Glutathione deficit impairs myelin maturation: relevance for white matter integrity in schizophrenia patients.

Schizophrenia pathophysiology implies both abnormal redox control and dysconnectivity of the prefrontal cortex, partly related to oligodendrocyte and myelin impairments. As oligodendrocytes are highly vulnerable to altered redox state, we investigated the interplay between glutathione and myelin. In control subjects, multimodal brain imaging revealed a positive association between medial prefrontal glutathione levels and both white matter integrity and resting-state functional connectivity along the cingulum bundle. In early psychosis patients, only white matter integrity was correlated with glutathione levels. On the other side, in the prefrontal cortex of peripubertal mice with genetically impaired glutathione synthesis, mature oligodendrocyte numbers, as well as myelin markers, were decreased. At the molecular levels, under glutathione-deficit conditions induced by short hairpin RNA targeting the key glutathione synthesis enzyme, oligodendrocyte progenitors showed a decreased proliferation mediated by an upregulation of Fyn kinase activity, reversed by either the antioxidant N-acetylcysteine or Fyn kinase inhibitors. In addition, oligodendrocyte maturation was impaired. Interestingly, the regulation of Fyn mRNA and protein expression was also impaired in fibroblasts of patients deficient in glutathione synthesis. Thus, glutathione and redox regulation have a critical role in myelination processes and white matter maturation in the prefrontal cortex of rodent and human, a mechanism potentially disrupted in schizophrenia

HIV-1 Vpu Neutralizes the Antiviral Factor Tetherin/BST-2 by Binding It and Directing Its Beta-TrCP2-Dependent Degradation

Host cells impose a broad range of obstacles to the replication of retroviruses. Tetherin (also known as CD317, BST-2 or HM1.24) impedes viral release by retaining newly budded HIV-1 virions on the surface of cells. HIV-1 Vpu efficiently counteracts this restriction. Here, we show that HIV-1 Vpu induces the depletion of tetherin from cells. We demonstrate that this phenomenon correlates with the ability of Vpu to counteract the antiviral activity of both overexpressed and interferon-induced endogenous tetherin. In addition, we show that Vpu co-immunoprecipitates with tetherin and β-TrCP in a tri-molecular complex. This interaction leads to Vpu-mediated proteasomal degradation of tetherin in a β-TrCP2-dependent manner. Accordingly, in conditions where Vpu-β-TrCP2-tetherin interplay was not operative, including cells stably knocked down for β-TrCP2 expression or cells expressing a dominant negative form of β-TrCP, the ability of Vpu to antagonize the antiviral activity of tetherin was severely impaired. Nevertheless, tetherin degradation did not account for the totality of Vpu-mediated counteraction against the antiviral factor, as binding of Vpu to tetherin was sufficient for a partial relief of the restriction. Finally, we show that the mechanism used by Vpu to induce tetherin depletion implicates the cellular ER-associated degradation (ERAD) pathway, which mediates the dislocation of ER membrane proteins into the cytosol for subsequent proteasomal degradation. In conclusion, we show that Vpu interacts with tetherin to direct its β-TrCP2-dependent proteasomal degradation, thereby alleviating the blockade to the release of infectious virions. Identification of tetherin binding to Vpu provides a potential novel target for the development of drugs aimed at inhibiting HIV-1 replication

Multilayered Mechanism of CD4 Downregulation by HIV-1 Vpu Involving Distinct ER Retention and ERAD Targeting Steps

A key function of the Vpu protein of HIV-1 is the targeting of newly-synthesized CD4 for proteasomal degradation. This function has been proposed to occur by a mechanism that is fundamentally distinct from the cellular ER-associated degradation (ERAD) pathway. However, using a combination of genetic, biochemical and morphological methodologies, we find that CD4 degradation induced by Vpu is dependent on a key component of the ERAD machinery, the VCP-UFD1L-NPL4 complex, as well as on SCFβ-TrCP-dependent ubiquitination of the CD4 cytosolic tail on lysine and serine/threonine residues. When degradation of CD4 is blocked by either inactivation of the VCP-UFD1L-NPL4 complex or prevention of CD4 ubiquitination, Vpu still retains the bulk of CD4 in the ER mainly through transmembrane domain interactions. Addition of a strong ER export signal from the VSV-G protein overrides this retention. Thus, Vpu exerts two distinct activities in the process of downregulating CD4: ER retention followed by targeting to late stages of ERAD. The multiple levels at which Vpu engages these cellular quality control mechanisms underscore the importance of ensuring profound suppression of CD4 to the life cycle of HIV-1

Genetic Diversity of EBV-Encoded LMP1 in the Swiss HIV Cohort Study and Implication for NF-Κb Activation

Epstein-Barr virus (EBV) is associated with several types of cancers including Hodgkin's lymphoma (HL) and nasopharyngeal carcinoma (NPC). EBV-encoded latent membrane protein 1 (LMP1), a multifunctional oncoprotein, is a powerful activator of the transcription factor NF-κB, a property that is essential for EBV-transformed lymphoblastoid cell survival. Previous studies reported LMP1 sequence variations and induction of higher NF-κB activation levels compared to the prototype B95-8 LMP1 by some variants. Here we used biopsies of EBV-associated cancers and blood of individuals included in the Swiss HIV Cohort Study (SHCS) to analyze LMP1 genetic diversity and impact of sequence variations on LMP1-mediated NF-κB activation potential. We found that a number of variants mediate higher NF-κB activation levels when compared to B95-8 LMP1 and mapped three single polymorphisms responsible for this phenotype: F106Y, I124V and F144I. F106Y was present in all LMP1 isolated in this study and its effect was variant dependent, suggesting that it was modulated by other polymorphisms. The two polymorphisms I124V and F144I were present in distinct phylogenetic groups and were linked with other specific polymorphisms nearby, I152L and D150A/L151I, respectively. The two sets of polymorphisms, I124V/I152L and F144I/D150A/L151I, which were markers of increased NF-κB activation in vitro, were not associated with EBV-associated HL in the SHCS. Taken together these results highlighted the importance of single polymorphisms for the modulation of LMP1 signaling activity and demonstrated that several groups of LMP1 variants, through distinct mutational paths, mediated enhanced NF-κB activation levels compared to B95-8 LMP1

Role of β-TrCP1, variant and homologue in HIV-1 life cycle

Summary The CD4 molecule plays a key role in AIDS pathogenesis, it is required for entry of the virus into permissive cells and its subsequent down-modulation of the cell surface is a hallmark of HN-1 infected cells. The virus encodes no less than three proteins that participate in this process: Nef, Vpu and Env. Vpu protein interacts with CD4 within the endoplasmic reticulum of infected cells, where it targets CD4 for degradation through the interaction with a cellular protein named ß-TrCP1. This F-box protein functions as the substrate recognition subunit of the SCF ß-Trcr E3 ubiquitin ligase, which normally induce the ubiquitination and subsequent degradation of various proteins such as ß-catenin and IxBa. Mammals possess a homologue of ß-TrCP1, HOS, also named ß-TrCP2 which has a cytoplasmic subcellular distribution. Structural analysis of the ligand-binding domain of both homologues shows striking surface similarities. Both F-box proteins have a redundant role in a number of cellular processes; however the potential role of ß-TrCP2 in HIV-1 infected cells has not been evaluated. In the present study, we assessed the existence of génetic variants of BRTC, encoding ß-TrCP1, and evaluated whether these variants would affect CD4 down-modulation. Additionally, we determined whether ß-TrCP2 shares with its homologue structural and functional properties that would allow it to bind Vpu, modulate CD4 expression, and thus participate in HN-1 pathogenesis. We identified a single nucleotide polymorphism present in the human population with an allelic frequency of 0.03 that leads to the substitution of alanine 507 by a serine. However, we showed by transient transfection in HeLa CD4+ cells that this variant behaves as ß-TrCP1 with respect to CD4 down-modulation. We established transient expression systems in HeLa CD4+ cells to test whether ß-TrCP2 is implicated in Vpu-mediated CD4 down-modulation. We show by coimmunoprecipitation experiments that ß-TrCP2 binds Vpu and is able to induce CD4 down-modulation as efficiently as ß-TrCP1. In two different cell lines, HeLa CD4+ and Jurkat, Vpu-mediated CD4 down-modulation could not be completely reversed through the silencing of endogenous ß-TrCP 1 or ß-TrCP2 individually, but required both genes to be silenced simultaneously. We evaluated the role of ß-TrCP1 and ß-TrCP2 in HIV-1 life cycle using silencing prior to actual viral infection. Both ß-TrCP1 and ß-TrCP2 contributed to CD4 down-modulation during aone-cycle viral infection iri Ghost cells. In addition, the combined silencing of both homologues in the absence of env and nef reversed CD4 down-modulation, showing that ß-TrCP 1 and ß-TrCP2 represent the main and additive effectors of HIV-1 encoded Vpu. In addition, we showed that silencing of ß-TrCPI but not ß-TrCP2 induced a decrease of HIV-1 LTR-driven expression. In a transient transfection system with Tat and a LTR luciferase reporter, both homologues modulated LTR-driven expression. The present study revealed that ß-TrCP2 represents a novel protein participating in HIV-1 cycle and complete comprehension of the complex interplay occurring between the two F-Box will improve our understanding of HIV-1 infection. Résumé La molécule CD4 joue un rôle clef dans la pathogenèse du SIDA ; elle est requise pour l'entrée du virus dans les cellules permissives et la diminution de sa concentration au niveau de la surface cellulaire est une importante caractéristique des cellules infectées par le VIH-1. Le virus encode pas moins de trois protéines qui participent à ce processus Nef, Vpu et Env. La protéine Vpu lie CD4 au niveau du réticulum endoplasmique et induit sa dégradation en interagissant avec une protéine cellulaire nommée ß-TrCP 1. Cette protéine de type F-Box est une sous unité du complexe ubiquitine-ligase E3 SCFß-TrCP. Elle permet la reconnaissance du substrat par le complexe qui induit l'ubiquitination et la subséquente dégradation de diverses protéines cellulaires comme la ß-catenin ou IκBα. Les mammifères possèdent un homologue à ß-TrCP1appelé ß-TrCP2 (ou HOS). L'analyse comparative du domaine permettant la reconnaissance des substrats des deux homologues montre de frappantes similarités. Le rôle de ß-TrCP2 dans le cycle viral du VIH-1 n'a pas encore été évalué. Lors de cette étude, nous avons recherché l'existence de variants génétique de BTRC (codant pour ß-TrCP1) et nous avons évalué si ces variants pourraient affecter la dégradation des molécules CD4 induite par le virus. Nous avons ainsi identifié un polymorphisme présent dans la population humaine avec une fréquence allélique de 0.03 qui consiste en une substitution de l'alanine 507 par une sérine. Nous avons cependant montré par transfection dans des cellules HeLa CD4+ que ce variant se comporte comme ß-TrCP 1 en ce qui concerne la modulation de CD4. De plus, nous avons déterminé si ß-TrCP2 partageait avec son homologue des propriétés structurelles et fonctionnelles qui lui permettraient de lier Vpu, moduler la concentration de CD4 et ainsi prendre part à la pathogenèse du SIDA. Pour ce faire, nous avons établi un système d'expression temporaire dans des cellules HeLa CD4+. Par co-immunoprécipitation, nous avons montré que ß-TrCP2 lie Vpu et est capable d'induire la dégradation de CD4 aussi efficacement que ß-TrCP1. Dans deux différentes lignées cellulaires, HeLa CD4+ et Jurkat, la dégradation de CD4 n'a pu être complètement inhibée par le silencing individuel de ß-TrCP 1 ou ß-TrCP2, mais nécessitait le silencing simultané des 2 gènes. Nous avons évalué le rôle des deux homologues dans le cycle viral du VIH-1 en infectant des cellules Ghost avec le virus après avoir effectué un silencing des deux protéines. Nous avons ainsi montré que ß-TrCP 1 et ß-TrCP2 contribuent de manière additive à la dégradation de CD4 induite par une infection du VIH-1. Le silencing combiné des deux homologues inhiba complètement cette dégradation en l'absence de env et nef, prouvant qu'aucune autre voie ne participe à ce processus: En outre, nous avons montré que le silencing de ß-TrCP 1 mais pas celui de ß-TrCP2 induisait une diminution de l'expression virale sous contrôle du LTR. Nous n'avons cependant pas été en mesure de reconstituer cet effet en exprimant Tat et un gène reporteur sous contrôle du LTR dans des cellules HeLa CD4+. Le présent travail révèle que ß-TrCP2 représente une nouvelle protéine participant dans le cycle viral du VIH-1. Une complète compréhension de l'effet de chacun des deux homologues sur le cycle viral permettra d'améliorer notre compréhension de l'infection par le VIH-1

Interaction of GAG trinucleotide repeat and C-129T polymorphisms impairs expression of the glutamate-cysteine ligase catalytic subunit gene.

Glutamate cysteine ligase (GCL) catalyzes the rate-limiting step in the de novo synthesis of glutathione (GSH). The catalytic subunit (GCLC) of GCL contains a GAG trinucleotide-repeat (TNR) polymorphism within the 5'-untranslated region (5'-UTR) that has been associated with various human disorders. Although several studies suggest that this variation influences GSH content, its implication for GCLC expression remains unknown. To better characterize its functional significance, we performed reporter gene assays with constructs containing the complete GCLC 5'-UTR upstream of a luciferase gene. Transfection of these vectors into various human cell lines did not reveal any significant differences between 7, 8, 9, or 10 GAG repeats, under either basal or oxidative stress conditions. To correlate these results with the previously described down-regulation induced by the C-129T GCLC promoter polymorphism, combinations of both variations were tested. Interestingly, the -129T allele down-regulates gene expression when combined with 7 GAG but not with 8, 9, or 10 GAG TNRs. This observation was confirmed in primary fibroblast cells, in which the combination of GAG TNR 7/7 and -129C/T genotypes decreased the GCLC protein level. These results provide evidence that interaction of the two variations can efficiently impair GCLC expression and thus suggest its involvement in the pathogenesis of diseases related to GSH metabolism

High frequency of alternative splicing of human genes participating in the HIV-1 life cycle: a model using TSG101, betaTrCP, PPIA, INI1, NAF1, and PML.

Alternative splicing may generate splice forms with different biologic roles or missing protein domains implicated in the interaction with HIV-1. To address this issue, 6 human genes were investigated-tumor suppressor gene 101 (TSG101), beta-transducin repeats containing protein (betaTrCP), peptidyl-proly cis-trans isomerase, cyclophilin A (PPIA), integrase interactor 1 protein (INI1), Nef-associated factor 1 (NAF1), and promyelacytic leukemia (PML)-involved in the viral life cycle and HIV-1 pathogenesis. All 6 genes presented alternative splicing, and a combined bioinformatic and reverse transcription polymerase chain reaction (RT-PCR) analysis identified 27 new variants for a total of 53 splice forms (an average of 9 variants per gene). The predicted frequency of the various splice forms based on expressed sequence tags (EST) analysis corresponded to the semiquantitative findings on RT-PCR analysis for the cell culture systems and for native CD4 cells investigated. Interindividual variation in the frequencies of various splice forms in CD4 T cells from blood donors was observed for INI1. Cell type-specific variation of splice pattern was observed for NAF1. Eight splice forms lacked or modified motifs implicated in the interaction with HIV-1, underscoring the potential interest of assessing alternative splicing when investigating viral cell biology and pathogenesis

Mutation screening of the glutamate cysteine ligase modifier (GCLM) gene in patients with schizophrenia.

BACKGROUND: Experimental evidences show that glutathione and its rate-limiting synthesizing enzyme, the glutamate-cysteine ligase (GCL), are involved in the pathogenesis of schizophrenia. Furthermore, genetic association has been previously reported between two single nucleotide polymorphisms lying in noncoding regions of glutamate cysteine ligase modifier (GCLM) gene, which specifies for the modifier subunit of GCL and schizophrenia. OBJECTIVE: We wanted to investigate the presence of GCLM true functional mutations, likely in linkage disequilibrium with the previously identified single nucleotide polymorphism alleles, in the same set of cases that allowed the detection of the original association signal. METHODS: We screened all the coding regions of GCLM and their intronic flanking vicinities in 353 patients with schizophrenia by direct DNA sequencing. RESULTS: Ten sequence variations were identified, five of which were not previously described. None of these DNA changes was within the GCLM coding sequence and in-silico analysis failed to indicate functional impairment induced by these variations. Furthermore, screening of normal controls and downstream statistical analyses revealed no significant relationship of any of these DNA variants with schizophrenia. CONCLUSION: It is unlikely that functional mutations in the GCLM gene could play a major role in genetic predisposition to schizophrenia and further studies will be required to assess its etiological function in the disease