signalisation de l'AMPc. La caféine a induit un "renforcement" de la paroi mais, contrairement aux autres drogues testées, cet effet ne s'exerce pas à travers une interaction directe. En effet, ce remodelage ne se produit pas à travers la voie habituelle Rlm1p, mais nécessite d'autres facteurs de transcription comme Crz1p, Swi4p et Msn2/4p. Ces résultats nous ont permis d'identifier de nouvelles relations entre les voies de maintien de l'intégrité pariétale, TOR, et la cascade de signalisation de l'AMPc, qui exercent un contrôle synergique sur la croissance des champignons en réponse aux stress.\ud ___________________________________________________________________This work is focused on the fungal response to the chemical compounds, which similarly to antifungal drugs, impair the cell wall or ergosterol biosynthesis. Both examined pathways are essential for survival of fungi. The cell wall protects them from the harmful environment whereas ergosterol ensures correct functioning of the plasma membrane. For investigation of those pathways, we chose the genetically tractable fungus, the yeast Saccharomyces cerevisiae, and studied its response to the cell wall damaging agents, i.e. Papulacandin B, Congo red, Calcofluor white and caffeine, and to the inhibitors of the ergosterol biosynthesis like lovastatin and zaragozic acid. A novel function of caffeine was discovered to inhibit Tor1p and to cause a rapid but transient decrease in the intracellular level of cAMP. We have identified Rom2p, a component of the cell wall integrity pathway, as a pivotal protein mediating caffeine-derived signaling to Mpk1 kinase as well as to the cAMP signaling pathway. Caffeine induced strengthening of the cell wall but, in contrast to other tested drugs, this effect was not exerted through a direct interaction with the cell wall structure. Moreover, the caffeine-induced remodeling of the cell wall did not occur through the usual Rlm1p-mediated way but it required some other transcription factors like Crz1p, Swi4p and Msn2/4p. Altogether, we report here a number of results that bear on the relationship between the cell wall integrity, the TOR pathway and the cAMP signaling cascade that together control the growth of the fungal cell in response to stressing conditions

In acute myeloid leukemia, B7-H1 (PD-L1) protection of blasts from cytotoxic T cells is induced by TLR ligands and interferon-gamma and can be reversed using MEK inhibitors

B7-H1 (PD-L1) is a B7-related protein that inhibits T-cell responses. B7-H1 participates in the immunoescape of cancer cells and is also involved in the long-term persistence of leukemic cells in a mouse model of leukemia. B7-H1 can be constitutively expressed by cancer cells, but is also induced by various stimuli. Therefore, we examined the constitutive and inducible expression of B7-H1 and the consequences of this expression in human acute myeloid leukemia (AML). We analyzed B7-H1 expression in a cohort of 79 patients with AML. In addition, we studied blast cells after incubation with interferon-gamma or toll-like receptors (TLR) ligands. Finally, we evaluated functionality of cytotoxic T-cell activity against blast cells. Expression of B7-H1 upon diagnosis was high in 18% of patients. Expression of TLR2, 4 and 9 was detected in one-third of AML samples. Expression of TLR2 and TLR4 ligands or IFN-γ induced by B7-H1 was found to protect AML cells from CTL-mediated lysis. Spontaneous B7-H1 expression was also found to be enhanced upon relapse in some patients. MEK inhibitors, including UO126 and AZD6244, reduced B7-H1 expression and restored CTL-mediated lysis of blast cells. In AML, B7-H1 expression by blasts represents a possible immune escape mechanism. The inducibility of B7-H1 expression by IFN-γ or TLR ligands suggests that various stimuli, either produced during the immune response against leukemia cells or released by infectious microorganisms, could protect leukemic cells from T cells. The efficacy of MEK inhibitors against B7-H1-mediated inhibition of CTLs suggests a possible cancer immunotherapy strategy using targeted drugs. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00262-010-0909-y) contains supplementary material, which is available to authorized users

Réseaux fonctionnels impliqués dans la biosynthèse de la paroi cellulaire et dans la voie des isoprénoïdes chez la levure Saccharomyces cerevisiae

TOULOUSE-INSA (315552106) / SudocSudocFranceF

Investigating the caffeine effects in the yeast Saccharomyces cerevisiae brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways

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The YTA7 gene is involved in the regulation of the isoprenoid pathway in the yeast Saccharomyces cerevisiae

International audienceThe isoprenoid pathway in yeasts is important not only for sterol biosynthesis but also for the production of nonsterol molecules, deriving from farnesyl diphosphate (FPP), implicated in N-glycosylation and biosynthesis of heme and ubiquinones. FPP formed from mevalonate in a reaction catalyzed by FPP synthase (Erg20p). In order to investigate the regulation of Erg20p in Saccharomyces cerevisiae, we searched for its protein partners using a two-hybrid screen, and identified five interacting proteins, among them Yta7p. Subsequently, we showed that Yta7p was a membrane-associated protein localized both to the nucleus and to the endoplasmic reticulum. Deletion of YTA7 affected the enzymatic activity of cis-prenyltransferase (the enzyme that utilizes FPP for dolichol biosynthesis) and the cellular levels of isoprenoid compounds. Additionally, it rendered cells hypersensitive to lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) that acts upstream of FPP synthase in the isoprenoid pathway. While HMGR is encoded by two genes, HMG1 and HMG2, only HMG2 overexpression was able to restore growth of the yta7Delta cells in the presence of lovastatin. Moreover, the expression level of the S. cerevisiae YTA7 gene was altered upon impairment of the isoprenoid pathway not only by lovastatin but also by zaragozic acid, an inhibitor of squalene synthase. Altogether, these results provide substantial evidence of Yta7p involvement in the regulation of isoprenoid biosynthesis

Exposure to wild-type AAV drives distinct capsid immunity profiles in humans

International audienceRecombinant adeno-associated virus (AAV) vectors have been broadly adopted as a gene delivery tool in clinical trials, owing to their high efficiency of transduction of several host tissues and their low immunogenicity. However, a considerable proportion of the population is naturally exposed to the WT virus from which AAV vectors are derived, which leads to the acquisition of immunological memory that can directly determine the outcome of gene transfer. Here, we show that prior exposure to AAV drives distinct capsid immunity profiles in healthy subjects. In peripheral blood mononuclear cells (PBMCs) isolated from AAV-seropositive donors, recombinant AAV triggered TNF-α secretion in memory CD8+ T cells, B cell differentiation into antibody-secreting cells, and anti-capsid antibody production. Conversely, PBMCs isolated from AAV-seronegative individuals appeared to carry a population of NK cells reactive to AAV. Further, we demonstrated that the AAV capsid activates IL-1β and IL-6 cytokine secretion in monocyte-related dendritic cells (moDCs). IL-1β and IL-6 blockade inhibited the anti-capsid humoral response in vitro and in vivo. These results provide insights into immune responses to AAV in humans, define a possible role for moDCs and NK cells in capsid immunity, and open new avenues for the modulation of vector immunogenicity

Early Phase Clinical Immunogenicity of Valoctocogene Roxaparvovec, an AAV5-Mediated Gene Therapy for Hemophilia A

International audienceEvaluation of immune responses to adeno-associated virus (AAV)-mediated gene therapies prior to and following dose administration plays a key role in determining therapeutic safety and efficacy. This report describes up to 3 years of immunogenicity data following administration of valoctocogene roxaparvovec (BMN 270), an AAV5-mediated gene therapy encoding human B domain-deleted FVIII (hFVIII-SQ) in a phase 1/2 clinical study of adult males with severe hemophilia A. Patients with pre-existing humoral immunity to AAV5 or with a history of FVIII inhibitors were excluded from the trial. Blood plasma and peripheral blood mononuclear cell (PBMC) samples were collected at regular intervals following dose administration for assessment of humoral and cellular immune responses to both the AAV5 vector and transgene-expressed hFVIII-SQ. The predominant immune response elicited by BMN 270 administration was largely limited to the development of antibodies against the AAV5 capsid that were cross-reactive with other common AAV serotypes. No FVIII inhibitor responses were observed within 3 years following dose administration. In a context of prophylactic or on-demand corticosteroid immunosuppression given after vector infusion, AAV5 and hFVIII-SQ peptide-specific cellular immune responses were intermittently detected by an interferon (IFN)-γ and tumor necrosis factor (TNF)-α FluoroSpot assay, but they were not clearly associated with detrimental safety events or changes in efficacy measures