Up-regulated 14-3-3β and 14-3-3ζ proteins in prefrontal cortex of subjects with schizophrenia: effect of psychotropic treatment.

14-3-3 is a family of conserved regulatory proteins that bind to a multitude of functionally diverse signalling proteins. Various genetic studies and gene expression and proteomic analyses have involved 14-3-3 proteins in schizophrenia (SZ). On the other hand, studies about the status of these proteins in major depressive disorder (MD) are still missing. Immunoreactivity values of cytosolic 14-3-3β and 14-3-3ζ proteins were evaluated by Western blot in prefrontal cortex (PFC) of subjects with schizophrenia (SZ; n=22), subjects with major depressive disorder (MD; n=21) and age-, gender- and postmortem delay-matched control subjects (n=52). The modulation of 14-3-3β and 14-3-3ζ proteins by psychotropic medication was also assessed. The analysis of both proteins in SZ subjects with respect to matched control subjects showed increased 14-3-3β (Δ=33±10%, p<0.05) and 14-3-3ζ (Δ=29±6%, p<0.05) immunoreactivity in antipsychotic-free but not in antipsychotic-treated SZ subjects. Immunoreactivity values of 14-3-3β and 14-3-3ζ were not altered in MD subjects. These results show the specific up-regulation of 14-3-3β and 14-3-3ζ proteins in PFC of SZ subjects and suggest a possible down-regulation of both proteins by antipsychotic treatment.

Identification of potential HIV restriction factors by combining evolutionary genomic signatures with functional analyses.

BACKGROUND: Known antiretroviral restriction factors are encoded by genes that are under positive selection pressure, induced during HIV-1 infection, up-regulated by interferons, and/or interact with viral proteins. To identify potential novel restriction factors, we performed genome-wide scans for human genes sharing molecular and evolutionary signatures of known restriction factors and tested the anti-HIV-1 activity of the most promising candidates. RESULTS: Our analyses identified 30 human genes that share characteristics of known restriction factors. Functional analyses of 27 of these candidates showed that over-expression of a strikingly high proportion of them significantly inhibited HIV-1 without causing cytotoxic effects. Five factors (APOL1, APOL6, CD164, TNFRSF10A, TNFRSF10D) suppressed infectious HIV-1 production in transfected 293T cells by >90% and six additional candidates (FCGR3A, CD3E, OAS1, GBP5, SPN, IFI16) achieved this when the virus was lacking intact accessory vpr, vpu and nef genes. Unexpectedly, over-expression of two factors (IL1A, SP110) significantly increased infectious HIV-1 production. Mechanistic studies suggest that the newly identified potential restriction factors act at different steps of the viral replication cycle, including proviral transcription and production of viral proteins. Finally, we confirmed that mRNA expression of most of these candidate restriction factors in primary CD4+ T cells is significantly increased by type I interferons. CONCLUSIONS: A limited number of human genes share multiple characteristics of genes encoding for known restriction factors. Most of them display anti-retroviral activity in transient transfection assays and are expressed in primary CD4+ T cells.

Quantitative proteomics of heat-treated human cells show an across-the-board mild depletion of housekeeping proteins to massively accumulate few HSPs.

Classic semiquantitative proteomic methods have shown that all organisms respond to a mild heat shock by an apparent massive accumulation of a small set of proteins, named heat-shock proteins (HSPs) and a concomitant slowing down in the synthesis of the other proteins. Yet unexplained, the increased levels of HSP messenger RNAs (mRNAs) may exceed 100 times the ensuing relative levels of HSP proteins. We used here high-throughput quantitative proteomics and targeted mRNA quantification to estimate in human cell cultures the mass and copy numbers of the most abundant proteins that become significantly accumulated, depleted, or unchanged during and following 4 h at 41 °C, which we define as mild heat shock. This treatment caused a minor across-the-board mass loss in many housekeeping proteins, which was matched by a mass gain in a few HSPs, predominantly cytosolic HSPCs (HSP90s) and HSPA8 (HSC70). As the mRNAs of the heat-depleted proteins were not significantly degraded and less ribosomes were recruited by excess new HSP mRNAs, the mild depletion of the many housekeeping proteins during heat shock was attributed to their slower replenishment. This differential protein expression pattern was reproduced by isothermal treatments with Hsp90 inhibitors. Unexpectedly, heat-treated cells accumulated 55 times more new molecules of HSPA8 (HSC70) than of the acknowledged heat-inducible isoform HSPA1A (HSP70), implying that when expressed as net copy number differences, rather than as mere "fold change" ratios, new biologically relevant information can be extracted from quantitative proteomic data. Raw data are available via ProteomeXchange with identifier PXD001666.

Protein loop compaction and the origin of the effect of arginine and glutamic acid mixtures on solubility, stability and transient oligomerization of proteins

Addition of a 50 mM mixture of l-arginine and l-glutamic acid (RE) is extensively used to improve protein solubility and stability, although the origin of the effect is not well understood. We present Small Angle X-ray Scattering (SAXS) and Nuclear Magnetic Resonance (NMR) results showing that RE induces protein compaction by collapsing flexible loops on the protein core. This is suggested to be a general mechanism preventing aggregation and improving resistance to proteases and to originate from the polyelectrolyte nature of RE. Molecular polyelectrolyte mixtures are expected to display long range correlation effects according to dressed interaction site theory. We hypothesize that perturbation of the RE solution by dissolved proteins is proportional to the volume occupied by the protein. As a consequence, loop collapse, minimizing the effective protein volume, is favored in the presence of RE.

Combined loss of the BH3-only proteins Bim and Bmf restores B-cell development and function in TACI-Ig transgenic mice.

Terminal differentiation of B cells depends on two interconnected survival pathways, elicited by the B-cell receptor (BCR) and the BAFF receptor (BAFF-R), respectively. Loss of either signaling pathway arrests B-cell development. Although BCR-dependent survival depends mainly on the activation of the v-AKT murine thymoma viral oncogene homolog 1 (AKT)/PI3-kinase network, BAFF/BAFF-R-mediated survival engages non-canonical NF-κB signaling as well as MAPK/extracellular-signal regulated kinase and AKT/PI3-kinase modules to allow proper B-cell development. Plasma cell survival, however, is independent of BAFF-R and regulated by APRIL that signals NF-κB activation via alternative receptors, that is, transmembrane activator and CAML interactor (TACI) or B-cell maturation (BCMA). All these complex signaling events are believed to secure survival by increased expression of anti-apoptotic B-cell lymphoma 2 (Bcl2) family proteins in developing and mature B cells. Curiously, how lack of BAFF- or APRIL-mediated signaling triggers B-cell apoptosis remains largely unexplored. Here, we show that two pro-apoptotic members of the 'Bcl2 homology domain 3-only' subgroup of the Bcl2 family, Bcl2 interacting mediator of cell death (Bim) and Bcl2 modifying factor (Bmf), mediate apoptosis in the context of TACI-Ig overexpression that effectively neutralizes BAFF as well as APRIL. Surprisingly, although Bcl2 overexpression triggers B-cell hyperplasia exceeding the one observed in Bim(-/-)Bmf(-/-) mice, Bcl2 transgenic B cells remain susceptible to the effects of TACI-Ig expression in vivo, leading to ameliorated pathology in Vav-Bcl2 transgenic mice. Together, our findings shed new light on the molecular machinery restricting B-cell survival during development, normal homeostasis and under pathological conditions. Our data further suggest that Bcl2 antagonists might improve the potency of BAFF/APRIL-depletion strategies in B-cell-driven pathologies.

Computational toolbox towards evolutionary domain mapping of membrane proteins

Membrane proteins account for about 20% to 30% of all proteins encoded in a typical genome. They play central roles in multiple cellular processes mediating the interaction of the cell with its surrounding. Over 60% of all drug targets contain a membrane domain. The experimental difficulties of obtaining a crystal structural severely limits our ability or understanding of membrane protein function. Computational evolutionary studies of proteins are crucial for the prediction of 3D structures. In this project, we construct a tool able to quantify the evolutionary positive selective pressure on each residue of membrane proteins through maximum likelihood phylogeny reconstruction. The conservation plot combined with a structural homology model is also a potent tool to predict those residues that have essentials roles in the structure and function of a membrane protein and can be very useful in the design of validation experiments.

Cell Cycle Proteins and Retinal Degeneration: Evidences of New Potential Therapeutic Targets.

During different forms of neurodegenerative diseases, including the retinal degeneration, several cell cycle proteins are expressed in the dying neurons from Drosophila to human revealing that these proteins are a hallmark of neuronal degeneration. This is true for animal models of Alzheimer's, and Parkinson's diseases, Amyotrophic Lateral Sclerosis and for Retinitis Pigmentosa as well as for acute injuries such as stroke and light damage. Longitudinal investigation and loss-of-function studies attest that cell cycle proteins participate to the process of cell death although with different impacts, depending on the disease. In the retina, inhibition of cell cycle protein action can result to massive protection. Nonetheless, the dissection of the molecular mechanisms of neuronal cell death is necessary to develop adapted therapeutic tools to efficiently protect photoreceptors as well as other neuron types.

Interleukin-1- and type I interferon-dependent enhanced immunogenicity of an NYVAC-HIV-1 Env-Gag-Pol-Nef vaccine vector with dual deletions of type I and type II interferon-binding proteins.

UNLABELLED: NYVAC, a highly attenuated, replication-restricted poxvirus, is a safe and immunogenic vaccine vector. Deletion of immune evasion genes from the poxvirus genome is an attractive strategy for improving the immunogenic properties of poxviruses. Using systems biology approaches, we describe herein the enhanced immunological profile of NYVAC vectors expressing the HIV-1 clade C env, gag, pol, and nef genes (NYVAC-C) with single or double deletions of genes encoding type I (ΔB19R) or type II (ΔB8R) interferon (IFN)-binding proteins. Transcriptomic analyses of human monocytes infected with NYVAC-C, NYVAC-C with the B19R deletion (NYVAC-C-ΔB19R), or NYVAC-C with B8R and B19R deletions (NYVAC-C-ΔB8RB19R) revealed a concerted upregulation of innate immune pathways (IFN-stimulated genes [ISGs]) of increasing magnitude with NYVAC-C-ΔB19R and NYVAC-C-ΔB8RB19R than with NYVAC-C. Deletion of B8R and B19R resulted in an enhanced activation of IRF3, IRF7, and STAT1 and the robust production of type I IFNs and of ISGs, whose expression was inhibited by anti-type I IFN antibodies. Interestingly, NYVAC-C-ΔB8RB19R induced the production of much higher levels of proinflammatory cytokines (tumor necrosis factor [TNF], interleukin-6 [IL-6], and IL-8) than NYVAC-C or NYVAC-C-ΔB19R as well as a strong inflammasome response (caspase-1 and IL-1β) in infected monocytes. Top network analyses showed that this broad response mediated by the deletion of B8R and B19R was organized around two upregulated gene expression nodes (TNF and IRF7). Consistent with these findings, monocytes infected with NYVAC-C-ΔB8RB19R induced a stronger type I IFN-dependent and IL-1-dependent allogeneic CD4(+) T cell response than monocytes infected with NYVAC-C or NYVAC-C-ΔB19R. Dual deletion of type I and type II IFN immune evasion genes in NYVAC markedly enhanced its immunogenic properties via its induction of the increased expression of type I IFNs and IL-1β and make it an attractive candidate HIV vaccine vector. IMPORTANCE: NYVAC is a replication-deficient poxvirus developed as a vaccine vector against HIV. NYVAC expresses several genes known to impair the host immune defenses by interfering with innate immune receptors, cytokines, or interferons. Given the crucial role played by interferons against viruses, we postulated that targeting the type I and type II decoy receptors used by poxvirus to subvert the host innate immune response would be an attractive approach to improve the immunogenicity of NYVAC vectors. Using systems biology approaches, we report that deletion of type I and type II IFN immune evasion genes in NYVAC poxvirus resulted in the robust expression of type I IFNs and interferon-stimulated genes (ISGs), a strong activation of the inflammasome, and upregulated expression of IL-1β and proinflammatory cytokines. Dual deletion of type I and type II IFN immune evasion genes in NYVAC poxvirus improves its immunogenic profile and makes it an attractive candidate HIV vaccine vector.

OmpA family proteins and Pmp-like autotransporter: new adhesins of Waddlia chondrophila.

Waddlia chondrophila is a obligate intracellular bacterium belonging to the Chlamydiales order, a clade that also includes the well-known classical Chlamydia responsible for a number of severe human and animal diseases. Waddlia is an emerging pathogen associated with adverse pregnancy outcomes in humans and abortion in ruminants. Adhesion to the host cell is an essential prerequisite for survival of every strict intracellular bacteria and, in classical Chlamydia, this step is partially mediated by polymorphic outer membrane proteins (Pmps), a family of highly diverse autotransporters that represent about 15% of the bacterial coding capacity. Waddlia chondrophila genome however only encodes one putative Pmp-like protein. Using a proteomic approach, we identified several bacterial proteins potentially implicated in the adhesion process and we characterized their expression during the replication cycle of the bacteria. In addition, we demonstrated that the Waddlia Pmp-like autotransporter as well as OmpA2 and OmpA3, two members of the extended Waddlia OmpA protein family, exhibit adhesive properties on epithelial cells. We hypothesize that the large diversity of the OmpA protein family is linked to the wide host range of these bacteria that are able to enter and multiply in various host cells ranging from protozoa to mammalian and fish cells.

Accessory phases as a tool to investigate magmatic underplating in polymetamorphic metasediments : an example from a fossil lower crust : Ivrea Verbano zne, NW Italy

The Ivrea and the Strona-Ceneri zones, NW italy and S Switzerland, offer the possibility to study the continental crust of the Southern Alps. Because of its high metamorphic degree and the abundant Permo- Carboniferous mafic intrusions, the Ivrea Zone is classically interpreted an exposed section trough the Permian lower crust. The present work is focused here on metasedimentary slices (septa) intercalated within Permian gabbro (mafic complex). In particular I studied the evolution of accessory phases such as rutile and zircon and the chemistry of the metasediments. The septa build an irregular and discontinuous band that cut obliquely the mafic complex from its deepest part (N) to its roof (S). The chemistry of the metasediments evolves along the band and the chemical evolution can be compared with that observed in the country-rock surrounding the mafic intrusion to the NE and overprinted by a main regional metamorphic event. This suggests that the degree of chemical depletion of the septa was mainly established during the same regional metamorphic event. Moreover it suggests that incorporation of the septa within the gabbro did not modify their original stratigraphie distribution within the crust. It implies that the mafic complex has been emplaced following a dynamic substantially different from the classic model of « gabbro glacier » (Quick et al., 1992; Quick et al., 1994). It is more likely that it has been emplaced by repeated injections of sills at different depths during a protracted period of time. Zircon trace elements and U-Pb ages suggest that regional metamorphism occurred 330-320Ma, the first sills in the deepest part of the Mafic Complex are injected at ~300Ma, the mafic magmas reached higher levels in the crust at 285Ma and the magmatic activity continued locally until 275Ma. The ages of detrital cores in zircons fix the maximal sedimentation age at ~370Ma, this age corresponds therefore with the maximal age of the incorporation of the Ivrea zone within the lower crust. I propose that the Ivrea zone has been accreted to the lower crust during the Hercynian orogeny sensu lato. The analysis of detrital ages suggests that the source terrains for the Ivrea zone and those for the Strona-Ceneri zone have a completely different Palaeozoic history. The systematic analysis of rutile in partially molten metasediments of the Ivrea zone reveals the occurrence of two generations. The two generations are characterized by a different chemistry and textural distribution. A first generation is formed during pro-grade metamorphism in the restitic counterpart. The second generation is formed in the melts during cooling at the same time that part of the first generation re-equilibrate. Re-equilibration of the first generation seems to be spatially controlled by the presence of fluids. Locally the second generation forms overgrowths on the first generation. Considered the different diffusivity of U and Pb in rutile, U heterogeneities have important implication for U-Pb dating of rutile. ID-TIMS and LA-ICPMS dating coupled with a careful textural investigation (SEM) suggest that rutile grains are characterized by multiple path along which Pb diffusion can occur: volume diffusion is an important process, but intragrain and subgrain boundaries provide additional high diffusivity pathways for Pb escape and reduce drastically the effective diffusion length. -- La zone d'Ivrea et la zone de Strona-Ceneri, en Italie nord-occidentale et Suisse méridionale, offrent la possibilité d'étudier la croûte continentale des Alpes du Sud. En raison du haut degré métamorphique et l'abondance d'intrusions mafiques d'âge Permo-Carbonifère [complexe mafique), la zone d'Ivrea est interprétée classiquement comme de la croûte inférieure permienne. Ce travail ce concentre sur des bandes metasédimentaires (septa) incorporées dans les magmas mafiques lors de l'intrusion. Les septa forment une bande irrégulière qui coupe obliquement le complexe mafique du bas (N) vers le haut (S). La chimie des septa évolue du bas vers le haut et l'évolution chimique se rapproche de l'évolution observé dans la roche encaissante l'intrusion affecté par un événement métamorphique régionale. Cette relation suggère que le degré d'appauvrissement chimique des septa a été établit principalement lors de l'événement métamorphique régional. De plus l'incorporation dans les gabbros n'a pas perturbée la distribution stratigraphique originelle des septa. Ces deux observations impliquent que le métamorphisme dans la roche encaissante précède la mise en place du gabbro et que cette dernière ne se fait pas selon le modèle classique (« gabbro glacier » de Quick et al., 1992, 1994), mais se fait plutôt par injections répétées de sills a différentes profondeurs. Les âges U-Pb et les éléments traces des zircons suggèrent que le métamorphisme régionale a eu lieu 330-320Ma, alors que les premiers sills dans la partie profonde du Mafic Complex s'injectent à ~300Ma, le magmatisme mafique atteigne des niveaux supérieurs à 285Ma et continue localement jusqu'à 270Ma. Les âges des coeurs détritiques des zircons permettent de fixer l'âge maximale de sédimentation à ~370Ma ce qui correspond donc à l'âge maximale de l'incorporation de la zone d'Ivrea dans la croûte inférieur. L'analyse systématique des rutiles, nous a permit de montrer l'existence de plusieurs générations qui ont une répartition texturale et une chimie différente. Une génération se forme lors de l'événement UHT dans les restites, une autre génération se forme dans les liquides lors du refroidissement, au même temps qu'une partie de la première génération se rééquilibre au niveau du Zr. Localement la deuxième génération peut former des surcroissances autour de la première génération. Dans ces cas, des fortes différences en uranium entre les deux générations ont des importantes implications pour la datation U-Pb sur rutile. Classiquement les ratios Pb/U dans le rutile sont interprétés comme indiquant l'âges du refroidissement du minéral sous une température à la quelle la diffusion du Pb dans le minéral n'est plus détectable et la diffusion à plus hautes températures est assumée se faire par «volume diffusion» dans le grain (Mezger et al., 1989). Par des datations ID-TIMS (sur grain entier) et LA-ICPMS (in-situ) et une analyse texturale (MEB) approfondie nous montrons que cette supposition est trop simpliste et que le rutile est repartie en sous-domaines. Chacun de ces domaines a ça propre longueur ou chemin de diffusion spécifique. Nous proposons donc une nouvelle approche plus cohérente pour l'interprétation des âges U-Pb sur rutile.

Neurofilament proteins in the postnatal rat hippocampus. Developmental expression and changes in experimental epilepsy

Neurofilament proteins (NFs) are the major components of the intermediate filaments of the neuronal cytoskeleton. The three different NF proteins; the low (NF-L), medium (NF-M),and dendrites.NF proteins play an important role in neuronal development, and plasticity,and seem to contribute to the pathophysiology of several diseases. However, the detailed expression patterns of NF proteins in the course of postnatal aturation, and in response to seizures in the rat have remained unknown. In this work, I have studied the developmental expression and cellular distribution of the three NF proteins in the rat hippocampus during the postnatal development. The reactivity of NF proteins in response to kainic acid (KA)-induced status epilepticus (SE)was studied in the hippocampus of 9-day-old rats, and using in vitro organotypic hippocampal slices cultures prepared from P6-7 rats. The results showed that NF-L and NF-M proteins are expressed already at the postnatal day 1, while the expression of NF-H mainly occurred during the second postnatal week. The immunoreactivity of NF proteins varied depending on the cell type and sub-cellular location in the hippocampus. In adult rats, KA-induced SE typically results in severe and permanent NF degradation. However, in our P9 rats KA-induced SE resulted in a transient increase in the expression of NF proteins during the first few hours but not degradation. No neuronal death or mossy fiber sprouting was observed at any time after SE. The in vitro studies with OHCs, which mimick the in vivo developing models where a local injection of KA is applied(e.g. intrahippocampal), indicated that NF proteins were rapidly degraded in response to KA treatment, this effect being effectively inhibited by the treatment with the AMPA receptor antagonist CNQX, and calpain inhibitor MDL-28170. These compounds also significantly ameliorated the KA-induced region-specific neuronal damage. The NMDA receptor antagonist and the L-type Ca2+ channel blocker did not have any significant effect. In conclusion, the results indicate that the developmental expression of NF in the rat hippocampus is differentially regulated and targeted in the different hippocampal cell types during the postnatal development. Furthermore, despite SE, the mechanisms leading to NF degradation and neuronal death are not activated in P9 rats unlike in adults. The reason for this remains unknown. The results in organotypic hippocampal cultures confirm the validity of this in vitro model to study development processes, and to perform pharmacological studies. The results also suggest that calpain proteases as interesting pharmacological targets to reduce neuronal damage after acute excitotoxic insults.