Biology subjects

Pesce, F.

Publications and source records attributed to Pesce, F..

9 recordsLinked to original sources

bioRxiv2023

Design of intrinsically disordered protein variants with diverse structural properties

Intrinsically disordered proteins (IDPs) perform a wide range of functions in biology, suggesting that the ability to design IDPs could help expand the repertoire of proteins with novel functions. Designing IDPs with specific structural or functional properties has, however, been diffcult, in part because determining accurate conformational ensembles of IDPs generally requires a combination of computational modelling and experiments. Motivated by recent advancements in effcient physics-based models for simulations of IDPs, we have developed a general algorithm for designing IDPs with specific structural properties. We demonstrate the power of the algorithm by generating variants of naturally occurring IDPs with different levels of compaction and that vary more than 100 fold in their propensity to undergo phase separation, even while keeping a fixed amino acid composition. We experimentally tested designs of variants of the low-complexity domain of hnRNPA1 and find high accuracy in our computational predictions, both in terms of single-chain compaction and propensity to undergo phase separation. We analyze the sequence features that determine changes in compaction and propensity to phase separate and find an overall good agreement with previous findings for naturally occurring sequences. Our general, physics-based method enables the design of disordered sequences with specified conformational properties. Our algorithm thus expands the toolbox for protein design to include also the most flexible proteins and will enable the design of proteins whose functions exploit the many properties afforded by protein disorder.

biophysics↗

bioRxiv2023

WWP2 MEDIATES THE METABOLIC REPROGRAMMING OF RENAL MYOFIBROBLASTS TO PROMOTE KIDNEY FIBROSIS

Renal fibrosis is a common pathological endpoint in chronic kidney disease (CKD) that is challenging to reverse. Although myofibroblasts are mainly responsible for the accumulation of a fibrillar collagen-rich extracellular matrix (ECM) in fibrotic kidney, recent studies have unveiled their diversity in terms of proliferative and fibrotic characteristics. This diversity could be linked with the existence of different metabolic states, and myofibroblast metabolic reprogramming may contribute to the pathogenesis and progression of renal fibrosis. Here, we reveal an unexpected role of the E3 ubiquitin-protein ligase WWP2 in the metabolic reprogramming of myofibroblasts during renal fibrosis. The tubulointerstitial expression of WWP2 contributes to the progression of fibrosis in CKD patients, and in pre-clinical murine models of CKD. WWP2 deficiency increases fatty acid oxidation and activates the pentose phosphate pathway, boosting mitochondrial respiration at the expense of glycolysis. This concurrently promotes myofibroblast proliferation and halts pro-fibrotic activation, reducing the severity of kidney fibrosis. Mechanistically, WWP2 suppresses the transcription of PGC-1, a metabolic mediator shaping myofibroblast fibrotic response. Pharmacological interventions targeting PGC-1 reverse the effects of WWP2 on fibrotic myofibroblasts. These findings demonstrate the influence of WWP2 on essential metabolic pathways involved in fibrogenesis, uncovering the WWP2-PGC-1 axis that orchestrates the metabolic reprogramming of myofibroblasts during renal fibrosis. Our study presents a potential novel target for therapeutic intervention in the treatment of chronic kidney disease. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=177 HEIGHT=200 SRC="FIGDIR/small/554242v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1847ffeorg.highwire.dtl.DTLVardef@1ef029aorg.highwire.dtl.DTLVardef@93e0f1org.highwire.dtl.DTLVardef@950d49_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIWWP2 expression is elevated in the tubulointerstitium of fibrotic kidneys and contributes to CKD pathogenesis and progression. C_LIO_LIWWP2 uncouples the pro-fibrotic activation and cell proliferation in renal myofibroblasts. C_LIO_LIWWP2 controls mitochondrial respiration in renal myofibroblasts through the metabolic regulator PGC-1 C_LIO_LIMyofibroblast metabolic reprogramming mediates the effect of WWP2 on fibrotic myofibroblasts. C_LI

cell biology↗

bioRxiv2023

Revisiting the use of dioxane as a reference compound for determination of the hydrodynamic radius of proteins by pulsed field gradient NMR spectroscopy

Measuring the compaction of a protein or complex is key to understand the interactions within and between biomolecules. Experimentally, protein compaction is often probed either by estimating the radius of gyration (Rg) obtained from small-angle X-ray scattering (SAXS) experiments or the hydrodynamic radius (Rh) obtained for example by pulsed field gradient nuclear magnetic resonance (PFG NMR) spectroscopy. PFG NMR experiments generally report on the translational diffusion coefficient, which in turn can be used to estimate Rh using an internal standard. Here, we examine the use of 1,4-dioxane as an internal NMR standard to account for sample viscosity and uncertainty about the gradient strength. Specifically, we revisit the basis for the commonly used reference value for the Rh of dioxane (2.12 [A]) that is used to convert measured diffusion coefficients into a hydrodynamic radius. We follow the same approach that was used to establish the current reference value for the Rh by measuring SAXS and PFG NMR data for a set of seven different proteins and using these as standards. Our analysis shows that the current Rh reference value for 1,4-dioxane Rh (2.12 [A]) is underestimated, and we instead suggest a new value of 2.27 [A] {+/-} 0.04 [A]. Using this updated reference value results in a [~]7% increase in Rh values for proteins whose hydrodynamic radius have been measured by PFG NMR. We discuss the implications for ensemble descriptions of intrinsically disordered proteins and evaluation of effect resulting from for example ligand binding, posttranslational modifications, or changes to the environment.

biophysics↗

bioRxiv2023

Conformational ensembles of the human intrinsically disordered proteome: Bridging chain compaction with function and sequence conservation

Intrinsically disordered proteins and regions (collectively IDRs) are pervasive across proteomes in all kingdoms of life, help shape biological functions, and are involved in numerous diseases. IDRs populate a diverse set of transiently formed structures, yet defy commonly held sequence-structure-function relationships. Recent developments in protein structure prediction have led to the ability to predict the three-dimensional structures of folded proteins at the proteome scale, and have enabled large-scale studies of structure-function relationships. In contrast, knowledge of the conformational properties of IDRs is scarce, in part because the sequences of disordered proteins are poorly conserved and because only few have been characterized experimentally. We have developed an efficient model to generate conformational ensembles of IDRs, and thereby to predict their conformational properties from sequence only. Here, we applied this model to simulate all IDRs of the human proteome. Examining conformational ensembles of 29,998 IDRs, we show how chain compaction is correlated with cellular function and localization, including in different types of biomolecular condensates. We train a model to predict compaction from sequence and use this to show conservation of structural properties across orthologs. Our results recapitulate observations from previous studies of individual protein systems, and enable us to study the relationship between sequence, conservation, conformational ensembles, biological function and disease variants at the proteome scale.

biophysics↗

bioRxiv2023

Combining experiments and simulations to examine the temperature-dependent behaviour of a disordered protein

Intrinsically disordered proteins are a class of proteins that lack stable folded conformations and instead adopt a range of conformations that determine their biochemical functions. The temperature-dependent behaviour of such disordered proteins is complex and can vary depending on the specific protein and environment. Here, we have used molecular dynamics simulations and previously published experimental data to investigate the temperature-dependent behaviour of Histatin 5, a 24-residue-long polypeptide. We examined the hypothesis that Histatin 5 undergoes a loss of polyproline II structure with increasing temperature, leading to more compact conformations. We found that the conformational ensembles generated by the simulations generally agree with small-angle X-ray scattering data for Histatin 5, but show some discrepancies with the hydrodynamic radius as probed by pulsed-field gradient nuclear magnetic resonance spectroscopy, and with the secondary structure information derived from circular dichroism. We attempted to reconcile these differences by reweighting the conformational ensembles against the scattering and NMR data. By doing so, we were in part able to capture the temperature-dependent behaviour of Histatin 5 and to link the observed decrease in hydrodynamic radius with increasing temperature to a loss of polyproline II structure. We were, however, unable to achieve agreement with both the scattering and NMR data within experimental errors. We discuss different possibilities for this outcome including inaccuracies in the force field, differences in conditions of the NMR and scattering experiments, and issues related to the calculation of the hydrodynamic radius from conformational ensembles. Our study highlights the importance of integrating multiple types of experimental data when modelling conformational ensembles of disordered proteins and how environmental factors such as the temperature influence them.

biophysics↗

bioRxiv2022

Deciphering the alphabet of disorder -- Glu and Asp act differently on local but not global properties

Compared to folded proteins, the sequences of intrinsically disordered proteins (IDPs) are enriched in polar and charged amino acids. Glutamate is one of the most enriched amino acids in IDPs, while the chemically similar amino acid aspartate is less enriched. So far, the underlying functional differences of glutamates and aspartates in IDPs remain poorly understood. In this study, we examine the differential effects of aspartate and glutamates in IDPs by comparing the function and conformational ensemble of glutamate and aspartate variants of the disordered protein Dss1, using a range of assays, including interaction studies, nuclear magnetic resonance spectroscopy, small angle X-ray scattering and molecular dynamics simulation. First, we analyze the sequences of the rapidly growing data base of experimentally verified IDPs (DisProt) and show that the glutamate enrichment is not caused by a taxonomy bias in IDPs. From analyses of local and global structural properties as well as cell growth and protein-protein interactions using a model acidic IDP from yeast and three Glu/Asp variants, we find that while Glu/Asp support similar function and global dimensions, the variants differ in their binding affinities and population of local transient structural elements. We speculate that these local structural differences may play roles in functional diversity where glutamates can support increased helicity important for folding and binding, while aspartates support extended structures and form helical caps, as well as playing more relevant roles in e.g., transactivation domains and ion-binding.

biophysics↗

bioRxiv2022

Assessment of models for calculating the hydrodynamic radius of intrinsically disordered proteins

Diffusion measurements by pulsed field gradient NMR and fluorescence correlation spectroscopy can be used to probe the hydrodynamic radius of proteins, which contains information about the overall dimension of a protein in solution. The comparison of this value with structural models of intrinsically disordered proteins is nonetheless impaired by the uncertainty of the accuracy of the methods for computing the hydrodynamic radius from atomic coordinates. To tackle this issue, we here build conformational ensembles of 11 intrinsically disordered proteins that we ensure are in agreement with measurements of compaction by small-angle X-ray scattering. We then use these ensembles to identify the forward model that more closely fits the radii derived from pulsed field gradient NMR diffusion experiments. Of the models we examined, we find that the Kirkwood-Riseman equation provides the best description of the hydrodynamic radius probed by pulsed field gradient NMR experiments. While some minor discrepancies remain, our results enable better use of measurements of the hydrodynamic radius in integrative modelling and for force field benchmarking and parameterization. SIGNIFICANCEAccurate models of the conformational properties of intrinsically disordered proteins rely on our ability to interpret experimental data that reports on the conformational ensembles of these proteins in solution. Methods to calculate experimental observables from conformational ensembles are central to link experiments and computation, for example in integrative modelling or the assessment of molecular force fields. Benchmarking such methods is, however, difficult for disordered proteins because it is difficult to construct accurate ensembles without using the data. We here circumvent this problem by combining independent measures of protein compaction to test several methods to calculate the hydrodynamic radius of a disordered protein, as measured by pulsed field gradient NMR diffusion experiments, and find the Kirkwood-Riseman model to be most accurate.

biophysics↗

bioRxiv2021

Improving the global dimensions of intrinsically disordered proteins in Martini 3

Coarse-grained molecular dynamics simulations are a useful tool to determine conformational ensembles of proteins. Here, we show that the coarse-grained force field Martini 3 underestimates the global dimensions of intrinsically disordered proteins (IDPs) and multidomain proteins when compared with small angle X-ray scattering (SAXS) data, and that increasing the strength of protein-water interactions favours more expanded conformations. We find that increasing the strength of interactions between protein and water by ca. 10% results in improved agreement with the SAXS data for IDPs and multi-domain proteins. We also show that this correction results in a more accurate description of self-association of IDPs and folded proteins and better agreement with paramagnetic relaxation enhancement data for most IDPs. While simulations with this revised force field still show deviations to experiments for some systems our results suggest that it is overall a substantial improvement for coarse-grained simulations of soluble proteins.

biophysics↗

bioRxiv2021

Refining conformational ensembles of flexible proteins against small-angle X-ray scattering data

Intrinsically disordered proteins and flexible regions in multi-domain proteins display substantial conformational heterogeneity. Characterizing the conformational ensembles of these proteins in solution typically requires combining one or more biophysical techniques with computational modelling or simulations. Experimental data can either be used to assess the accuracy of a computational model or to refine the computational model to get a better agreement with the experimental data. In both cases, one generally needs a so-called forward model, i.e. an algorithm to calculate experimental observables from individual conformations or ensembles. In many cases, this involve one or more parameters that need to be set, and it is not always trivial to determine the optimal values or to understand the impact on the choice of parameters. For example, in the case of small-angle X-ray scattering (SAXS) experiments, many forward models include parameters that describe the contribution of the hydration layer and displaced solvent to the background-subtracted experimental data. Often, one also needs to fit a scale factor and a constant background for the SAXS data, but across the entire ensemble. Here, we present a protocol to dissect the effect of free-parameters on the calculated SAXS intensities, and to identify a reliable set of values. We have implemented this procedure in our Bayesian/Maximum Entropy framework for ensemble refinement, and demonstrate the results on four intrinsically disordered proteins and a three-domain protein connected by flexible linkers. Our results show that the resulting ensembles can depend on the parameters used for solvent effects, and suggests that these should be chosen carefully. We also find a set of parameters that work robustly across all proteins. SIGNIFICANCEThe flexibility of a protein is often key to its biological function, yet understanding and characterizing its conformational heterogeneity is difficult. We here describe a robust protocol for combining small-angle X-ray scattering experiments with computational modelling to obtain a conformational ensemble. In particular, we focus on the contribution of protein hydration to the experiments and how this is included in modelling the data. Our resulting algorithm and software should make modelling intrinsically disordered proteins and multi-domain proteins more robust, thus aiding in understanding the relationship between protein dynamics and biological function.

biophysics↗