Biology subjects

Collepardo-Guevara, R.

Publications and source records attributed to Collepardo-Guevara, R..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Analysis of long-range contacts across cell types outlines a core sequence determinant of 3D genome organisation

The sequence-driven organising principles of the 3D genome are crucial for interpreting the core effects of genomic variation and for understanding the evolution of genome organisation and function. We investigated these by isolating and analysing cell-type-persistent contacts, heavily dependent on the similarly cell-type-persistent genomic sequence. We stratified long-range contacts from a diverse group of human tissues and cell lines based on contact persistence, cp, reflecting their presence across cell or tissue types, presenting them as an atlas of contacts and the cell-type invariant (CETI) hubs they form across human chromosomes. Our survey of more than 300 chromatin and genome features revealed their association with cp, contrasting variable from persistent contacts in terms of co-localisation with genes, 3D architectural domains, epigenetic and sequence elements. We found persistent contacts to be predominantly comprised of AT-rich sequences and related to heterochromatin. A key outcome is finding a link between the experimental genomic contacts and the complementarity between pairs of contacting DNA loci. This work provides evidence for a sequence determinant of genomic contacts contributing to the decoding of the relationship between sequence and structure that is crucial for functional and evolutionary studies concerning the 3D genome organisation.

genomics↗

bioRxiv2025

Molecular insights on the mechanism of α1-antitrypsin condensate formation and maturation

The deficiency of 1-antitrypsin protein is a genetic disorder characterized by the accumulation of misfolded protein aggregates within hepatocytes, leading to liver dysfunction. In the lung, it is found in macrophages, bronchial and epithelial alveolar cells type 2, leading to pulmonary emphysema. Despite extensive research, the precise mechanism underlying the formation of 1-antitrypsin inclusion bodies remain elusive. In this study, we combine equilibrium and non-equilibrium molecular dynamics simulations to elucidate the intricate process of 1-antitrypsin condensate formation and maturation. Our mechanistic model explains cluster accumulation--specifically the onset of this pathogenesis--through the emergence of phase-separated liquid-like protein droplets, which subsequently undergo inter-protein {beta}-sheet transitions between misfolded variants, resulting in solid-like clusters. We find that this mechanism only applies to the misfolded variant, Z-1-antitrypsin, which phase-separates driven by its disordered C-terminus. In contrast, the native protein, M-1-antitrypsin, shows much lower propensity to phase-separate and later form kinetically trapped aggregates. Furthermore, we explore how Z-1-antitrypsin exhibits an increased capacity to form condensates near external walls with different types of interactions. Such conditions can be similar to those found within the endoplasmic reticulum membrane, where phase separation and hardening take place. Overall, our results shed light on the molecular basis of 1-antitrypsin-related disorders and provide valuable microscopic insights for the development of therapeutic strategies targeting protein misfolding and aggregation-related disorders. Author summaryAlpha-1 antitrypsin deficiency is a genetic disorder that can cause liver and lung disease. It occurs when the protein 1-antitrypsin folds incorrectly and accumulates in harmful clumps inside cells. Exactly how these clumps form has been unclear. In this study, we used computer simulations to reveal the steps behind this process. We found that the faulty version of the protein first collects into liquid-like droplets that later harden into solid clusters. This behavior is driven by a flexible tail region of the misfolded protein. In contrast, the normal version is far less likely to clump. Our findings provide new insight into disease onset and may guide the development of therapies.

biophysics↗

bioRxiv2025

Linker histone H1 functions as a liquid-like glue to organize chromatin in living human cells

Linker histone H1, the most abundant chromatin protein, condenses chromatin, modulates DNA transactions such as transcription and DNA replication/repair, and participates in differentiation, development and tumorigenesis. While recent studies indicate that nucleosomes are clustered as condensed chromatin domains in higher eukaryotic cells, how histone H1 mechanically condenses chromatin remains unclear. Here, using a combination of direct visualization of single-H1 molecules in living human cells and multiscale molecular dynamics simulations, we demonstrate that the majority of H1 behaves like a liquid inside chromatin domains, rather than binding stably to nucleosomes as suggested by the traditional model. H1 functions as a liquid-like "glue", mediating dynamic multivalent electrostatic interactions between nucleosomes within chromatin domains. Consistently, rapid-depletion of H1.2 leads to decondensed chromatin domains both in cells and in silico. Our findings suggest that the H1 "glue" condenses chromatin domains while keeping them fluid and accessible, thereby supporting essential DNA transactions.

cell biology↗

bioRxiv2025

Compositional control of ageing kineticsin TDP-43 condensates

Biomolecular compartments orchestrate the spatiotemporal organisation of cells. The spontaneous assembly of proteins and nucleic acids through liquid-liquid phase separation into biomolecular condensates has been shown to ubiquitously contribute to the functional compartmentalisation of the cytoplasm and nucleoplasm. However, some condensates can undergo an additional phase transition from functional liquid states to pathological solid-like assemblies (i.e., ageing). This liquid-to-solid transition, driven by the accumulation of protein cross-{beta}-sheet structures, represents a hallmark of multiple neurodegenerative disorders. In this study, we employ Molecular Dynamics simulations to explore the role of various biomolecules in regulating the ageing kinetics of condensates scaffolded by TDP-43, a key RNA-binding protein linked to amyotrophic lateral sclerosis and frontotemporal dementia. We find that the recruitment of arginine-rich peptides, such as those produced by the C9orf72 gene, accelerates the nucleation of cross-{beta}-sheet structures. In contrast, the inclusion of poly-Uridine RNA and the HSP70 chaperone significantly slows the emergence of these structures. Remarkably, we observe a correlation between the compactness of the low-complexity domain of TDP-43-- which drives the transition to cross-{beta}-sheet structures--and the condensate ageing kinetics as we vary the composition of the condensates. Moreover, we find that near-interfacial regions of TDP-43 condensates exhibit faster {beta}-sheet transitions than the bulk core of the condensate. Together, our findings underscore the critical role of client biomolecules in modulating protein conformational ensembles and intermolecular interactions, thereby controlling the propensity of condensates to transition into harmful solid-like states.

biophysics↗

bioRxiv2025

Cold-induced nucleosome dynamics linked to silencing of Arabidopsis FLC

Temperature influences nucleosome dynamics, and thus chromatin, to regulate gene expression. Such mechanisms underlie the epigenetic silencing of Arabidopsis FLOWERING LOCUS C (FLC) by prolonged cold. Here, we show a temperature-dependent transition in local chromatin structure at the H3K27me3 nucleation region, from a modality active for transcription to a state that can be Polycomb silenced. In vivo chromatin measurements and coarse-grained simulations at near-atomistic resolution show the active transcription state is characterized by a highly dynamic nucleosome arrangement that exposes the FLC TSS. Cold exposure then changes the chromatin structure by reducing nucleosome dynamics and re-positioning the +1 nucleosome, leading to transcriptional repression. This local chromatin transition partially depends on VRN1, a non-sequence-specific DNA-binding protein. Loss of VRN1 results in hyperaccumulation of H2A.Z, more dynamic nucleosomes and an inability to accumulate H2Aub and H3K27me3. Our work highlights how local nucleosome dynamics link to chromatin structure transitions to integrate temperature inputs into epigenetic switching mechanisms in plants.

molecular biology↗

bioRxiv2025

Multi-scale structure of chromatin condensates rationalizes phase separation and material properties

Biomolecular condensates, compartments that concentrate molecules without surrounding membranes, are integral to numerous cellular processes. The structure and interaction networks of molecules within condensates remain poorly understood. Using cryo-electron tomography and molecular dynamics simulations we elucidated the structure of phase separated chromatin condensates across scales, from individual amino acids to network architecture. We found that internucleosomal DNA linker length controls nucleosome arrangement and histone tail interactions, shaping the structure of individual chromatin molecules both within and outside condensates. This structural modulation determines the balance between intra- and intermolecular interactions, which in turn governs the molecular network, thermodynamic stability, and material properties of chromatin condensates. Mammalian nuclei contain dense clusters of nucleosomes whose non-random organization is mirrored by the reconstituted condensates. Our work explains how the structure of individual chromatin molecules ultimately determines physical properties of chromatin condensates, with implications for cellular chromatin organization.

biophysics↗

bioRxiv2024

Nucleosome Spacing Can Fine-Tune Higher Order Chromatin Assembly

Cellular chromatin displays heterogeneous structure and dynamics, properties that control diverse nuclear processes. Models invoke phase separation of conformational ensembles of chromatin fibers as a mechanism regulating chromatin organization in vivo. Here we combine biochemistry and molecular dynamics simulations to examine, at single base-pair resolution, how nucleosome spacing controls chromatin phase separation. We show that as DNA linkers extend from 25 bp to 30 bp, as examplars of 10N+5 and 10N (integer N) bp lengths, chromatin condensates become less thermodynamically stable and nucleosome mobility increases. Simulations reveal that this is due to trade-offs between inter- and intramolecular nucleosome stacking, favored by rigid 10N+5 and 10N bp linkers, respectively. A remodeler can induce or inhibit phase separation by moving nucleosomes, changing the balance between intra- and intermolecular stacking. The intrinsic phase separation capacity of chromatin enables fine tuning of compaction and dynamics, likely contributing to heterogeneous chromatin organization in vivo.

biophysics↗

bioRxiv2024

Charged peptides enriched in aromatic residues decelerate condensate ageing driven by cross-beta-sheet formation

Biomolecular condensates, formed through liquid-liquid phase separation, play wide-ranging roles in cellular compartmentalization and biological processes. However, their transition from a functional liquid-like phase into a solid-like state--usually termed as condensate ageing--represents a hallmark associated with the onset of multiple neurodegenerative diseases. In this study, we design a computational pipeline to explore potential candidates, in the form of small peptides, to regulate ageing kinetics in biomolecular condensates. By combining equilibrium and non-equilibrium simulations of a sequence-dependent residue-resolution force field, we investigate the impact of peptide insertion--with different composition, patterning, and net charge--in the condensate phase diagram and ageing kinetics of archetypal proteins driving condensate ageing: TDP-43 and FUS. We reveal that small peptides composed of a specific balance of aromatic and charged residues can substantially decelerate ageing over an order of magnitude. The mechanism is controlled through condensate density reduction induced by peptide self-repulsive electrostatic interactions that specifically target protein regions prone to form cross-{beta}-sheet fibrils. Our work proposes an efficient computational framework to rapidly scan the impact of small molecule insertion in condensate ageing and develop novel pathways for controlling phase transitions relevant to disease prevention.

biophysics↗

bioRxiv2024

Dynamically arrested condensate fusion creates complex structures with varying material properties

The cell nucleus and cytosol contain numerous biomolecular condensates which dynamically reshape, fuse and split to accomplish precise compartmentalization of the cell material. While it has been observed that some condensates rapidly coalesce, some others only attach to each other, or do not establish persistent interactions over time. Here, we explain these observations through optical tweezers and Molecular Dynamics simulations focusing on two condensate-forming, RNA-binding proteins--FUS and G3BP1--strongly involved in RNA metabolism and stress responses. We find that the fusion of pure droplets formed by these proteins can give rise to multiphase single-component condensates exhibiting notably different densities, architectures, and material properties. Such behaviour is dictated by the relative timescales of condensate fusion and protein internal mixing. A critical parameter controlling this interplay is the extent of ageing that condensates display; e.g., their progressive hardening driven by the accumulation of inter-protein {beta}-sheet assemblies over time. Strikingly, different degrees of ageing in fusing droplets can lead single-component condensates to form diverse architectures including concentric drops or two-sided condensates. Overall, our results highlight a mechanism, based on the temporal coupling between ageing, fusion, and mixing rate, by which biomolecular condensates form multiphasic structures with markedly different material properties, and hence potentially distinct biological roles.

biophysics↗

bioRxiv2024

Capturing single-molecule properties does not ensure accurate prediction of biomolecular phase diagrams

Intracellular liquid-liquid phase separation of proteins and nucleic acids represents a fundamental mechanism by which cells organise their components into biomolecular condensates that perform multiple biological tasks. Computer simulations provide powerful tools to investigate biomolecular phase separation, offering microscopic insights into the physicochemical principles that regulate these systems. In this study, we investigate the phase behaviour of the low-complexity domain (LCD) of hnRNPA1 and several mutants via Molecular Dynamics simulations. We systematically compare the performance of five state-of-the-art residue-resolution coarse-grained protein models: HPS, HPS-cation-{pi}, CALVADOS2, Mpipi, and Mpipi-Recharged. Our evaluation focuses on how well these models reproduce experimental coexistence densities and single-protein radii of gyration for the LCD-hnRNPA1 set of mutants. While most models yield similar intramolecular contact maps and reasonable estimates of the single-protein radius of gyration compared to in vitro measurements, only Mpipi-Recharged, Mpipi, and CALVADOS2 accurately predict phase diagrams that align with experimental data. This suggests that force field parameterizations optimized solely to reproduce single-protein properties may not always capture the phase behaviour of protein solutions. Additionally, our findings reveal that some residue-resolution coarse-grained models can lead to significant discrepancies in predicting the roles of individual intermolecular interactions, even for relatively simple intrinsically disordered proteins like the low-complexity domain of hnRNPA1. Our work highlights the importance of balancing both single-molecule and collective properties of proteins to accurately predict condensate formation and material properties.

biophysics↗

bioRxiv2024

Phase behaviour of hnRNPA1 low-complexity domain mutants described by different sequence-dependent models

Intracellular liquid-liquid phase separation (LLPS) of proteins and nucleic acids is a fundamental mechanism by which cells compartmentalize their components and perform essential biological functions. Molecular simulations play a crucial role in providing microscopic insights into the physicochemical processes driving this phenomenon. In this study, we systematically compare six state-of-the-art sequence-dependent, residue-resolution models to evaluate their performance in reproducing the phase behaviour and material properties of condensates formed by seven variants of the low-complexity domain (LCD) of the hnRNPA1 protein (A1-LCD)--a protein implicated in the pathological liquid-to-solid transition of stress granules. Specifically, we assess the HPS, HPS-cation-{pi}, HPS-Urry, CALVADOS2, Mpipi, and Mpipi-Recharged models in their predictions of the condensate saturation concentration, critical solution temperature, and condensate viscosity for the A1-LCD variants. Our analyses demonstrate that, among the tested models, Mpipi, Mpipi-Recharged, and CALVADOS2 provide accurate descriptions of the critical solution temperatures and saturation concentrations for the various A1-LCD variants tested. Regarding the prediction of material properties for condensates of A1-LCD and its variants, Mpipi-Recharged stands out as the most reliable model. Overall, this study benchmarks a range of residue-resolution coarse-grained models for the study of the thermodynamic stability and material properties of condensates and establishes a direct link between their performance and the ranking of intermolecular interactions these models consider.

biophysics↗

bioRxiv2024

Chemically-informed coarse-graining of electrostatic forces in charge-richbiomolecular condensates

Biomolecular condensates composed of highly charged biomolecules like DNA, RNA, chromatin, and nucleic-acid binding proteins are ubiquitous in the cell nucleus. The biophysical properties of these charge-rich condensates are largely regulated by electrostatic interactions. Residue-resolution coarse-grained models that describe solvent and ions implicitly are widely used to gain mechanistic insights into the biophysical properties of condensates, offering transferability, computational efficiency, and accurate predictions for many systems. However, their predictive accuracy diminishes for charge-rich condensates due to the implicit treatment of solvent and ions. Here, we present the Mpipi-Recharged model, a residue-resolution coarse-grained model that improves the description of charge effects in biomolecular condensates containing disordered proteins, multi-domain proteins, and/or disordered RNAs. Mpipi-Recharged maintains the computational efficiency of its predecessor--the Mpipi model--by still treating solvent and ions implicitly, but improves its accuracy by incorporating a pair-specific asymmetric electrostatic potential informed by atomistic simulations in explicit solvent and ions. We show that such asymmetric coarse-graining of electrostatic forces is needed to recapitulate the stronger mean-field impact of associative interactions between opposite-charge pairs over the repulsion among equally charged pairs revealed by our atomistic simulations. Mpipi-Recharged shows excellent agreement with the experimental phase behavior of highly charged systems, capturing subtle effects challenging to model without explicit solvation, such as the impact of charge blockiness, stoichiometry changes, and salt concentration variation. By offering improved predictions for charge-rich biomolecular condensates, Mpipi-Recharged extends the computational tools available to investigate the physicochemical mechanisms regulating biomolecular condensates.

biophysics↗

bioRxiv2024

Tandem-repeat proteins introduce tuneable properties to engineered biomolecular condensates

The cells ability to rapidly partition biomolecules into biomolecular condensates is linked to a diverse range of cellular functions. Understanding how the structural attributes of biomolecular condensates are linked with their biological roles can be facilitated by the development synthetic condensate systems that can be manipulated in a controllable and predictable way. Here, we design and characterise a tuneable synthetic biomolecular condensate platform fusing modular consensus-designed tetratricopeptide repeat (CTPR) proteins to intrinsically-disordered domains. Trends between the CTPR structural attributes and condensate propensity were recapitulated across different experimental conditions and by in silico modelling, demonstrating that the CTPR domain can systematically affect the condensates in a predictable manner. Moreover, we show that incorporating short binding motifs into the CTPR domain results in specific target-protein recruitment into the condensates. Our model system can be rationally designed in a versatile manner to both tune condensate propensity and endow the condensates with new functions.

biophysics↗

bioRxiv2023

Mechano-dependent sorbitol accumulation supports biomolecular condensate

Biomolecular condensates regulate a wide range of cellular functions from signaling to RNA metabolism1, 2, yet, the physiologic conditions regulating their formation remain largely unexplored. Biomolecular condensate assembly is tightly regulated by the intracellular environment. Changes in the chemical or physical conditions inside cells can stimulate or inhibit condensate formation3-5. However, whether and how the external environment of cells can also regulate biomolecular condensation remain poorly understood. Increasing our understanding of these mechanisms is paramount as failure to control condensate formation and dynamics can lead to many diseases6, 7. Here, we provide evidence that matrix stiffening promotes biomolecular condensation in vivo. We demonstrate that the extracellular matrix links mechanical cues with the control of glucose metabolism to sorbitol. In turn, sorbitol acts as a natural crowding agent to promote biomolecular condensation. Using in silico simulations and in vitro assays, we establish that variations in the physiological range of sorbitol, but not glucose, concentrations, are sufficient to regulate biomolecular condensates. Accordingly, pharmacologic and genetic manipulation of intracellular sorbitol concentration modulates biomolecular condensates in breast cancer - a mechano-dependent disease. We propose that sorbitol is a mechanosensitive metabolite enabling protein condensation to control mechano-regulated cellular functions. Altogether, we uncover molecular driving forces underlying protein phase transition and provide critical insights to understand the biological function and dysfunction of protein phase separation.

cell biology↗

bioRxiv2023

Universal predictive scaling laws of phase separation of prion-like low complexity domains

Proteins containing prion-like low complexity domains (PLDs) are common drivers of the formation of biomolecular condensates and are prone to misregulation due to amino acid mutations. Here, we exploit the accuracy of our residue-resolution coarse-grained model, Mpipi, to quantify the impact of amino acid mutations on the stability of 140 PLD mutants from six proteins (hnRNPA1, TDP43, FUS, EWSR1, RBM14, and TIA1). Our simulations reveal the existence of scaling laws that quantify the range of change in the critical solution temperature of PLDs as a function of the number and type of amino acid sequence mutations. These rules are consistent with the physicochemical properties of the mutations and extend across the entire family tested, suggesting that scaling laws can be used as tools to predict changes in the stability of PLD condensates. Our work offers a quantitative lens into how the emergent behavior of PLD solutions varies in response to physicochemical changes of single PLD molecules.

biophysics↗

bioRxiv2023

Energy landscapes and heat capacity signatures for peptides correlate with phase separation propensity

Phase separation plays an important role in the formation of membraneless compartments within the cell, and intrinsically disordered proteins with low-complexity sequences can drive this compartmentalisation. Various intermolecular forces, such as aromatic-aromatic and cation-aromatic interactions, promote phase separation. However, little is known about how the ability of proteins to phase separate under physiological conditions is encoded in their energy landscapes, and this is the focus of the present investigation. Our results provide a first glimpse into how the energy landscapes of minimal peptides that contain{pi} -{pi} and cation-{pi} interactions differ from the peptides that lack amino acids with such interactions. The peaks in the heat capacity (CV) as a function of temperature report on alternative low-lying conformations that differ significantly in terms of their enthalpic and entropic contributions. The CV analysis and subsequent quantification of frustration of the energy landscape suggest that the interactions that promote phase separation leads to features (peaks or inflection points) at low temperatures in CV, more features may occur for peptides containing residues with better phase separation propensity and the energy landscape is more frustrated for such peptides. Overall, this work links the features in the underlying single-molecule potential energy landscapes to their collective phase separation behaviour, and identifies quantities (CV and frustration metric) that can be utilised in soft material design.

biophysics↗

bioRxiv2023

Aromatic and arginine content drives multiphasic condensation of protein-RNA mixtures

Multiphasic architectures are found ubiquitously in biomolecular condensates and are thought to have important implications for the organisation of multiple chemical reactions within the same compartment. Many of these multiphasic condensates contain RNA in addition to proteins. Here, we investigate the importance of different interactions in multiphasic condensates comprising two different proteins and RNA using computer simulations with a residue-resolution coarse-grained model of proteins and RNA. We find that in multilayered condensates containing RNA in both phases, protein-RNA interactions dominate, with aromatic residues and arginine forming the key stabilising interactions. The total aromatic and arginine content of the two proteins must be appreciably different for distinct phases to form, and we show that this difference increases as the system is driven towards greater multiphasicity. Using the trends observed in the different interaction energies of this system, we demonstrate that we can also construct multilayered condensates with RNA preferentially concentrated in one phase. The rules identified can thus enable the design of synthetic multiphasic condensates to facilitate further study of their organisation and function.

biophysics↗

bioRxiv2022

Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation

PRC1 (Polycomb repressive complex 1) plays a significant role in cellular differentiation and development by repressing lineage-inappropriate genes. PRC1 proteins phase separate to form Polycomb condensates (bodies) that are multi-component hubs for silencing Polycomb target genes; however, the molecular principles that underpin the condensate assembly and biophysical properties remain unknown. Here, by using biochemical reconstitution, cellular imaging, and multiscale molecular simulations, we show that PRC1 condensates are assembled via a scaffold-client liquid-liquid phase separation (LLPS) model by which Chromobox 2 (CBX2) is the scaffold and other subunits of the CBX2-PRC1 complex act as clients. The clients induce a reentrant phase transition of CBX2 condensates in a concentration-dependent manner. The composition of the multi-component, heterotypic LLPS systems directs the assembly and biophysical properties of CBX2-PRC1 condensates and selectively promotes the formation of CBX4-PRC1 condensates, but specifically dissolves condensates of CBX6-, CBX7-, and CBX8-PRC1. Additionally, the composition of CBX2-PRC1 condensates controls the enrichment of CBX4-, CBX7-, and CBX8-PRC1 into condensates but the exclusion of CBX6-PRC1 from condensates. Our results show the composition- and stoichiometry-dependent scaffold-client assembly of multi-component PRC1 condensates and supply a conceptual framework underlying the molecular basis and dynamics of Polycomb condensate assembly.

biochemistry↗