Biology subjects

Vilar, M.

Publications and source records attributed to Vilar, M..

8 recordsLinked to original sources

bioRxiv2025

High conformational flexibility of phosphomannomutase 2: Implications for functioning mechanisms, stability and pharmacological chaperone design

Phosphomannomutase 2 (PMM2) is a critical enzyme in the N-glycosylation pathway, and its defect is the cause of the most common congenital disorder of glycosylation. Despite its biological relevance, the understanding of PMM2 is limited, as the catalytic mechanism and required conformational dynamics remain unknown. In this study, we investigated murine PMM2 (Pmm2) to elucidate its structural flexibility and functional insights. High-resolution crystal structures of Pmm2 in both apo and activator-bound forms provided a more detailed model of the protein, underscoring the role of three ionic cofactors that are essential for dimerization, catalysis, and stability. The Pmm2 structures also provided eight distinct conformations of the protein subunit, highlighting its dynamic nature. Structural comparisons among Pmm2, human PMM2 and other phosphomannomutases helped define the architecture of the enzyme as a dimer assembled by the rigid association of the cap domains, which provide a flat platform from which the core domains of each subunit protrude in a flexible manner. Molecular dynamics (MD) simulations of the human and murine PMM2s further emphasized the enzymes substantial conformational flexibility, revealing extensive core domain movements and suggesting potential inter-subunit communication within the dimer. This study refines the model of PMM2 function, demonstrating its dynamic role in substrate binding, intermediate reorientation, and product release. The observed flexibility provides new opportunities to target specific enzyme states, enabling the development of pharmacological chaperones.

biochemistry↗

bioRxiv2023

Alzheimer's disease linked Aβ42 exerts product feedback inhibition on γsecretase impairing downstream cell signaling

Amyloid {beta} (A{beta}) peptides accumulating in the brain are proposed to trigger Alzheimers disease (AD). However, molecular cascades underlying their toxicity are poorly defined. Here, we explored a novel hypothesis for A{beta}42 toxicity that arises from its proven affinity for {gamma}-secretases. We hypothesized that the reported increases in A{beta}42, particularly in the endolysosomal compartment, promote the establishment of a product feedback inhibitory mechanism on {gamma}-secretases, and thereby impair downstream signaling events. We show that human A{beta}42 peptides, but neither murine A{beta}42 nor human A{beta}17-42 (p3), inhibit {gamma}-secretases and trigger accumulation of unprocessed substrates in neurons, including C-terminal fragments (CTFs) of APP, p75 and pan-cadherin. Moreover, A{beta}42 treatment dysregulated cellular homeostasis, as shown by the induction of p75-dependent neuronal death in two distinct cellular systems. Our findings raise the possibility that pathological elevations in A{beta}42 contribute to cellular toxicity via the {gamma}-secretase inhibition, and provide a novel conceptual framework to address A{beta} toxicity in the context of {gamma}-secretase-dependent homeostatic signaling.

biochemistry↗

bioRxiv2023

Structural Basis for the transmembrane signaling and antidepressant-induced activation of the receptor tyrosine kinase TrkB

Neurotrophin receptors of the Trk family are involved in the regulation of brain development and neuroplasticity, and therefore can serve as targets for anti-cancer and stroke-recovery drugs, antidepressants, and many others. The structures of Trk protein domains in various states upon activation need to be elucidated to allow rational drug design. However, little is known about the conformations of the transmembrane and juxtamembrane domains of Trk receptors. In the present study, we employed NMR spectroscopy to solve the structure of the TrkB dimeric transmembrane domain in the lipid environment. We verified the structure using mutagenesis and confirmed that the conformation corresponds to the active state of the receptor. Subsequent study of TrkB interaction with the antidepressant drug fluoxetine, and the antipsychotic drug chlorpromazine, provided a clear self-consistent model, describing the mechanism by which fluoxetine activates the receptor by binding to its transmembrane domain.

biochemistry↗

bioRxiv2022

Identification of a guanine-specific pocket in the protein N of SARS-CoV-2

The SARS-CoV-2 nucleocapsid protein (N) is responsible for RNA binding. Here we report the crystal structure of the C-terminal domain (NCTD) in open and closed conformations and in complex with guanine triphosphate, GTP. The crystal structure and biochemical studies reveals a specific interaction between the guanine, a nucleotide enriched in the packaging signals regions of coronaviruses, and a highly conserved tryptophan residue (W330). In addition, EMSA assays with SARS-CoV-2 derived RNA hairpin loops from a putative viral packaging sequence showed the preference interaction of the N-CTD to RNA oligonucleotides containing G and the loss of the specificity in the mutant W330A. Here we propose that this interaction may facilitate the viral assembly process. In summary we have identified a specific guanine-binding pocket in the N protein that may be used to design viral assembly inhibitors.

biophysics↗

bioRxiv2022

Functional and structural deficiencies of Gemin5 variants associated with neurological disease

Dysfunction of RNA-binding proteins are often linked to a wide range of human disease in general, and particularly with neurological conditions. Gemin5 is a member of the survival of motor neuron (SMN) complex, a ribosome-binding protein and a translation reprogramming factor. Recently, pathogenic mutations in Gemin5 protein have been reported but the functional consequences of these variants remain elusive. Here we report functional and structural deficiencies associated with compound heterozygosity variants within the Gemin5 gene found in patients with neurodevelopmental disorders. These clinical variants are located in key domains of Gemin5, the tetratricopeptide repeat (TPR)-like dimerization module and the non-canonical RNA-binding site 1 (RBS1). We show that the TPR-like variants disrupt protein dimerization while the RBS1 variant confers protein instability. All mutants are defective in the interaction with protein networks involved in translation and RNA-driven pathways. Importantly, the TPR-like variants fail to associate with native ribosomes, abolishing the cap-dependent and selective translation control of Gemin5, and establishing a functional difference with the wild type protein. Our study provides a molecular basis of disease associated with malfunction of Gemin5 protein.

molecular biology↗

bioRxiv2021

Neurotrophin signaling is modulated by specific transmembrane domain interactions

The neurotrophin receptors p75 and TrkA play an important role in the development and survival of the nervous system. Biochemical data suggest that p75 and TrkA regulate the activities of each other. For instance, p75 is able to regulate the response of TrkA to lower concentrations of NGF and TrkA promotes p75 shedding by -secretases in a ligand-dependent manner. The current model is that p75 and TrkA are regulated by means of a physical direct interaction, however the nature of such interaction has been elusive so far. Here using NMR in micelles, multiscale molecular dynamics (MD), FRET and functional studies we identified and characterized the direct interaction between TrkA and p75 through the transmembrane domains (TMDs). MD of p75-TMD mutants suggests that although the interaction between TrkA and p75 TMDs is maintained, a specific protein interface is required to facilitate TrkA active homodimerization in the presence of NGF. The same mutations in the TMD protein interface of p75 reduced the activation of TrkA by NGF and cell differentiation. In summary we provide a structural model of the p75/TrkA receptor complex stabilized by transmembrane domain interactions.

biochemistry↗

bioRxiv2019

Structural Basis and Mechanism of TrkA Activation by NGF through Ligand-Induced Rotation of Transmembrane Domain Dimers.

Trk receptors are essential for the nervous system development. The molecular mechanism of TrkA activation by its ligand NGF is still unsolved. Recent data indicates that at endogenous levels most of TrkA is in an equilibrium monomer-dimer and the binding of NGF induces an increase of the dimer and oligomer forms of the receptor. An unsolved issue is the role of the transmembrane domain (TMD) in the dimerization of TrkA and the structural details of the TMD in the active dimer receptor. We found that TrkA-TMD can form dimers, identified the structural determinants of the dimer interface in the active receptor and validated this interface using site-directed mutagenesis together with functional and cell differentiation studies. Using in vivo crosslinking we identified a reordering of the extracellular juxtamembrane (JTM) region after ligand binding. Replacement of some residues in the JTM region with cysteine form ligand-independent active dimers and reveal a preferred dimer interface. In addition to that, insertion of leucine residues into the TMD helix induces a ligand-independent TrkA activation suggesting that a rotation of the TMD dimers could be behind TrkA activation by NGF. Altogether our data indicates that the transmembrane and juxtamembrane regions of the receptor play a key role in the dimerization and activation of TrkA by NGF.

biochemistry↗

bioRxiv2019

Inter-individual genomic heterogeneity within European population isolates

A number of studies carried out since the early 70s has investigated the effects of isolation on genetic variation within and among human populations in diverse geographical contexts. However, no extensive analysis has been carried out on the heterogeneity among genomes within isolated populations. This issue is worth exploring since events of recent admixture and/or subdivision could potentially disrupt the genetic homogeneity which is to be expected when isolation is prolonged and constant over time. Here, we analyze literature data relative to 87,818 autosomal single-nucleotide polymorphisms, which were obtained from a total of 28 European populations. Our results challenge the traditional paradigm of population isolates as genetically (and genomically) uniform entities. In fact, focusing on the distribution of variance of intra-population diversity measures across individuals, we show that the inter-individual heterogeneity of isolated populations is at least comparable to the open ones. More in particular, three small and highly inbred isolates (Sappada, Sauris and Timau in Northeastern Italy) were found to be characterized by levels of this parameter largely exceeding that of all other populations, possibly due to relatively recent events of genetic introgression. Finally, we propose a way to monitor the effects of inter-individual heterogeneity in disease-gene association studies.

genomics↗