Biology subjects

Requena, J. R.

Publications and source records attributed to Requena, J. R..

4 recordsLinked to original sources

bioRxiv2020

All-Atom Simulation of the HET-s Prion Replication

Prions are self-replicative protein particles lacking nucleic acids. Originally discovered for causing infectious neurodegenerative disorders, they have also been found to play several physiological roles in a variety of species. Functional and pathogenic prions share a common mechanism of replication, characterized by the ability of an amyloid conformer to propagate by inducing the conversion of its physiological, soluble counterpart. In this work, we focus on the propagation of the prion forming domain of HET-s, a physiological fungal prion for which high-resolution structural data are available. Since time-resolved biophysical experiments cannot yield a full reconstruction of prion replication, we resort to computational methods. To overcome the computational limitations of plain Molecular Dynamics (MD) simulations, we adopt a special type of biased dynamics called ratchet-and-pawl MD (rMD). The accuracy of this enhanced path sampling protocol strongly depends on the choice of the collective variable (CV) used to define the biasing force. Since for prion propagation a reliable reaction coordinate (RC) is not yet available, we resort to the recently developed Self-Consistent Path Sampling (SCPS). Indeed, in such an approach the CV where the biasing force is applied is not heuristically postulated but is calculated through an iterative refinement procedure. Our atomistic reconstruction of the HET-s replication shows remarkable similarities with a previously reported mechanism of mammalian PrPSc propagation obtained with a different computational protocol. Together, these results indicate that the propagation of prions generated by evolutionary distant proteins shares common features. In particular, in both these cases, prions propagate their conformation through a very similar templating mechanism.

biophysics↗

bioRxiv2020

Structural features of an infectious recombinant PrPSc prion using solid state NMR

PrPSc, the first described and most notorious prion, is the only protein known to cause epidemics of deadly disease. Its properties are encoded in its unique structure. Here we report a first solid state NMR study of a uniformly labelled (U-13C,15N)-Bank vole (BV) infectious recombinant PrPSc prion. C-C, C-H and N-H spectra were obtained with MAS rotation of the sample at up to 60 kHz. We obtained amino acid-type secondary structure information and used it to challenge a physically plausible atomistic model of PrPSc consisting of a 4-rung {beta} solenoid recently proposed by us. In all cases, our model was compatible with the data. This study shows that elucidation of the structure of PrPSc is within reach using recombinant PrPSc, NMR, and our model as a guiding tool.

biochemistry↗

bioRxiv2020

PHARMACOLOGICAL PROTEIN INACTIVATION BY TARGETING FOLDING INTERMEDIATES

Recent computational advancements in the simulation of biochemical processes allow investigating the mechanisms involved in protein regulation with realistic physics-based models, at an atomistic level of resolution. Using these techniques to study the negative regulation of the androgen receptor (AR), we discovered a key functional role played by non-native metastable states appearing along the folding pathway of this protein. This unexpected observation inspired us to design a completely novel drug discovery approach, named Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT), based on the rationale of negatively regulating protein expression by targeting folding intermediates. Here, PPI-FIT was tested for the first time on the cellular prion protein (PrP), a cell surface glycoprotein playing a key role in fatal and transmissible neurodegenerative pathologies known as prion diseases. We predicted the all-atom structure of an intermediate appearing along the folding pathway of PrP, and identified four different small molecule ligands for this conformer, all capable of selectively lowering the expression of the protein by promoting its degradation. Our data support the notion that the level of target proteins could be modulated by acting on their folding pathways, implying a previously unappreciated role for folding intermediates in the biological regulation of protein expression.

biochemistry↗

bioRxiv2018

Full Atomistic Model Of Prion Structure And Conversion

Prions are unusual protein assemblies that propagate their conformationally-encoded information in absence of nucleic acids. The first prion identified, the scrapie isoform (PrPSc) of the cellular prion protein (PrPC), is the only one known to cause epidemic and epizootic episodes(1). Most aggregates of other misfolding-prone proteins are amyloids, often arranged in a Parallel-In-Register-{beta}-Sheet (PIRIBS)(2) or {beta}-solenoid conformations(3). Similar folding models have also been proposed for PrPSc, although none of these have been confirmed experimentally. Recent cryo-electron microscopy (cryo-EM) and X-ray fiber-diffraction studies provided evidence that PrPSc is structured as a 4-rung {beta}-solenoid (4R{beta}S)(4, 5). Here, we combined different experimental data and computational techniques to build the first physically-plausible, atomic resolution model of mouse PrPSc, based on the 4R{beta}S architecture. The stability of this new PrPSc model, as assessed by Molecular Dynamics (MD) simulations, was found to be comparable to that of the prion forming domain of Het-s, a naturally-occurring {beta}-solenoid. Importantly, the 4R{beta}S arrangement allowed the first simulation of the sequence of events underlying PrPC conversion into PrPSc. Our results provide the most updated, experimentally-driven and physically-coherent model of PrPSc, together with an unprecedented reconstruction of the mechanism underlying the self-catalytic propagation of prions.\n\nSignificanceSince the original hypothesis by Stanley Prusiner, prions have represented enigmatic agents diverging from the classical concept of genetic inheritance. However, the structure of PrPSc, the infectious isoform of the cellular prion protein (PrPC), has so far remained elusive, mostly due to technical challenges posed by its aggregation propensity. Here, we present a new high resolution model of PrPSc derived from the integration of a wide array of recent experimental constraints. By coupling the information of such model with a newly developed computational method, we reconstructed for the first time the conformational transition of PrPC to PrPSc. This study offers a unique workbench for designing therapeutics against prion diseases, and a physically-plausible mechanism explaining how protein conformation could self-propagate.

biochemistry↗