The checkpoint Saccharomyces cerevisiae Rad9 protein contains a tandem tudor domain that recognizes DNA.

International audienceDNA damage checkpoints are signal transduction pathways that are activated after genotoxic insults to protect genomic integrity. At the site of DNA damage, 'mediator' proteins are in charge of recruiting 'signal transducers' to molecules 'sensing' the damage. Budding yeast Rad9, fission yeast Crb2 and metazoan 53BP1 are presented as mediators involved in the activation of checkpoint kinases. Here we show that, despite low sequence conservation, Rad9 exhibits a tandem tudor domain structurally close to those found in human/mouse 53BP1 and fission yeast Crb2. Moreover, this region is important for the resistance of Saccharomyces cerevisiae to different genotoxic stresses. It does not mediate direct binding to a histone H3 peptide dimethylated on K79, nor to a histone H4 peptide dimethylated on lysine 20, as was demonstrated for 53BP1. However, the tandem tudor region of Rad9 directly interacts with single-stranded DNA and double-stranded DNAs of various lengths and sequences through a positively charged region absent from 53BP1 and Crb2 but present in several yeast Rad9 homologs. Our results argue that the tandem tudor domains of Rad9, Crb2 and 53BP1 mediate chromatin binding next to double-strand breaks. However, their modes of chromatin recognition are different, suggesting that the corresponding interactions are differently regulated

Sub-Nucleocapsid Nanoparticles: A Nasal Vaccine against Respiratory Syncytial Virus

Background: Bronchiolitis caused by the respiratory syncytial virus (RSV) in infants less than two years old is a growing public health concern worldwide, and there is currently no safe and effective vaccine. A major component of RSV nucleocapsid, the nucleoprotein (N), has been so far poorly explored as a potential vaccine antigen, even though it is a target of protective anti-viral T cell responses and is remarkably conserved between human RSV A and B serotypes. We recently reported a method to produce recombinant N assembling in homogenous rings composed of 10–11 N subunits enclosing a bacterial RNA. These nanoparticles were named sub-nucleocapsid ring structure (N SRS). Methodology and Principal Findings: The vaccine potential of N SRS was evaluated in a well-characterized and widely acknowledged mouse model of RSV infection. BALB/c adult mice were immunized intranasally with N SRS adjuvanted with the detoxified E. coli enterotoxin LT(R192G). Upon RSV challenge, vaccinated mice were largely protected against virus replication in the lungs, with a mild inflammatory lymphocytic and neutrophilic reaction in their airways. Mucosal immunization with N SRS elicited strong local and systemic immunity characterized by high titers of IgG1, IgG2a and IgA anti-N antibodies, antigen-specific CD8+ T cells and IFN-c-producing CD4+ T cells. Conclusions/Significance: This is the first report of using nanoparticles formed by the recombinant nucleocapsid protein as an efficient and safe intra-nasal vaccine against RSV

Circulating microRNAs in sera correlate with soluble biomarkers of immune activation but do not predict mortality in ART treated individuals with HIV-1 infection: A case control study

Introduction: The use of anti-retroviral therapy (ART) has dramatically reduced HIV-1 associated morbidity and mortality. However, HIV-1 infected individuals have increased rates of morbidity and mortality compared to the non-HIV-1 infected population and this appears to be related to end-organ diseases collectively referred to as Serious Non-AIDS Events (SNAEs). Circulating miRNAs are reported as promising biomarkers for a number of human disease conditions including those that constitute SNAEs. Our study sought to investigate the potential of selected miRNAs in predicting mortality in HIV-1 infected ART treated individuals. Materials and Methods: A set of miRNAs was chosen based on published associations with human disease conditions that constitute SNAEs. This case: control study compared 126 cases (individuals who died whilst on therapy), and 247 matched controls (individuals who remained alive). Cases and controls were ART treated participants of two pivotal HIV-1 trials. The relative abundance of each miRNA in serum was measured, by RTqPCR. Associations with mortality (all-cause, cardiovascular and malignancy) were assessed by logistic regression analysis. Correlations between miRNAs and CD4+ T cell count, hs-CRP, IL-6 and D-dimer were also assessed. Results: None of the selected miRNAs was associated with all-cause, cardiovascular or malignancy mortality. The levels of three miRNAs (miRs -21, -122 and -200a) correlated with IL-6 while miR-21 also correlated with D-dimer. Additionally, the abundance of miRs -31, -150 and -223, correlated with baseline CD4+ T cell count while the same three miRNAs plus miR- 145 correlated with nadir CD4+ T cell count. Discussion: No associations with mortality were found with any circulating miRNA studied. These results cast doubt onto the effectiveness of circulating miRNA as early predictors of mortality or the major underlying diseases that contribute to mortality in participants treated for HIV-1 infection

Development and Validation of a Risk Score for Chronic Kidney Disease in HIV Infection Using Prospective Cohort Data from the D:A:D Study

Ristola M. on työryhmien DAD Study Grp ; Royal Free Hosp Clin Cohort ; INSIGHT Study Grp ; SMART Study Grp ; ESPRIT Study Grp jäsen.Background Chronic kidney disease (CKD) is a major health issue for HIV-positive individuals, associated with increased morbidity and mortality. Development and implementation of a risk score model for CKD would allow comparison of the risks and benefits of adding potentially nephrotoxic antiretrovirals to a treatment regimen and would identify those at greatest risk of CKD. The aims of this study were to develop a simple, externally validated, and widely applicable long-term risk score model for CKD in HIV-positive individuals that can guide decision making in clinical practice. Methods and Findings A total of 17,954 HIV-positive individuals from the Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D) study with >= 3 estimated glomerular filtration rate (eGFR) values after 1 January 2004 were included. Baseline was defined as the first eGFR > 60 ml/min/1.73 m2 after 1 January 2004; individuals with exposure to tenofovir, atazanavir, atazanavir/ritonavir, lopinavir/ritonavir, other boosted protease inhibitors before baseline were excluded. CKD was defined as confirmed (>3 mo apart) eGFR In the D:A:D study, 641 individuals developed CKD during 103,185 person-years of follow-up (PYFU; incidence 6.2/1,000 PYFU, 95% CI 5.7-6.7; median follow-up 6.1 y, range 0.3-9.1 y). Older age, intravenous drug use, hepatitis C coinfection, lower baseline eGFR, female gender, lower CD4 count nadir, hypertension, diabetes, and cardiovascular disease (CVD) predicted CKD. The adjusted incidence rate ratios of these nine categorical variables were scaled and summed to create the risk score. The median risk score at baseline was -2 (interquartile range -4 to 2). There was a 1: 393 chance of developing CKD in the next 5 y in the low risk group (risk score = 5, 505 events), respectively. Number needed to harm (NNTH) at 5 y when starting unboosted atazanavir or lopinavir/ritonavir among those with a low risk score was 1,702 (95% CI 1,166-3,367); NNTH was 202 (95% CI 159-278) and 21 (95% CI 19-23), respectively, for those with a medium and high risk score. NNTH was 739 (95% CI 506-1462), 88 (95% CI 69-121), and 9 (95% CI 8-10) for those with a low, medium, and high risk score, respectively, starting tenofovir, atazanavir/ritonavir, or another boosted protease inhibitor. The Royal Free Hospital Clinic Cohort included 2,548 individuals, of whom 94 individuals developed CKD (3.7%) during 18,376 PYFU (median follow-up 7.4 y, range 0.3-12.7 y). Of 2,013 individuals included from the SMART/ESPRIT control arms, 32 individuals developed CKD (1.6%) during 8,452 PYFU (median follow-up 4.1 y, range 0.6-8.1 y). External validation showed that the risk score predicted well in these cohorts. Limitations of this study included limited data on race and no information on proteinuria. Conclusions Both traditional and HIV-related risk factors were predictive of CKD. These factors were used to develop a risk score for CKD in HIV infection, externally validated, that has direct clinical relevance for patients and clinicians to weigh the benefits of certain antiretrovirals against the risk of CKD and to identify those at greatest risk of CKD.Peer reviewe

Etudes par radiocristallographie de l'interaction entre les superantigènes des cellules B et la région Fab des récepteurs des lymphocytes B

La compréhension des mécanismes développés par les micro-organismes pathogènes pour coloniser l'organisme humain et échapper à son système immunitaire représente un enjeu majeur depuis l'émergence des phénomènes de résistance aux antibiotiques. Certains organismes pathogènes utilisent des protéines de surface capables d'induire le dysfonctionnement des cellules B du système immunitaire. C'est le cas de la protéine A de Staphylococcus aureus (SpA) et de la protéine L de Peptostreptococcus magnus (PpL) qui reconnaissent un nombre anormalement élevé (50 % contre 0,01 % dans le cas des antigènes classiques) d'immunoglobulines ancrées à la surface des cellules B. Ce travail présente les structures cristallographiques de complexes entre la région Fab des immunoglobulines et un domaine de SpA ou de PpL. Ces structures mettent en évidence les résidus de chaque partenaire qui sont impliqués dans les interactions. Elles révèlent les bases moléculaires qui permettent à ces deux protéines de fixer un large répertoire d'immunoglobulines. Ces protéines fixent les immunoglobulines en dehors de leur site de reconnaissance des antigènes, elles ne sont donc pas reconnues comme des antigènes classiques. La propriété de fixation de ces protéines bactériennes aux immunoglobulines sans empêcher la fixation des antigènes peut être utilisée pour obtenir des cristaux de complexes Fab-antigènes qui seraient délicats à cristalliser. En effet, elles peuvent moduler la surface de l'assemblage protéique à cristalliser et conduire à la formation de cristaux. Cette stratégie a été utilisée pour obtenir pour la première fois, des cristaux du complexe entre un Fab et un antigène parasitaire: la protéine P30 de Toxoplasma gondii.The comprehension of the mechanisms developed by pathogenic microorganisms to colonise the human body and to escape its immune system is of great importance since the emergence of the phenomena of resistance to antibiotics. Some pathogenic organisms produce surface proteins able to induce the dysfunction of the B cells of the immune system. It is the case of the Staphylococcus aureus protein A (SpA) and of the Peptostreptococcus magnus protein L (PpL) which recognise an unusually high number (50% against 0,01 % in the case of the conventional intigens) of immunoglobulins anchored to the surface of the B cells. This work presents the crystallographic structures of complexes between the Fab region of the immunoglobulins and SpA or PpL domains. These structures highlight the residues of each partner, which are involved in the interactions. They reveal the molecular basis responsible for the property of these two proteins to bind a broad population of immunoglobulins. These proteins interact with the immunoglobulins apart from their combining site; they are thus not recognised as conventional antigens. The property of these bacterial proteins to bind the immunoglobulins without preventing the recognition of the antigens can be used to obtain crystals of Fab-antigens complexes that would be difficult crystallise. Indeed, these proteins can modulate the surface of the molecular assembly to crystallise and lead to the crystal formation. This strategy was used to obtain for the first time, crystals of the complex between a Fab and a parasitic antigen: the Toxoplasma gondii protein P30.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Analyse structurale de protéines de l'enveloppe nucléaire impliquées dans des pathologies génétiques

Les protéines de l enveloppe nucléaire comme MAN1 et la lamine A sont mutées dans des maladies héréditaires. Notre objectif est de déterminer la structure 3D de ces protéines en complexe avec leurs partenaires afin de proposer des mécanismes moléculaires pour les pathologies correspondantes. La protéine MAN1 se lie aux régulateurs de la transcription Smads et par là est impliquée dans la régulation de la prolifération cellulaire. J ai montré par RMN que le fragment C-terminal de MAN1 est composé d un domaine globulaire adoptant un repliement de type Winged Helix et d une région en échange conformationnel. Ce fragment interagit avec l ADN et les Smads, et jouerait ainsi un rôle de cofacteur des Smads. J ai aussi travaillé à l élucidation des conséquences structurales d une nouvelle mutation pathogène R439C de la lamine A/C. L introduction d une cystéine supplémentaire provoque la formation d oligomères perturbant les capacités de liaison du domaine muté in vitro.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Structure and dynamics of the anticodon arm binding domain of Bacillus stearothermophilus Tyrosyl-tRNA synthetase.

International audienceThe structure of a recombinant protein, TyrRS(delta4), corresponding to the anticodon arm binding domain of Bacillus stearothermophilus tyrosyl-tRNA synthetase, has been solved, and its dynamics have been studied by nuclear magnetic resonance (NMR). It is the first structure described for such a domain of a tyrosyl-tRNA synthetase. It consists of a five-stranded beta sheet, packed against two alpha helices on one side and one alpha helix on the other side. A large part of the domain is structurally similar to other functionally unrelated RNA binding proteins. The basic residues known to be essential for tRNA binding and charging are exposed to the solvent on the same face of the molecule. The structure of TyrRS(delta4), together with previous mutagenesis data, allows one to delineate the region of interaction with tRNATyr

Internal motion time scales of a small, highly stable and disulfide rich protein. An 15N, 13C NMR and molecular dynamics study.

International audienceMotions of the backbone C alpha H alpha and threonine C beta H beta bonds of toxin alpha were investigated using natural abundance 13C NMR and molecular dynamics. Measurement of the 13C longitudinal and transverse relaxation rates employed ACCORDION techniques together with coherence selection by pulsed field gradients and sensitivity enhancement through the use of preservation of equivalent pathway, thus allowing a considerable reduction of the required spectrometer time. 13C R1, R2, 1H-->13C NOE were obtained, as well as the variations of R1 rho (90 degrees) as a function of the rf field strength. These data were compared to those recorded by 1H and 15N NMR on a labelled sample of the toxin [Guenneugues et al. (1997) Biochemistry, 36, 16097-16108]. Both sets of data showed that picosecond to nanosecond time scale motions are well correlated to the secondary structure of the protein. This was further reinforced by the analysis of a 1 ns molecular dynamics simulation in water. Several C alpha H alpha and threonine C beta H beta experimentally exhibit fast motions with a correlation time longer than 500 ps, that cannot be sampled along the simulation. In addition, the backbone exhibits motions on the microsecond to millisecond time scale on more than half of its length. Thus, toxin alpha, a highly stable protein (Tm = 75 degrees C at acidic pH) containing 61 amino acids and 4 disulfides, shows important internal motions on time scales ranging from 0.1-0.5 ps, to 10-100 ps, 1 ns, and about 30 microseconds to 10 ms