Laurent Héliot

NS2 Protein of Hepatitis C Virus Interacts with Structural and Non-Structural Proteins towards Virus Assembly

Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly.

Concurrent Fast and Slow Cycling of a Transcriptional Activator at an Endogenous Promoter

For gene regulation, some transcriptional activators bind periodically to promoters with either a fast (∼1 minute) or a slow (∼15 to 90 minutes) cycle. It is uncertain whether the fast cycle occurs on natural promoters, and the function of either cycle in transcription remains unclear. We report that fast and slow cycling can occur simultaneously on an endogenous yeast promoter and that slow cycling in this system reflects an oscillation in the fraction of accessible promoters rather than the recruitment and release of stably bound transcriptional activators. This observation, combined with single-cell measurements of messenger RNA (mRNA) production, argues that fast cycling initiates transcription and that slow cycling regulates the quantity of mRNA produced. These findings counter the prevailing view that slow cycling initiates transcription.

Rab6-interacting Protein 1 Links Rab6 and Rab11 Function

Rab11 and Rab6 guanosine triphosphatases are associated with membranes of the recycling endosomes (REs) and Golgi complex, respectively. Evidence indicates that they sequentially regulate a retrograde transport pathway between these two compartments, suggesting the existence of proteins that must co‐ordinate their functions. Here, we report the characterization of two isoforms of a protein, Rab6‐interacting protein 1 (R6IP1), originally identified as a Rab6‐binding protein. R6IP1 also binds to Rab11A in its GTP‐bound conformation. In interphase cells, R6IP1 is targeted to the Golgi in a Rab6‐dependent manner but can associate with Rab11‐positive compartments when the level of Rab11A is increased within the cells. Fluorescence resonance energy transfer analysis using fluorescence lifetime imaging shows that the overexpression of R6IP1 promotes an interaction between Rab11A and Rab6 in living cells. Accordingly, the REs marked by Rab11 and transferrin receptor are depleted from the cell periphery and accumulate in the pericentriolar area. However, endosomal and Golgi membranes do not appear to fuse with each other. We also show that R6IP1 function is required during metaphase and cytokinesis, two mitotic steps in which a role of Rab6 and Rab11 has been previously documented. We propose that R6IP1 may couple Rab6 and Rab11 function throughout the cell cycle.

Weak but Uniform Enrichment of the Histone Variant macroH2A1 along the Inactive X Chromosome

ABSTRACT We studied the enrichment and distribution of the histone variant mH2A1 in the condensed inactive X (Xi) chromosome. By using highly specific antibodies against mH2A1 and stable HEK 293 cell lines expressing either green fluorescent protein (GFP)-mH2A1 or GFP-H2A, we found that the Xi chromosome contains ∼1.5-fold more mH2A1 than the autosomes. To determine the in vivo distribution of mH2A1 along the X chromosome, we used a native chromatin immunoprecipitation-on-chip technique. DNA isolated from mH2A1-immunoprecipitated nucleosomes from either male or female mouse liver were hybridized to tiling microarrays covering 5 kb around most promoters or the entire X chromosome. The data show that mH2A1 is uniformly distributed across the entire Xi chromosome. Interestingly, a stronger mH2A1 enrichment along the pseudoautosomal X chromosome region was observed in both sexes. Our results indicate a potential role for macroH2A in large-scale chromosome structure and genome stability.

Plasmonic photothermal destruction of uropathogenic E. coli with reduced graphene oxide and core/shell nanocomposites of gold nanorods/reduced graphene oxide

The development of non-antibiotic based treatments against bacterial infections by Gram-negative uropathogenic E. coli is a complex task. New strategies to treat such infections are thus urgently needed. This report illustrates the development of pegylated reduced graphene oxide nanoparticles (rGO-PEG) and gold nanorods (Au NRs) coated with rGO-PEG (rGO-PEG-Au NRs) for the selective killing of uropathogenic E. coli UTI89. We took advantage of the excellent light absorption properties of rGO-PEG and Au NR particles in the near-infrared (NIR) region to photothermally kill Gram-negative pathogens up to 99% in 10 min by illumination of solutions containing the bacteria. The rGO-PEG-Au NRs demonstrated better photothermal efficiency towards E. coli than rGO-PEG. Targeted killing of E. coli UTI89 could be achieved with rGO-PEG-Au NRs functionalized with multimeric heptyl α-d-mannoside probes. This currently offers a unique biocompatible method for the ablation of pathogens with the opening of probably a new possibility for clinical treatments of patients with urinary infections.

Impact of Bronchial Epithelium on Dendritic Cell Migration and Function: Modulation by the Bacterial Motif KpOmpA

Mucosal immune response depends on the surveillance network established by dendritic cells (DC), APC localized within the epithelium. Bronchial epithelial cells (BEC) play a pivotal role both in the host defense and in the pathogenesis of inflammatory airway disorders. We previously showed that the outer membrane protein A from Klebsiella pneumoniae (KpOmpA), a pathogen-associated molecular pattern (PAMP) derived from Klebsiella pneumoniae, activates BEC. In this study, we evaluated the consequences of this activation on DC traffic and functions. KpOmpA significantly increased the production of CCL2, CCL5, CXCL10, and CCL20 by BEC. Stimulation of BEC increased their chemotactic activity for monocyte-derived DC (MDDC) precursors, through CCL5 and CXCL10 secretion. BEC/MDDC precursor coculture leads to an ICAM-1-dependent accelerated differentiation and enhanced maturation of MDDC. BEC/DC interactions did not affect the capacity of DC to induce T cell proliferation. However, DC preincubated with BEC increased significantly the IL-10 production by autologous T cells. Basolateral and intraepithelial DC differently enhance IL-4 and/or IL-10 synthesis according to the condition of stimulation. In vivo, intranasal injections of KpOmpA into BALB/c mice induced the recruitment of CD11c+ and I-Ad+ myeloid DC associated with bronchial epithelium activation as evidenced by CCL20 expression. These data show that KpOmpA-exposed BEC participate in the homeostasis of myeloid DC network, and regulate the induction of local immune response.

Compounds Triggering ER Stress Exert Anti-Melanoma Effects and Overcome BRAF Inhibitor Resistance

We have discovered and developed a series of molecules (thiazole benzenesulfonamides). HA15, the lead compound of this series, displayed anti-cancerous activity on all melanoma cells tested, including cells isolated from patients and cells that developed resistance to BRAF inhibitors. Our molecule displayed activity against other liquid and solid tumors. HA15 also exhibited strong efficacy in xenograft mouse models with melanoma cells either sensitive or resistant to BRAF inhibitors. Transcriptomic, proteomic, and biochemical studies identified the chaperone BiP/GRP78/HSPA5 as the specific target of HA15 and demonstrated that the interaction increases ER stress, leading to melanoma cell death by concomitant induction of autophagic and apoptotic mechanisms.

A Functional γδTCR/CD3 Complex Distinct from γδT Cells Is Expressed by Human Eosinophils

Background Eosinophils are effector cells during parasitic infections and allergic responses. However, their contribution to innate immunity has been only recently unravelled. Methodology/Principal Findings Here we show that human eosinophils express CD3 and γδ T Cell Receptor (TCR) but not αβ TCR. Surface expression of γδTCR/CD3 is heterogeneous between eosinophil donors and inducible by mycobacterial ligands. Surface immunoprecipitation revealed expression of the full γδTCR/CD3 complex. Real-time PCR amplification for CD3, γ and δ TCR constant regions transcripts showed a significantly lower expression in eosinophils than in γδT cells. Limited TCR rearrangements occur in eosinophils as shown by spectratyping analysis of CDR3 length profiles and in situ hybridization. Release by eosinophils of Reactive Oxygen Species, granule proteins, Eosinophil Peroxidase and Eosinophil-Derived Neurotoxin and cytokines (IFN-γ and TNF-α) was observed following activation by γδTCR-specific agonists or by mycobacteria. These effects were inhibited by anti-γδTCR blocking antibodies and antagonists. Moreover, γδTCR/CD3 was involved in eosinophil cytotoxicity against tumor cells. Conclusions/Significance Our results provide evidence that human eosinophils express a functional γδTCR/CD3 with similar, but not identical, characteristics to γδTCR from γδT cells. We propose that this receptor contributes to eosinophil innate responses against mycobacteria and tumors and may represent an additional link between lymphoid and myeloid lineages.

A Rab11A/Myosin Vb/Rab11-FIP2 Complex Frames Two Late Recycling Steps of Langerin from the ERC to the Plasma Membrane

A large body of knowledge relating to the constitution of Rab GTPase/Rab effector complexes and their impact on both membrane domain organization and overall membrane trafficking has been built up in recent years. However in the context of the live cell there are still many questions that remain to be answered, such as where and when these complexes assemble and where they perform their primary function(s). We describe here the dynamic processes that take place in the final steps of the Rab11A dependent recycling pathway, in the context of the membrane platform constituted by Myosin Vb, Rab11A, and Rab11‐FIP2. We first confirm that a series of previously reported observations obtained during the study of a number of trafficking cargoes also apply to langerin. Langerin is a cargo molecule that traffics through Rab11A‐positive membrane domains of the endosomal recycling pathway. In order to explore the relative dynamics of this set of partners, we make extensive use of a combinatory approach of Live‐FRET, fast FRAP video, fast confocal and TIRF microscopy modalities. Our data show that the Myosin Vb/Rab11A/Rab11‐FIP2 platform is spatially involved in the regulation of langerin trafficking at two distinct sites within live cells, first at the sorting site in the endosomal recycling compartment (ERC) where transport vesicles are formed, and subsequently, in a strict time‐defined order, at the very late stage of docking/tethering and fusion of these langerin recycling vesicles to the plasma membrane.

Impairment of GABAB receptor dimer by endogenous 14‐3‐3ζ in chronic pain conditions

In the central nervous system, the inhibitory GABAB receptor is the archetype of heterodimeric G protein‐coupled receptors (GPCRs). However, the regulation of GABAB dimerization, and more generally of GPCR oligomerization, remains largely unknown. We propose a novel mechanism for inhibition of GPCR activity through de‐dimerization in pathological conditions. We show here that 14‐3‐3ζ, a GABAB1‐binding protein, dissociates the GABAB heterodimer, resulting in the impairment of GABAB signalling in spinal neurons. In the dorsal spinal cord of neuropathic rats, 14‐3‐3ζ is overexpressed and weakens GABAB inhibition. Using anti‐14‐3‐3ζ siRNA or competing peptides disrupts 14‐3‐3ζ/GABAB1 interaction and restores functional GABAB heterodimers in the dorsal horn. Importantly, both strategies greatly enhance the anti‐nociceptive effect of intrathecal Baclofen in neuropathic rats. Taken together, our data provide the first example of endogenous regulation of a GPCR oligomeric state and demonstrate its functional impact on the pathophysiological process of neuropathic pain sensitization.