Luc Camoin

Molecular Characterisation of Endogenous Vangl2/Vangl1 Heteromeric Protein Complexes

Background Mutations in the Planar Cell Polarity (PCP) core gene Vangl2 cause the most severe neural tube defects (NTD) in mice and humans. Genetic studies show that the Vangl2 gene genetically interacts with a close homologue Vangl1. How precisely Vangl2 and Vangl1 proteins interact and crosstalk has remained a difficult issue to address, with the main obstacle being the accurate discrimination of the two proteins, which share close sequence homology. Experimental evidence previously presented has been sparse and addressed with ectopically expressed proteins or with antibodies unable to biochemically discriminate Vangl1 from Vangl2, therefore giving rise to unclear results. Methodology and Main Findings A highly specific monoclonal anti-Vangl2 antibody was generated and rigorously tested on both recombinant and extracted Vangl2 using surface plasmon resonance (SPR) analysis, western blot, and immunoprecipitation experiments. This antibody efficiently affinity-purified Vangl2 from cell lysates and allowed the unambiguous identification of endogenous Vangl2 by proteomic analysis. Vangl1 was also present in Vangl2 immunoprecipitates, establishing the first biochemical evidence for the existence of Vangl2/Vangl1 heterodimers at an endogenous level. Epitope-tagged Vangl2 and Vangl1 confirmed that both proteins interact and colocalize at the plasma membrane. The Vangl2 antibody is able to acutely assess differential expression levels of Vangl2 protein in culture cell lines, as corroborated with gene expression analysis. We characterised Vangl2 expression in the cochlea of homozygous and heterozygous Lp mutant mice bearing a point mutation within the C-terminal Vangl2 region that leads to profound PCP defects. Our antibody could detect much lower levels of Vangl2Lp protein in mutant mice compared to the wild type mice. Conclusion Our results provide an in-depth biochemical characterisation of the interaction observed between Vangl paralogues.

Essential and nonredundant roles for Diaphanous formins in cortical microtubule capture and directed cell migration

The Diaphanous formins mDia1, mDia2, and mDia3 are involved in the capture of cortical microtubules and ErbB2-dependent directed migration. These functions are independent of actin. They are mediated by mDia FH2 domains, which associate with distinct sets of proteins. Rab6IP2 is a novel interactor of mDia1 that contributes to microtubule tethering.

Kinetic Analysis of Mouse Brain Proteome Alterations Following Chikungunya Virus Infection before and after Appearance of Clinical Symptoms

Recent outbreaks of Chikungunya virus (CHIKV) infection have been characterized by an increasing number of severe cases with atypical manifestations including neurological complications. In parallel, the risk map of CHIKV outbreaks has expanded because of improved vector competence. These features make CHIKV infection a major public health concern that requires a better understanding of the underlying physiopathological processes for the development of antiviral strategies to protect individuals from severe disease. To decipher the mechanisms of CHIKV infection in the nervous system, a kinetic analysis on the host proteome modifications in the brain of CHIKV-infected mice sampled before and after the onset of clinical symptoms was performed. The combination of 2D-DIGE and iTRAQ proteomic approaches, followed by mass spectrometry protein identification revealed 177 significantly differentially expressed proteins. This kinetic analysis revealed a dramatic down-regulation of proteins before the appearance of the clinical symptoms followed by the increased expression of most of these proteins in the acute symptomatic phase. Bioinformatic analyses of the protein datasets enabled the identification of the major biological processes that were altered during the time course of CHIKV infection, such as integrin signaling and cytoskeleton dynamics, endosome machinery and receptor recycling related to virus transport and synapse function, regulation of gene expression, and the ubiquitin-proteasome pathway. These results reveal the putative mechanisms associated with severe CHIKV infection-mediated neurological disease and highlight the potential markers or targets that can be used to develop diagnostic and/or antiviral tools.

Fibronectin-guided migration of carcinoma collectives

Functional interplay between tumour cells and their neoplastic extracellular matrix plays a decisive role in malignant progression of carcinomas. Here we provide a comprehensive data set of the human HNSCC-associated fibroblast matrisome. Although much attention has been paid to the deposit of collagen, we identify oncofetal fibronectin (FN) as a major and obligate component of the matrix assembled by stromal fibroblasts from head and neck squamous cell carcinomas (HNSCC). FN overexpression in tumours from 435 patients corresponds to an independent unfavourable prognostic indicator. We show that migration of carcinoma collectives on fibrillar FN-rich matrices is achieved through αvβ6 and α9β1 engagement, rather than α5β1. Moreover, αvβ6-driven migration occurs independently of latent TGF-β activation and Smad-dependent signalling in tumour epithelial cells. These results provide insights into the adhesion-dependent events at the tumour–stroma interface that govern the collective mode of migration adopted by carcinoma cells to invade surrounding stroma in HNSCC.

The scaffolding function of the RLTPR protein explains its essential role for CD28 co-stimulation in mouse and human T cells

In two complementary papers, Casanova, Malissen, and collaborators report the discovery of human RLTPR deficiency, the first primary immunodeficiency of the human CD28 pathway in T cells. Together, the two studies elucidate the largely (but not completely) overlapping roles of RLTPR in CD28 signaling in T and B cells of humans and mice.

Hepatocyte pathway alterations in response to in vitro Crimean Congo hemorrhagic fever virus infection

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus responsible for hemorrhagic manifestations and multiple organ failure, with a high mortality rate. In infected humans, damage to endothelial cells and vascular leakage may be a direct result of virus infection or an immune response-mediated indirect effect. The main target cells are mononuclear phagocytes, endothelial cells and hepatocytes; the liver being a key target for the virus, which was described as susceptible to interferon host response and to induce apoptosis. To better understand the early liver cell alterations due to virus infection, the protein profile of in vitro CCHFV-infected HepG2 cells was analyzed using two quantitative proteomic approaches, 2D-DIGE and iTRAQ. A set of 243 differentially expressed proteins was identified. Bioinformatics analysis (Ingenuity Pathways Analysis) revealed multiple host cell pathways and functions altered after CCHFV infection, with notably 106 proteins related to cell death, including 79 associated with apoptosis. Different protein networks emerged with associated pathways involved in inflammation, oxidative stress and apoptosis, ubiquitination/sumoylation, regulation of the nucleo-cytoplasmic transport, and virus entry. Collectively, this study revealed host liver protein abundances that were modified at the early stages of CCHFV infection, offering an unparalleled opportunity of the description of the potential pathogenesis processes and of possible targets for antiviral research.

Altered Protein Networks and Cellular Pathways in Severe West Nile Disease in Mice

Background The recent West Nile virus (WNV) outbreaks in developed countries, including Europe and the United States, have been associated with significantly higher neuropathology incidence and mortality rate than previously documented. The changing epidemiology, the constant risk of (re-)emergence of more virulent WNV strains, and the lack of effective human antiviral therapy or vaccines makes understanding the pathogenesis of severe disease a priority. Thus, to gain insight into the pathophysiological processes in severe WNV infection, a kinetic analysis of protein expression profiles in the brain of WNV-infected mice was conducted using samples prior to and after the onset of clinical symptoms. Methodology/Principal Findings To this end, 2D-DIGE and gel-free iTRAQ labeling approaches were combined, followed by protein identification by mass spectrometry. Using these quantitative proteomic approaches, a set of 148 proteins with modified abundance was identified. The bioinformatics analysis (Ingenuity Pathway Analysis) of each protein dataset originating from the different time-point comparisons revealed that four major functions were altered during the course of WNV-infection in mouse brain tissue: i) modification of cytoskeleton maintenance associated with virus circulation; ii) deregulation of the protein ubiquitination pathway; iii) modulation of the inflammatory response; and iv) alteration of neurological development and neuronal cell death. The differential regulation of selected host protein candidates as being representative of these biological processes were validated by western blotting using an original fluorescence-based method. Conclusion/Significance This study provides novel insights into the in vivo kinetic host reactions against WNV infection and the pathophysiologic processes involved, according to clinical symptoms. This work offers useful clues for anti-viral research and further evaluation of early biomarkers for the diagnosis and prevention of severe neurological disease caused by WNV.

Quantitative proteomic analysis exploring progression of colorectal cancer: Modulation of the serpin family

UNLABELLEDnColorectal cancer (CRC) remains a major cause of cancer related-death in developed countries. The mortality risk is correlated with the stage of CRC determined at the primary diagnosis and early diagnosis is associated with enhanced survival rate. Currently, only faecal occult blood tests are used to screen for CRC. Consequently, there is an incentive to identify specific markers of CRC. We used quantitative proteomic analysis of serum samples to characterize protein profiles in adenoma, CRC and healthy control samples. We identified 89 distinct proteins modulated between normal, colorectal adenoma and carcinoma patients. This list emphasizes proteins involved in enzyme regulator activities and in particular the serpin family. In serum samples, protein profiles of three members of the serpin family (SERPINA1, SERPINA3 and SERPINC1) were confirmed by ELISA assays. We obtained sensitivity/specificity values of 95%/95% for both SERPINA1 and SERPINC1, and 95%/55% for SERPINA3. This study supports the idea that serum proteins can discriminate adenoma and CRC patients from unaffected patients and reveals a panel of regulated proteins that might be useful for selecting patients for colonoscopy. By evaluating SERPINA1, SERPINA3 and SERPINC1, we highlight the potential role of the serpin family during the development and progression of CRC.nnnSIGNIFICANCEnColorectal cancer (CRC) remains a major cause of cancer mortality throughout the world. However, very few CRC biomarkers have satisfactory sensitivity and specificity in clinical practice. To the best of our knowledge our study is the first to profile sera proteomes between adenoma, CRC and healthy patients. We report a comprehensive list of proteins that may be used as early diagnostic biomarkers of CRC. It is noteworthy that 17% of these modulated proteins have been previously described as candidate biomarkers in CRC. Enzyme regulator activity was found to be the main molecular function among these proteins and, in particular, there was an enrichment of members of the serpin family. The subsequent verification on a new cohort by ELISA demonstrates that these serpins could be useful to discriminate healthy from colorectal carcinoma patients with a high sensitivity and specificity. The combination of these biomarkers should increase predictive powers of CRC diagnosis. The remaining candidates form a reserve for further evaluation of additional biomarkers for CRC diagnosis.

Circulating vascular endothelial growth factor (VEGF) as predictive factor of progression-free survival in patients with advanced chordoma receiving sorafenib: an analysis from a phase II trial of the french sarcoma group (GSF/GETO)

Background Patients with advanced chordoma are often treated with tyrosine kinase inhibitors without any predictive factor to guide decision. We report herein an ancillary analysis of the the Angionext phase II trial (NCT 00874874). Results From May 2011 to January 2014, 26 were sampled. The 9-month PFS rate was 72.9% (95%-CI: 45.9-87.9). During sorafenib treatment, a significant increase in PlGF (18.4 vs 43.8 pg/mL, p<0.001) was noted along with a non-significant increase in VEGF (0.7 vs 1.0 ng/mL, p=0.07). VEGF at D1 >1.04 ng/mL (HR=12.5, 95%-CI: 1.37-114, p=0.025) and VEGF at D7 >1.36 ng/mL (HR=10.7, 95%-CI: 1.16-98, p=0.037) were associated with shorter PFS. The 9-month PFS rate was 92.3% (95%-CI: 56.6-98.9) when VEGF at D1 was ≤1.04 ng/mL versus 23.3% (95%-CI: 1.0-63.2) when >1.04 ng/mL. Patients and Methods Chordoma patients were treated with sorafenib 800 mg/day for 9 months, unless earlier occurrence of progression or toxicities. Six biomarkers (sE-Selectin, VEGF, VEGF-C, placental growth factor (PlGF), Thrombospondin, Stem Cell Factor (SCF)) were measured at baseline (day 1: D1) and day 7 (D7). Conclusion High levels of VEGF was associated with poor outcome.

Cerebrospinal fluid biomarker candidates associated with human WNV neuroinvasive disease

During the last decade, the epidemiology of WNV in humans has changed in the southern regions of Europe, with high incidence of West Nile fever (WNF) cases, but also of West Nile neuroinvasive disease (WNND). The lack of human vaccine or specific treatment against WNV infection imparts a pressing need to characterize indicators associated with neurological involvement. By its intimacy with central nervous system (CNS) structures, modifications in the cerebrospinal fluid (CSF) composition could accurately reflect CNS pathological process. Until now, few studies investigated the association between imbalance of CSF elements and severity of WNV infection. The aim of the present study was to apply the iTRAQ technology in order to identify the CSF proteins whose abundances are modified in patients with WNND. Forty-seven proteins were found modified in the CSF of WNND patients as compared to control groups, and most of them are reported for the first time in the context of WNND. On the basis of their known biological functions, several of these proteins were associated with inflammatory response. Among them, Defensin-1 alpha (DEFA1), a protein reported with anti-viral effects, presented the highest increasing fold-change (FC>12). The augmentation of DEFA1 abundance in patients with WNND was confirmed at the CSF, but also in serum, compared to the control individual groups. Furthermore, the DEFA1 serum level was significantly elevated in WNND patients compared to subjects diagnosed for WNF. The present study provided the first insight into the potential CSF biomarkers associated with WNV neuroinvasion. Further investigation in larger cohorts with kinetic sampling could determine the usefulness of measuring DEFA1 as diagnostic or prognostic biomarker of detrimental WNND evolution.