Patients carrying autosomal dominant mutations in the histone/lysine acetyl transferases CBP or EP300 develop a neurodevelopmental disorder: Rubinstein-Taybi syndrome (RSTS). The biological pathways underlying these neurodevelopmental defects remain elusive. Here, we unravel the contribution of a stress-responsive pathway to RSTS. We characterize the structural and functional interaction between CBP/EP300 and heat-shock factor 2 (HSF2), a tuner of brain cortical development and major player in prenatal stress responses in the neocortex: CBP/EP300 acetylates HSF2, leading to the stabilization of the HSF2 protein. Consequently, RSTS patient-derived primary cells show decreased levels of HSF2 and HSF2-dependent alteration in their repertoire of molecular chaperones and stress response. Moreover, we unravel a CBP/EP300-HSF2-N-cadherin cascade that is also active in neurodevelopmental contexts, and show that its deregulation disturbs neuroepithelial integrity in 2D and 3D organoid models of cerebral development, generated from RSTS patient-derived iPSC cells, providing a molecular reading key for this complex pathology.
CREB-Binding Protein , Heat-Shock Proteins , Neurodevelopmental Disorders , Rubinstein-Taybi Syndrome , Transcription Factors , Humans , CREB-Binding Protein/genetics , CREB-Binding Protein/metabolism , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Histones/genetics , Mutation , Neurodevelopmental Disorders/genetics , Neurodevelopmental Disorders/pathology , Rubinstein-Taybi Syndrome/genetics , Rubinstein-Taybi Syndrome/pathology , Transcription Factors/genetics , Transcription Factors/metabolism , E1A-Associated p300 Protein/genetics , E1A-Associated p300 Protein/metabolism
Influenza A viruses are highly contagious RNA viruses that cause respiratory tract infections in humans and animals. Their non-structural protein NS1, a homodimer of two 230-residue chains, is the main viral factor in counteracting the antiviral defenses of the host cell. Its RNA-binding domain is an obligate dimer that is connected to each of the two effector domains by a highly flexible unstructured linker region of ten amino acids. The flexibility of NS1 is a key property that allows its effector domains and its RNA binding domain to interact with several protein partners or RNAs. The three-dimensional structures of full-length NS1 dimers revealed that the effector domains could adopt three distinct conformations as regards their mutual interactions and their orientation relative to the RNA binding domain (closed, semi-open and open). The origin of this structural polymorphism is currently being investigated and several hypotheses are proposed, among which one posits that it is a strain-specific property. In the present study, we explored through computational molecular modeling the dynamic and flexibility properties of NS1 from three important influenza virus A strains belonging to three distinct subtypes (H1N1, H6N6, H5N1), for which at least one conformation is available in the Protein Data Bank. In order to verify whether NS1 is stable in three forms for the three strains, we constructed homology models if the corresponding forms were not available in the Protein Data Bank. Molecular dynamics simulations were performed in order to predict the stability over time of the three distinct sequence variants of NS1, in each of their three distinct conformations. Our results favor the co-existence of three stable structural forms, regardless of the strain, but also suggest that the length of the linker, along with the presence of specific amino acids, modulate the dynamic properties and the flexibility of NS1.
Influenza A Virus, H1N1 Subtype , Influenza A Virus, H5N1 Subtype , Influenza A virus , Influenza, Human , Amino Acids/metabolism , Animals , Humans , Influenza A Virus, H1N1 Subtype/metabolism , Influenza A Virus, H5N1 Subtype/genetics , Influenza A virus/genetics , Influenza A virus/metabolism , Molecular Dynamics Simulation , Viral Nonstructural Proteins/metabolism
Influenza A viruses still represent a major health issue, for both humans and animals. One of the main viral proteins of interest to target is the NS1 protein, which counters the host immune response and promotes viral replication. NS1 is a homodimer composed of a dimeric RNA-binding domain (RBD), which is structurally stable and conserved in sequence, and two effector domains that are tethered to the RBD by linker regions. This linker flexibility leads to NS1 polymorphism and can therefore exhibit different forms. Previously, we identified a putative drug-binding site, located in the RBD interface in a crystal structure of NS1. This pocket could be targeted to block RNA binding and inhibit NS1 activities. The objective of the present study is to confirm the presence of this druggable site, whatever the sequence variants, in order to develop a universal therapeutic compound that is insensitive to sequence variations and structural flexibility. Using a set of four NS1 full-length structures, we combined different bioinformatics approaches such as pocket tracking along molecular dynamics simulations, druggability prediction and classification. This protocol successfully confirmed a frequent large binding-site that is highly druggable and shared by different NS1 forms, which is promising for developing a robust NS1-targeted therapy.
Influenza A virus , RNA , Animals , Humans , RNA/metabolism , Influenza A virus/genetics , Influenza A virus/metabolism , Viral Nonstructural Proteins/chemistry , Binding Sites , Protein Domains
The aim of our study was to identify novel molecules able to induce an adaptative response against oxidative stress during the first stages of metabolic syndrome. A cellular survival in vitro test against H2O2-based test was applied after pretreatment with various natural bitter Asteraceae extracts. This screening revealed potent protection from burdock leaf extract. Using chromatography and LC-MS-RMN, we then isolated and identified an original sesquiterpene lactone bioactive molecule: the Xanthatin-13-(pyrrolidine-2-carboxylic acid) (XPc). A real-time RT-qPCR experiment was carried out on three essential genes involved in oxidative stress protection: GPx, SOD, and G6PD. In presence of XPc, an over-expression of the G6PD gene was recorded, whereas no modification of the two others genes could be observed. A biochemical docking approach demonstrated that XPc had a high probability to directly interact with G6PD at different positions. One of the most probable docking sites corresponds precisely to the binding site of AG1, known to stabilize the G6PD dimeric form and enhance its activity. In conclusion, this novel sesquiterpene lactone XPc might be a promising prophylactic bioactive agent against oxidative stress and inflammation in chronic diseases such as metabolic syndrome or type 2 diabetes.
