Biology subjects

Figueiredo, A. M.

Publications and source records attributed to Figueiredo, A. M..

4 recordsLinked to original sources

bioRxiv2026

PDGFD-induced pericyte contractility drives pathological neoangiogenesis

Neovascular tufts (NVTs) are a defining feature of proliferative retinopathies such as diabetic retinopathy and retinopathy of prematurity, in which they compromise retinal integrity and vision. Excessive endothelial cell (EC) sprouting underlies their formation, yet the cellular and mechanical mechanisms that shape NVTs, and the contribution of pericytes (PCs) to this process, remain unclear. In this work, we investigated the role of actomyosin contractility in ECs and PCs during pathological angiogenesis and NVT formation. We identified non-muscle myosin IIA (NMIIA) and phosphorylated myosin light chains (MLCs) as a key signature of PCs and ECs in retinopathy and NVTs. Using inducible deletion of Myh9 in the oxygen-induced retinopathy mouse model, we uncover distinct and opposing roles for NMIIA in the two cell types. Endothelial NMIIA promotes neovascularisation and prevents enlargement of NVTs, whilst PC NMIIA drives neovascular tuft formation. Mechanistically, we identify platelet-derived growth factor D (PDGFD), but not PDGFB, as a driver of pathological PC actomyosin contractility through Rho-associated coiled-coil kinase (ROCK). Genetic deletion of Pdgfd partially phenocopies the loss of NMIIA in PCs, reducing NVT formation and lowering NMIIA and phosphorylated MLC levels within NVTs. Our findings establish cell-type-specific actomyosin contractility as a determinant of NVT pathogenesis and identify PDGFD-ROCK-NMIIA signalling in PCs as a therapeutic target in proliferative retinopathies.

cell biology↗

bioRxiv2026

NMR reveals specific remodelling of protein folding landscapes in ionic liquids

Ionic liquids (ILs) are designer solvents with tunable properties that have emerged as powerful cosolutes in protein science and biotechnology. While ILs are known to stabilize or destabilize proteins, their molecular mechanisms remain poorly defined, particularly regarding effects on the unfolded ensemble. Here, we use the metastable SH3 domain of the Drosophila adaptor protein Drk, which coexists in a slow two-state equilibrium between folded and unfolded states, as a model to dissect how ILs modulate protein conformational equilibria. Using nuclear magnetic resonance (NMR) spectroscopy, we show that cholinium glutamate ([Ch][Glu]) stabilizes the folded state by preferential exclusion from the protein surface, raising the barrier to unfolding in a manner reminiscent of osmolytes and crowding agents. In contrast, 1-butyl-3-methylimidazolium dicyanamide ([Bmim][dca]) shifts the equilibrium toward the unfolded state, not by destabilizing the native fold, but by stabilizing a compact, non-native -helical conformation within the unfolded ensemble. Notably, this concentration-dependent mechanism differs from traditional denaturants such as urea or guanidinium chloride, which promote random-coil unfolded states. Kinetic measurements further reveal that [Ch][Glu] slows unfolding, whereas [Bmim][dca] slows folding, indicating that the two ILs reshape the folding landscape in opposite directions. These findings challenge the prevailing view that cosolutes act primarily on the folded state and establish unfolded-state stabilization as a critical determinant of protein stability. More broadly, this work provides a molecular framework for understanding how ILs reshape protein folding landscapes and offers design principles for tailoring ILs as stabilizers or destabilizers in biotechnology and therapeutic applications. Significance StatementProteins exist in a delicate balance between folded and unfolded states, and cosolutes such as salts are generally viewed as acting only on the folded state. Here, we show that ionic liquids (ILs) are able to reshape this balance and uniquely modulate both folded and unfolded protein states through distinct molecular mechanisms. These results challenge the conventional view of protein stabilization/destabilization and demonstrate that unfolded-state modulation is a critical determinant of protein behavior. By establishing principles for how ILs alter protein folding landscapes, this work provides a framework for designing ILs with tailored effects, with implications for biocatalysis, protein engineering, and therapeutic formulation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/700775v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@372d3borg.highwire.dtl.DTLVardef@7a7fadorg.highwire.dtl.DTLVardef@543f4eorg.highwire.dtl.DTLVardef@1084836_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

An integrative characterisation of proline cis and trans conformers in a disordered peptide

Intrinsically disordered proteins (IDPs) often contain proline residues, which undergo cis/trans isomerisation. While molecular dynamics (MD) simulations have the potential to fully characterise the proline cis and trans sub-ensembles, they are limited by the slow timescales of isomerisation and force field inaccuracies. Nuclear magnetic resonance (NMR) spectroscopy can report on ensemble-averaged observables for both the cis and trans proline states, but a full atomistic characterisation of these sub-ensembles is challenging. Given the importance of proline cis/trans isomerisation for influencing the conformational sampling of disordered proteins, we employed a combination of all-atom MD simulations with enhanced sampling (metadynamics), NMR, and small-angle X-ray scattering (SAXS) to characterise the two sub-ensembles of the ORF6 C-terminal region (ORF6CTR) from SARS-CoV-2 corresponding to the proline-57 (P57) cis and trans states. We performed MD simulations in three distinct force fields: AMBER03ws, AMBER99SB-disp, and CHARMM36m, which are all optimised for disordered proteins. Each simulation was run for an accumulated time of 180-220 {micro}s until convergence was reached, as assessed by blocking analysis. A good agreement between the cis-P57 populations predicted from metadynamics simulations in AMBER03ws was observed with populations obtained from experimental NMR data. Moreover, we observed good agreement between the radius of gyration predicted from the metadynamics simulations in AMBER03ws and that measured using SAXS. Our findings suggest that both the cis-P57 and trans-P57 conformations of ORF6CTR are extremely dynamic and that interdisciplinary approaches combining both multi-scale computations and experiments offer avenues to explore highly dynamic states that cannot be reliably characterised by either approach in isolation. SIGNIFICANCEThis study employs MD simulations (with metadynamics), NMR spectroscopy, and SAXS to elucidate the individual cis and trans proline conformations of ORF6CTR from SARS-CoV-2. The good agreement on proline cis/trans populations observed in experiments (NMR) and those calculated from simulations in the AMBER03ws force field (with SAXS reweighting) showcases the efficiency of this interdisciplinary approach, which can be used to characterise highly dynamic disordered protein states, even for very slow processes. Furthermore, our study emphasises the importance of considering both computational and experimental methodologies to gain a more holistic understanding of highly dynamic proteins. The presented integrative approach sets a precedent for future studies aiming to explore complex and dynamic biological systems with slow transitions such as proline isomerisations.

biophysics↗

bioRxiv2023

Small-molecule binding to an intrinsically disordered protein revealed by experimental NMR 19F transverse spin-relaxation

Intrinsically disordered proteins are highly dynamic biomolecules that rapidly interconvert between many structural conformations. Traditionally, these proteins have been considered un-druggable because of their lack of classical long-lived binding pockets. Recent evidence suggests that intrinsically disordered proteins can bind small, drug-like molecules, however, there are limited approaches to characterize these interactions experimentally. Here we demonstrate that ligand-detected 19F transverse relaxation rates (R2) obtained from Nuclear Magnetic Resonance spectroscopy are highly sensitive to the interaction between a small-molecule and an intrinsically disordered protein, in contrast to chemical shift perturbations which are minimally sensitive for this interaction. With this method, we show that the small molecule, 5-fluoroindole, interacts with the disordered domains of non-structural protein 5A from hepatitis C virus with a Kd of 260 {+/-} 110 M. We also demonstrate that 5-fluoroindole remains highly dynamic in the bound form. Our findings suggest that ligand-detected 19F transverse relaxation measurements could represent a highly effective screening strategy to identify molecules capable of interacting with these traditionally elusive, dynamic biomolecules.

biophysics↗