Molecular dynamics studies on the NMR and X-ray structures of rabbit prion protein wild-type and mutants

Prion diseases are invariably fatal and highly infectious neurodegenerative diseases that affect a wide variety of mammalian species such as sheep, goats, mice, humans, chimpanzees, hamsters, cattle, elks, deer, minks, cats, chicken, pigs, turtles, etc. These neurodegenerative diseases are caused by the conversion from a soluble normal cellular protein into insoluble abnormally folded infectious prions and the conversion is believed to involve conformational change from a predominantly alpha-helical protein to one rich in beta-sheet structure. Such conformational changes may be amenable to study by molecular dynamics (MD) techniques. For rabbits, classical studies show they have a low susceptibility to be infected, but in 2012 it was reported that rabbit prion can be generated (though not directly) and the rabbit prion is infectious and transmissible (Proceedings of the National Academy of Sciences USA 109(13): 5080-5). This paper studies the NMR and X-ray molecular structures of rabbit prion protein wild-type and mutants by MD techniques, in order to understand the specific mechanism of rabbit prion protein and rabbit prions.Comment: (The 2nd version of arXiv1304.7633

Characterizaion of conformation-dependent prion protein epitopes

Whereas prion replication involves structural rearrangement of cellular prion protein (PrP(C)), the existence of conformational epitopes remains speculative and controversial, and PrP transformation is monitored by immunoblot detection of PrP(27–30), a protease-resistant counterpart of the pathogenic scrapie form (PrP(Sc)) of PrP. We now describe the involvement of specific amino acids in conformational determinants of novel monoclonal antibodies (mAbs) raised against randomly chimeric PrP. Epitope recognition of two mAbs depended on polymorphisms controlling disease susceptibility. Detection by one, referred to as PRC5, required alanine and asparagine at discontinuous mouse PrP residues 132 and 158, which acquire proximity when residues 126–218 form a structured globular domain. The discontinuous epitope of glycosylation-dependent mAb PRC7 also mapped within this domain at residues 154 and 185. In accordance with their conformational dependence, tertiary structure perturbations compromised recognition by PRC5, PRC7, as well as previously characterized mAbs whose epitopes also reside in the globular domain, whereas conformation-independent epitopes proximal or distal to this region were refractory to such destabilizing treatments. Our studies also address the paradox of how conformational epitopes remain functional following denaturing treatments and indicate that cellular PrP and PrP(27–30) both renature to a common structure that reconstitutes the globular domain

Replication properties of a contemporary Zika virus from West Africa

Zika virus (ZIKV) has become a global health problem over the past decade due to the extension of the geographic distribution of the Asian/American genotype. Recent epidemics of Asian/American ZIKV have been associated with developmental disorders in humans. There is mounting evidence that African ZIKV may be associated with increased fetal pathogenicity necessitating to pay a greater attention towards currently circulating viral strains in sub-Saharan Africa. Here, we generated an infectious molecular clone GUINEA-18 of a recently transmitted human ZIKV isolate from West Africa, ZIKV-15555. The available infectious molecular clone MR766MC of historical African ZIKV strain MR766-NIID was used for a molecular clone-based comparative study. Viral clones GUINEA-18 and MR766MC were compared for their ability to replicate in VeroE6, A549 and HCM3 cell lines. There was a lower replication rate for GUINEA-18 associated with weaker cytotoxicity and reduced innate immune system activation compared with MR766MC. Analysis of chimeric viruses between viral clones stressed the importance of NS1 to NS4B proteins, with a particular focus of NS4B on GUINEA-18 replicative properties. ZIKV has developed strategies to prevent cytoplasmic stress granule formation which occurs in response to virus infection. GUINEA-18 was greatly efficient in inhibiting stress granule assembly in A549 cells subjected to a physiological stressor, with NS1 to NS4B proteins also being critical in this process. The impact of these GUINEA-18 proteins on viral replicative abilities and host-cell responses to viral infection raises the question of the role of nonstructural proteins in the pathogenicity of currently circulating ZIKV in sub-Saharan Africa

The NS1 protein of contemporary West African Zika virus potentiates viral replication and reduces innate immune activation.

Mosquito-borne Zika virus (ZIKV) from sub-Saharan Africa has recently gained attention due to its epidemic potential and its capacity to be highly teratogenic. To improve our knowledge on currently circulating strains of African ZIKV, we conducted protein sequence alignment and identified contemporary West Africa NS1 (NS1CWA) protein as a highly conserved viral protein. Comparison of NS1CWA with the NS1 of the historical African ZIKV strain MR766 (NS1MR766), revealed seven amino acid substitutions. The effects of NS1 mutations on protein expression, virus replication, and innate immune activation were assessed in human cells using recombinant NS1 proteins and a chimeric viral clone MR766 with NS1CWA replacing NS1MR766. Our data indicated higher secretion efficiency of NS1CWA compared to NS1MR766 associated with a change in subcellular distribution. A chimeric MR766 virus with NS1CWA instead of authentic protein displayed a greater viral replication efficiency, leading to more pronounced cell death compared to parental virus. Enhanced viral growth was associated with reduced activation of innate immunity. Our data raise questions of the importance of NS1 protein in the pathogenicity of contemporary ZIKV from sub-Saharan Africa and point to differences within viral strains of African lineage

Epitope shaving promotes fungal immune evasion

This is the final version. Available on open access from the American Society for Microbiology via the DOI in this recordData availability. The data supporting the findings in this study are available within the paper and accompanying supplementary files. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (44) partner repository with the dataset identifiers: PXD018027and PXD018044. The source data for cytometry, enzyme activity, and immune interaction studies are provided as a source data file.The cell wall provides a major physical interface between fungal pathogens and their mammalian host. This extracellular armour is critical for fungal cell homeostasis and survival. Fungal-specific cell wall moieties, such as β-1,3-glucan, are recognised as pathogen-associated molecular patterns (PAMPs) that activate immune-mediated clearance mechanisms. We have reported that the opportunistic human fungal pathogen, Candida albicans, masks β-1,3-glucan following exposure to lactate, hypoxia or iron depletion. However, the precise mechanism(s) by which C. albicans masks β-1,3-glucan have remained obscure. Here we identify a secreted exoglucanase, Xog1, that is induced in response to lactate or hypoxia. Xog1 functions downstream of the lactate-induced β-glucan “masking” pathway to promote β-1,3-glucan “shaving”. Inactivation of XOG1 blocks most, but not all β-1,3-glucan masking in response to lactate, suggesting that other activities contribute to this phenomenon. Nevertheless, XOG1 deletion attenuates the lactate-induced reductions in phagocytosis and cytokine stimulation normally observed for wild type cells. We also demonstrate that the pharmacological inhibition of exoglucanases undermines β-glucan shaving, enhances the immune visibility of the fungus, and attenuates its virulence. Our study establishes a new mechanism underlying environmentally-induced PAMP remodelling that can be manipulated pharmacologically to influence immune recognition and infection outcomes.Medical Research Council (MRC)Wellcome TrustUniversity of AberdeenEuropean Research Council (ERC)Netherlands Organization for Scientific Researc

A Simple, Versatile and Sensitive Cell-Based Assay for Prions from Various Species

Detection and quantification of prion infectivity is a crucial step for various fundamental and applied aspects of prion research. Identification of cell lines highly sensitive to prion infection led to the development of cell-based titration procedures aiming at replacing animal bioassays, usually performed in mice or hamsters. However, most of these cell lines are only permissive to mouse-adapted prions strains and do not allow titration of prions from other species. In this study, we show that epithelial RK13, a cell line permissive to mouse and bank vole prion strains and to natural prion agents from sheep and cervids, enables a robust and sensitive detection of mouse and ovine-derived prions. Importantly, the cell culture work is strongly reduced as the RK13 cell assay procedure designed here does not require subcultivation of the inoculated cultures. We also show that prions effectively bind to culture plastic vessel and are quantitatively detected by the cell assay. The possibility to easily quantify a wider range of prions, including rodent experimental strains but also natural agents from sheep and cervids, should prompt the spread of cell assays for routine prion titration and lead to valuable information in fundamental and applied studies

In Vitro Amplification of Misfolded Prion Protein Using Lysate of Cultured Cells

Protein misfolding cyclic amplification (PMCA) recapitulates the prion protein (PrP) conversion process under cell-free conditions. PMCA was initially established with brain material and then with further simplified constituents such as partially purified and recombinant PrP. However, availability of brain material from some species or brain material from animals with certain mutations or polymorphisms within the PrP gene is often limited. Moreover, preparation of native PrP from mammalian cells and tissues, as well as recombinant PrP from bacterial cells, involves time-consuming purification steps. To establish a convenient and versatile PMCA procedure unrestricted to the availability of substrate sources, we attempted to conduct PMCA with the lysate of cells that express cellular PrP (PrPC). PrPSc was efficiently amplified with lysate of rabbit kidney epithelial RK13 cells stably transfected with the mouse or Syrian hamster PrP gene. Furthermore, PMCA was also successful with lysate of other established cell lines of neuronal or non-neuronal origins. Together with the data showing that the abundance of PrPC in cell lysate was a critical factor to drive efficient PrPSc amplification, our results demonstrate that cell lysate in which PrPC is present abundantly serves as an excellent substrate source for PMCA

P2X7 receptor: Death or life?

The P2X7 plasma membrane receptor is an intriguing molecule that is endowed with the ability to kill cells, as well as to activate many responses and even stimulate proliferation. Here, the authors give an overview on the multiplicity and complexity of P2X7-mediated responses, discussing recent information on this receptor. Particular attention has been paid to early and late signs of apoptosis and necrosis linked to activation of the receptor and to the emerging field of P2X7 function in carcinogenesis

Reconstitution dosimétrique physique d’accident radiologique par simulations numériques à l’aide d’outils associant un modèle anthropomorphe à un code de calcul Monte Carlo

In case of radiological accident involving an external source, several techniques are used in order to determine the dose received by the victim and to allow a better medical management of the victim. One of them is the numerical dosimetric reconstruction. To perform numerical dosimetric reconstructions, the Ionizing Radiation Dosimetry Laboratory from IRSN has developed since ten years a tool named SESAME which combines voxel phantoms and the Monte Carlo computer code MCNP(X). The aim of this thesis is to develop new implementations in SESAME to take into account the morphology and the posture of the victim and to allow the reconstruction of accidents which occured in external radiotherapy treatment. At first a new procedure, which takes into account the posture of the victim at the moment of the accident has been developed in SESAME. This procedure is based on the NURBS format to create modified voxel phantoms and has been numerically and experimentally validated. The second part of the work deals with a feasibility study in order to implement in SESAME tool a functionality dedicated to the reconstruction of accidents in external radiotherapy. Two modelling of a linear accelerator Clinac 2100C with 6MV and 25 MV nominal voltage have been performed with the MCNPX computer code and have been validated. Then, they have been optimised in order to reduce the calculation time to make them compatible with a reconstruction of accident.Lors d’un accident radiologique dû à une source externe, plusieurs techniques sont associées afin de déterminer la dose reçue par la victime et ainsi permettre une meilleure prise en charge de la victime. L’une d’entre elles est la reconstitution dosimétrique physique numérique. Afin de procéder à ces reconstitutions dosimétriques numériques, le Laboratoire de Dosimétrie des Rayonnements Ionisants de l’IRSN développe depuis une dizaine d’année un outil nommé SESAME qui associe des fantômes voxélisés au code de calcul Monte Carlo MCNP(X). L’objectif de cette thèse était de développer de nouvelles fonctionnalités dans SESAME permettant en particulier de prendre en compte la morphologie et la posture de la victime et de reconstituer des accidents survenant en radiothérapie externe. Tout d’abord une nouvelle procédure, permettant de prendre compte la posture de la victime au moment de l’accident, a été développée dans SESAME. Cette procédure permet la transformation par l’intermédiaire du format NURBS des fantômes voxélisés et a été validée numériquement et expérimentalement à l’aide d’un fantôme physique anthropomorphe. Le second thème du travail a porté sur une étude de faisabilité pour implémenter dans SESAME une fonctionnalité dédiée à la reconstitution d’accident en radiothérapie externe. Deux modélisations d’un accélérateur Clinac 2100C avec une tension nominale de 6 MV et 25 MV ont été réalisées à l’aide du code Monte Carlo MCNPX et validées. Elles ont ensuite été optimisées afin de réduire le temps de calcul pour les rendre compatible avec une reconstitution d’accident