Identification of a Novel Drug Lead That Inhibits HCV Infection and Cell-to-Cell Transmission by Targeting the HCV E2 Glycoprotein

Hepatitis C Virus (HCV) infects 200 million individuals worldwide. Although several FDA approved drugs targeting the HCV serine protease and polymerase have shown promising results, there is a need for better drugs that are effective in treating a broader range of HCV genotypes and subtypes without being used in combination with interferon and/or ribavirin. Recently, two crystal structures of the core of the HCV E2 protein (E2c) have been determined, providing structural information that can now be used to target the E2 protein and develop drugs that disrupt the early stages of HCV infection by blocking E2’s interaction with different host factors. Using the E2c structure as a template, we have created a structural model of the E2 protein core (residues 421–645) that contains the three amino acid segments that are not present in either structure. Computational docking of a diverse library of 1,715 small molecules to this model led to the identification of a set of 34 ligands predicted to bind near conserved amino acid residues involved in the HCV E2: CD81 interaction. Surface plasmon resonance detection was used to screen the ligand set for binding to recombinant E2 protein, and the best binders were subsequently tested to identify compounds that inhibit the infection of Huh-7 cells by HCV. One compound, 281816, blocked E2 binding to CD81 and inhibited HCV infection in a genotype-independent manner with IC50’s ranging from 2.2 µM to 4.6 µM. 281816 blocked the early and late steps of cell-free HCV entry and also abrogated the cell-to-cell transmission of HCV. Collectively the results obtained with this new structural model of E2c suggest the development of small molecule inhibitors such as 281816 that target E2 and disrupt its interaction with CD81 may provide a new paradigm for HCV treatment

Alterations of EDEM1 functions enhance ATF6 pro-survival signaling

International audienceActivating transcription factor 6 alpha (referred to as ATF6 hereafter) is an endoplasmic reticulum (ER)-resident glycoprotein and one of the three sensors of the unfolded protein response (UPR). Upon ER stress, ATF6 is exported to the Golgi complex where it is cleaved by the S1P and S2P proteases thus releasing ATF6 cytosolic fragment and leading to the transcription of ATF6 target genes. In this study, we performed a phenotypic small-interfering RNA (siRNA) screening to better characterize the ER mechanisms involved in ATF6 activation upon ER stress. This revealed that silencing of ER-degradation-enhancing alpha-mannosidase-like protein-1 (EDEM1) increased the bioavailability of ER stress-induced ATF6 export to the Golgi complex through the stabilization of the natively unstable ATF6 protein. Moreover, we characterized a somatic variant of EDEM1 (N198I) found in hepatocellular carcinoma that alters ATF6 signaling and might provide a selective advantage to the transforming cells. Hence, our work confirms the natively unstable nature of ATF6 and links this property to potentially associated pro-oncogenic functions

Le harcèlement sexuel : une dépénalisation qui fait mauvais genre

Commentaire collectif de la décision du Conseil constitutionnel QPC n° 2012-240 du 4 mai 2012, publié sur le blog du programme de Recherche et d’Études sur le Genre et les Inégalités dans les Normes en Europe (REGINE), http://regine-droit.blogspot.fr/2012/05/le-harcelement-sexuel-une.htm

ATF6 alpha regulates morphological changes associated with senescence in human fibroblasts

International audienceCellular senescence is known as an anti-tumor barrier and is characterized by a number of determinants including cell cycle arrest, senescence associated beta-galactosidase activity and secretion of pro-inflammatory mediators. Senescent cells are also subjected to enlargement, cytoskeleton-mediated shape changes and organelle alterations. However, the underlying molecular mechanisms responsible for these last changes remain still uncharacterized. Herein, we have identified the Unfolded Protein Response (UPR) as a player controlling some morphological aspects of the senescent phenotype. We show that senescent fibroblasts exhibit ER expansion and mild UPR activation, but conserve an ER stress adaptive capacity similar to that of exponentially growing cells. By genetically invalidating the three UPR sensors in senescent fibroblasts, we demonstrated that ATF6 alpha signaling dictates senescence-associated cell shape modifications. We also show that ER expansion and increased secretion of the pro-inflammatory mediator IL6 were partly reversed by silencing ATF6a in senescent cells. Moreover, ATF6a drives the increase of senescence associated-beta-galactosidase activity. Collectively, these findings unveil a novel and central role for ATF6a in the establishment of morphological features of senescence in normal human primary fibroblasts

New insights into the ORF2 capsid protein, a key player of the hepatitis E virus lifecycle

Abstract Hepatitis E Virus (HEV) genome encodes three proteins including the ORF2 capsid protein. Recently, we demonstrated that HEV produces three different forms of ORF2: (i) the ORF2i form (infectious ORF2) which is the component of infectious particles, (ii) the secreted ORF2g (glycosylated ORF2) and ORF2c (cleaved ORF2) forms that are not associated with infectious particles, but are the major antigens in HEV-infected patient sera. The ORF2 protein sequence contains three highly conserved potential N-glycosylation sites (N1, N2 and N3). The status and biological relevance of ORF2 N-glycosylation in HEV lifecycle remain to be elucidated. Here, we generated and extensively characterized a series of ORF2 mutants in which the three N-glycosylation sites were mutated individually or in combination. We demonstrated that the ORF2g/c protein is N-glycosylated on N1 and N3 sites but not on the N2 site. We showed that N-glycosylation of ORF2 protein does not play any role in replication and assembly of infectious HEV particles. We found that glycosylated ORF2g/c forms are very stable proteins which are targeted by patient antibodies. We also demonstrated that the ORF2i protein is translocated into the nucleus of infected cells. Hence, our study led to new insights into the molecular mechanisms of ORF2 expression

LXR agonist treatment of blastic plasmacytoid dendritic cell neoplasm restores cholesterol efflux and triggers apoptosis

International audienceBlastic plasmacytoid dendritic cell (PDC) neoplasm (BPDCN) is an aggressive hematological malignancy with a poor prognosis that derives from PDCs. No consensus for optimal treatment modalities is available today and the full characterization of this leukemia is still emerging. We identified here a BPDCN-specific transcriptomic profile when compared with those of acute myeloid leukemia and T-acute lymphoblastic leukemia, as well as the transcriptomic signature of primary PDCs. This BPDCN gene signature identified a dysregulation of genes involved in cholesterol homeostasis, some of them being liver X receptor (LXR) target genes. LXR agonist treatment of primary BPDCN cells and BPDCN cell lines restored LXR target gene expression and increased cholesterol efflux via the upregulation of adenosine triphosphate-binding cassette (ABC) transporters, ABCA1 and ABCG1. LXR agonist treatment was responsible for limiting BPDCN cell proliferation and inducing intrinsic apoptotic cell death. LXR activation in BPDCN cells was shown to interfere with 3 signaling pathways associated with leukemic cell survival, namely: NF-kB activation, as well as Akt and STAT5 phosphorylation in response to the BPDCN growth/survival factor interleukin-3. These effects were increased by the stimulation of cholesterol efflux through a lipid acceptor, the apolipoprotein A1. In vivo experiments using a mouse model of BPDCN cell xenograft revealed a decrease of leukemic cell infiltration and BPDCN-induced cytopenia associated with increased survival after LXR agonist treatment. This demonstrates that cholesterol homeostasis is modified in BPDCN and can be normalized by treatment with LXR agonists which can be proposed as a new therapeutic approach

BPDCN: When polychemotherapy does not compromise allogeneic CD123 CAR‐T cell cytotoxicity

Abstract Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematological malignancy with poor prognosis and no treatment consensus. Combining chemotherapy and immunotherapy is a promising strategy to enhance therapeutic effect. Before combining these therapies, the influence of one on the other has to be explored. We set up a model to test the combination of polychemotherapy ‐ named methotrexate, idarubicine, dexamethasone, and L‐asparaginase (MIDA) ‐ and CD123 CAR‐T cell therapy. We showed that CD123 CAR‐T cells exert the same effect on BPDCN models alone, or after MIDA regimen. These data support a preclinical rationale to use immunotherapy after a treatment with polychemotherapy for BPDCN patients

The out-of-field dose in radiation therapy induces delayed tumorigenesis by senescence evasion

International audienceA rare but severe complication of curative-intent radiation therapy is the induction of second primary cancers. These cancers preferentially develop not inside the planning target volume (PTV) but around, over several centimeters, after a latency period of 1–40 years. We show here that normal human or mouse dermal fibroblasts submitted to the out-of-field dose scattering at the margin of a PTV receiving a mimicked patient’s treatment do not die but enter in a long-lived senescent state resulting from the accumulation of unrepaired DNA single-strand breaks, in the almost absence of double-strand breaks. Importantly, a few of these senescent cells systematically and spontaneously escape from the cell cycle arrest after a while to generate daughter cells harboring mutations and invasive capacities. These findings highlight single-strand break-induced senescence as the mechanism of second primary cancer initiation, with clinically relevant spatiotemporal specificities. Senescence being pharmacologically targetable, they open the avenue for second primary cancer prevention

Involvement and prognosis value of CD8+ T cells in giant cell arteritis

IF 7.760International audienceCD8(+) T cells participate in the pathogenesis of some vasculitides. However, little is known about their role in Giant Cell Arteritis (GCA). This study was conducted to investigate CD8(+) T cell involvement in the pathogenesis of GCA.Analyses were performed at diagnosis and after 3 months of glucocorticoid treatment in 34 GCA patients and 26 age-matched healthy volunteers . Percentages of CD8(+) T-cell subsets, spectratype analysis of the TCR v beta families of CD8(+) T cells, levels of cytokines and chemokines and immunohistochemistry of temporal artery biopsies (TAB) were assessed.Among total CD8+ T cells, percentages of circulating cytotoxic CD8 T lymphocytes (CTL, CD3(+)CD8(+)perforin(+)granzymeB(+)), Tc17 (CD3(+)CD8(+)IL-17(+)), CD63(+)CD8(+) T cells and levels of soluble granzymes A and B were higher in patients than in controls, whereas the percentage of Tc1 cells (CD3(+)CD8(+)IFN-gamma(+)) was similar. Moreover, CD8+ T cells displayed a restricted TCR repertoire in GCA patients. Percentages of circulating CTL, Tc17 and soluble levels of granzymes A and B decreased after treatment. CXCR3 expression on CD8(+) T cells and its serum ligands (CXCL9, -10, -11) were higher in patients. Analyses of TAB revealed high expression of CXCL9 and -10 associated with infiltration by CXCR3(+)CD8(+) T cells expressing granzyme B and TiA1. The intensity of the CD8 T-cell infiltrate in TAB was predictive of the severity of the disease.This study demonstrates the implication and the prognostic value of CD8(+) T-cells in GCA and suggests that CD8(+) T-cells are recruited within the vascular wall through an interaction between CXCR3 and its ligands. (C) 2016 Elsevier Ltd. All rights reserved

Comparison of the crystal structure of E2c with the homology model.

<p>Structural superposition between E2c crystal structure from the PDB chain 4mwf_D (red) and the homology model (black) is illustrated using a ribbon representation. The crystal structure and homology model overlap in most of the regions, except the fragments where coordinates in the experimental structure are missing (red dashed lines).</p