Regulation of cellular zinc balance as a potential mechanism of EVER-mediated protection against pathogenesis by cutaneous oncogenic human papillomaviruses

Epidermodysplasia verruciformis (EV) is a genodermatosis associated with skin cancers that results from a selective susceptibility to related human papillomaviruses (EV HPV). Invalidating mutations in either of two genes (EVER1 and EVER2) with unknown functions cause most EV cases. We report that EVER1 and EVER2 proteins form a complex and interact with the zinc transporter 1 (ZnT-1), as shown by yeast two-hybrid screening, GST pull-down, and immunoprecipitation experiments. In keratinocytes, EVER and ZnT-1 proteins do not influence intracellular zinc concentration, but do affect intracellular zinc distribution. EVER2 was found to inhibit free zinc influx to nucleoli. Keratinocytes with a mutated EVER2 grew faster than wild-type keratinocytes. In transiently and stably transfected HaCaT cells, EVER and ZnT-1 down-regulated transcription factors stimulated by zinc (MTF-1) or cytokines (c-Jun and Elk), as detected with luciferase assays. To get some insight into the control of EV HPV infection, we searched for interaction between EVER and ZnT-1 and oncoproteins of cutaneous (HPV5) and genital (HPV16) genotypes. HPV16 E5 protein binds to EVER and ZnT-1 and blocks their negative regulation. The lack of a functional E5 protein encoded by EV HPV genome may account for host restriction of these viruses

AI-guided pipeline for protein-protein interaction drug discovery identifies a SARS-CoV-2 inhibitor.

peer reviewedProtein-protein interactions (PPIs) offer great opportunities to expand the druggable proteome and therapeutically tackle various diseases, but remain challenging targets for drug discovery. Here, we provide a comprehensive pipeline that combines experimental and computational tools to identify and validate PPI targets and perform early-stage drug discovery. We have developed a machine learning approach that prioritizes interactions by analyzing quantitative data from binary PPI assays or AlphaFold-Multimer predictions. Using the quantitative assay LuTHy together with our machine learning algorithm, we identified high-confidence interactions among SARS-CoV-2 proteins for which we predicted three-dimensional structures using AlphaFold-Multimer. We employed VirtualFlow to target the contact interface of the NSP10-NSP16 SARS-CoV-2 methyltransferase complex by ultra-large virtual drug screening. Thereby, we identified a compound that binds to NSP10 and inhibits its interaction with NSP16, while also disrupting the methyltransferase activity of the complex, and SARS-CoV-2 replication. Overall, this pipeline will help to prioritize PPI targets to accelerate the discovery of early-stage drug candidates targeting protein complexes and pathways.LabEx IBEID (Biologie Intégrative des Maladies Infectieuses Emergentes

Études de néo-égyptien. Les temps seconds i-sḏm.f et i-ri.f sḏm : entre syntaxe et sémantique

Cassonnet Patricia. Études de néo-égyptien. Les temps seconds i-sḏm.f et i-ri.f sḏm : entre syntaxe et sémantique. In: École pratique des hautes études. 4e section, sciences historiques et philologiques. Livret 12. 1996-1997. 1998. pp. 269-271

Action de petits peptides, fragments d'ACTH, sur l'incorporation de P32 dans les protéines cérébrales in vitro.

Source gallica.bnf.fr / Bibliothèque nationale de FranceInternational audienceSome small peptides, ACTH sequences, are able to modify the 32P incorporation in brain proteins in vitro. The possibility that these peptides play a role in the regulation mechanism of some brain functions through differentiated protein-kinases could be considered

Protéines cellulaires et voies de signalisation cibles des oncoprotéines E6 et E7 du papillomavirus humain de type 5

International audienceLes papillomavirus humains (PVH) sont des virus à ADN double brin d’environ 8 kb. Certains génotypes de PVH occupent une place de premier ordre en tant qu’agents infectieux responsables de cancer, notamment de cancers anogénitaux (PVH16 et 18) et de carcinomes cutanés (PVH5 et 8), dans le cadre d’une génodermatose rare, l’épidermodysplasie verruciforme (EV). Nos travaux récents ont permis l’identification de deux gènes cellulaires (EVER1 et EVER2) dont des mutations à l’état homozygote confèrent la sensibilité aux PVH de l’EV, en particulier le PVH oncogène de type 5. Notre objectif est de comprendre le rôle de ces deux gènes dans le contrôle de l’infection par le PVH5 et les mécanismes pathogéniques sous-jacents. En préalable à cette étude nous avons cherché à identifier les partenaires cellulaires des oncoprotéines virales E6 et E7.Une banque d’ADN complémentaires des ARN messagers de la lignée de kératinocytes humains HaCaT a été construite et établie chez la levure S. cerevisiae. Les protéines cellulaires partenaires des oncoprotéines E6 et E7 ont été identifiées par la technique du double hybride chez la levure. Les différentes interactions observées ont été confirmées par colocalisation cellulaire en microscopie confocale et par des expériences de GST pull-down, d’immunocapture. L’impact de ces interactions sur les voies de signalisation a été analysé par l’étude de la régulation d’un gène rapporteur.Plusieurs protéines cellulaires interagissant avec les protéines E6 et E7 ont été identifiées. Elles sont impliquées dans les voies de signalisation du TNFβ, du TGFα et du calcium ou dans le contrôle du cycle cellulaire. Nous étudions actuellement le mécanisme d’action de la protéine E6 dans la voie de signalisation du TGFâ1. Cette voie aboutit à la synthèse de protéines de régulation négative du cycle cellulaire (p16, p17, p21, p27,…). Certaines de ces protéines bloquent le passage des cellules de la phase G1 à la phase S qui est nécessaire à la réplication du génome viral.L’étude de ces interacteurs devrait apporter une meilleure compréhension des mécanismes cellulaires perturbés par les oncoprotéines E6 et E7 du PVH5 et plus généralement, sur les processus de transformation induits par les PVH oncogènes

The EVER Proteins as a Natural Barrier against Papillomaviruses: a New Insight into the Pathogenesis of Human Papillomavirus Infections

Summary: Infections by human papillomaviruses (HPVs) are the most frequently occurring sexually transmitted diseases. The crucial role of genital oncogenic HPV in cervical carcinoma development is now well established. In contrast, the role of cutaneous HPV in skin cancer development remains a matter of debate. Cutaneous beta-HPV strains show an amazing ubiquity. The fact that a few oncogenic genotypes cause cancers in patients suffering from epidermodysplasia verruciformis is in sharp contrast to the unapparent course of infection in the general population. Our recent investigations revealed that a natural barrier exists in humans, which protects them against infection with these papillomaviruses. A central role in the function of this HPV-specific barrier is played by a complex of the zinc-transporting proteins EVER1, EVER2, and ZnT-1, which maintain cellular zinc homeostasis. Apparently, the deregulation of the cellular zinc balance emerges as an important step in the life cycles not only of cutaneous but also of genital HPVs, although the latter viruses have developed a mechanism by which they can break the barrier and impose a zinc imbalance. Herein, we present a previously unpublished list of the cellular partners of EVER proteins, which points to future directions concerning investigations of the mechanisms of action of the EVER/ZnT-1 complex. We also present a general overview of the pathogenesis of HPV infections, taking into account the latest discoveries regarding the role of cellular zinc homeostasis in the HPV life cycle. We propose a potential model for the mechanism of function of the anti-HPV barrier

Human Papillomavirus Type 5 E6 Oncoprotein Represses the Transforming Growth Factor β Signaling Pathway by Binding to SMAD3

Mechanisms of cellular transformation associated with human papillomavirus type 5 (HPV5), which is responsible for skin carcinomas in epidermodysplasia verruciformis (EV) patients, are poorly understood. Using a yeast two-hybrid screening and molecular and cellular biology experiments, we found that HPV5 oncoprotein E6 interacts with SMAD3, a key component in the transforming growth factor β1 (TGF-β1) signaling pathway. HPV5 E6 inhibits SMAD3 transactivation by destabilizing the SMAD3/SMAD4 complex and inducing the degradation of both proteins. Interestingly, the E6 protein of nononcogenic EV HPV9 failed to interact with SMAD3, suggesting that downregulation of the TGF-β1 signaling pathway could be a determinant in HPV5 skin carcinogenesis

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

International audienceSignificant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors. This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells. A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays

Epidermodysplasie verruciforme et infection plurifocale extensive à papillomavirus humains au cours d'un déficit acquis en CD4

International audienceEpidermodysplasie verruciforme et infection plurifocale extensive à papillomavirus humains au cours d'un déficit acquis en CD4

A Cottontail Rabbit Papillomavirus Strain (CRPVb) with Strikingly Divergent E6 and E7 Oncoproteins: An Insight in the Evolution of Papillomaviruses

International audienceWe previously observed that warts induced by an isolate of cottontail rabbit papillomavirus (CRPV) showed incomplete instead of systemic regression in some domestic rabbits. We report that the viral isolate contained, as a major component, a CRPV strain (CRPVb) showing an unexpectedly high divergence in the E6 and E7 open reading frames (ORFs), compared to the prototype CRPVa present in the isolate as a minor component. The E6 and E7 oncoproteins of CRPVa and -b disclosed only 87.5% identical amino acids and differed in size by three and two amino acids, respectively. This divergence involved (i) a great number (4.4%) of nucleotide substitutions and a high rate (83.3%) of nonsynonymous mutations; (ii) mutations changing the E6 and E7 stop codons; and (iii) in-frame sequence insertions in the E6 ORF (18 nucleotides) and downstream of the mutated E7 stop codon (6 nucleotides), both likely to result from a duplication of adjacent sequences. These extensive differences could account for distinct biological and antigenic properties. Strikingly, only four (0.8%) amino acids of the L1 major capsid protein were variable. Thus, it seems likely that sequence duplications and mutations affecting stop codons exert a strong selection pressure on the fixation of nonsynonymous mutations and that phylogenetic calculations based only on point mutations may misevaluate the time scale of the evolution of papillomaviruses