Biology subjects

Mashaghi, A.

Publications and source records attributed to Mashaghi, A..

11 recordsLinked to original sources

bioRxiv2026

Topological Investigation of Protein Folding and Intrinsic Disorder

Mapping protein conformations into a space of fold topologies offers an unprecedented perspective on the long-standing protein folding problem. In this study, we apply circuit topology to investigate the folding landscape of both stably folded and intrinsically disordered proteins. This topological approach quantifies intra-chain contact arrangements within a polypeptide chain. We demonstrate that ordered and disordered proteins can be distinguished by their topological organization, and that a topology-based model can predict chain compaction and folding state. Furthermore, topology relates to folding and unfolding kinetics and thermodynamics. These findings establish topology as a fundamental concept for understanding protein folding and disorder.

biophysics↗

bioRxiv2025

Modulation of Nur77-DNA Interactions by the Glucocorticoid Receptor

Nuclear receptors (NRs) comprise a superfamily of (ligand-)regulated transcription factors that are pivotal in orchestrating gene networks essential for development, metabolism, and cellular homeostasis. Their activity is critical for normal physiology, and consequently, dysregulation of NR signaling is implicated in a wide array of human diseases. Within this superfamily, the orphan nuclear receptor Nur77 and the glucocorticoid receptor (GR) are key regulators that exhibit significant crosstalk, primarily antagonistic, which is crucial for modulating inflammatory and stress responses. Despite the recognized importance of their interplay, the precise molecular mechanisms by which GR modulates Nur77s engagement with DNA remain incompletely defined. This study elucidates the direct impact of GR and its ligand, dexamethasone (Dex), on the DNA binding dynamics of Nur77. Single-molecule DNA tightrope assays revealed that Nur77 employs a three-dimensional diffusion-based search mechanism on non-specific DNA, characterized by transient interactions with two distinct dissociation kinetic profiles. GR significantly stabilizes Nur77-DNA interactions, evidenced by a shift towards longer residence times, primarily achieved by slowing the dissociation of the more transiently interacting Nur77 population. Conversely, single-molecule analysis and biochemical assays demonstrated that Dex alone markedly reduces Nur77s overall DNA binding affinity kinetics and frequency in a sequence-dependent manner, to such an extent that accurate quantification was unfeasible. These findings delineate distinct modulatory effects of the GR protein and its ligand on Nur77-DNA interactions, providing crucial biophysical insights into their complex regulatory interplay and revealing a direct, GR-independent impact of dexamethasone on Nur77s DNA engagement. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/685035v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@1207cdorg.highwire.dtl.DTLVardef@1be29eborg.highwire.dtl.DTLVardef@1b23eaaorg.highwire.dtl.DTLVardef@13030b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

PyMOL plugin for Protein Circuit Topology

Circuit Topology (CT) provides a fundamental framework for analysing folded polymer chains, with applications in functional annotation, protein engineering and drug development. We present a protein CT analysis plugin for PyMOL v3.1.6.1 with a graphical user interface (GUI), automatic installation, and novel features developed through integration with PyMOL's application programming interface (API). The plugin integrates various previously developed CT methodologies for studying structured proteins and their complexes as well as the dynamics of disordered proteins. Analysis of a representative protein and a molecular dynamics trajectory demonstrates the plugin's three analysis modes and their outputs. The plugin reproduces the reference ProteinCT implementation exactly on the structures tested, and is distributed with a versioned release, a pinned environment and a one-command reproduction of every result reported here.

bioinformatics↗

bioRxiv2025

Optically driven control of mechanochemistry and fusion dynamics of biomolecular condensates via thymine dimerization

Phase-separated biomolecular condensates serve as functional elements of biological cells, contribute to protocell formation in prebiotic systems during early life, and represent a distinct class of soft matter with a broad range of potential applications. Understanding and controlling condensate mechanochemistry is critical for their function and material properties. Photochemical processes, such as UV-induced chemical modifications, are ubiquitous in nature and can have both detrimental and constructive impacts on living systems, and are also readily implemented in engineering applications. However, how phase-separated condensate formation influences photochemical processes, and conversely, how photochemical reactions impact condensate dynamics, remains an open question. Combining scanning probe microscopy with optical imaging and control, we developed assays that enable the study of mechanical transitions and fusion dynamics in condensate droplets, revealing that UV-induced thymine dimerization alters condensate nucleation and coalescence. Depending on the frequency and topological arrangement of thymine dimers, particularly the balance between inter- and intrachain crosslinks, UV can induce a transition from liquid-like to solid-like behaviours or lead to aggregate formation. UV treatment also leads to compartmentalization in condensate systems by e.g., promoting the formation of arrested fusion droplets, which are stable against environmental changes. UV illumination can thus be leveraged to program the architecture and material properties of DNA-based biomolecular condensates, with implications for prebiotic chemistry, and bio-inspired engineering.

biophysics↗

bioRxiv2025

Synthesis and Characterization of Phase-Separated Extracellular Condensates in Interactions with Cells

Biomolecular condensates formed through liquid-liquid phase separation play key roles in intracellular organization and signaling, yet their function in extracellular environments remains largely unexplored. Here, we establish a model using heparan sulfate, a key component of the extracellular matrix, to study extracellular condensate-cell interactions. We demonstrate that heparan sulfate can form condensates with a positively charged counterpart in serum-containing solutions, mimicking the complexity of extracellular fluid, and supporting cell viability. We observe that these condensates adhere to cell membranes and remain stable, enabling a versatile platform for examining extracellular condensate dynamics and quantifying their rheological properties as well as their adhesion forces with cellular surfaces. Our findings and methodology open new avenues for understanding the organizational roles of condensates beyond cellular boundaries.

biophysics↗

bioRxiv2024

Polyglutamine expansion induced dynamic misfolding of Androgen Receptor

Spinal bulbar muscular atrophy (SBMA) is caused by a polyglutamine expansion (pQe) in the N-terminal transactivation domain of human androgen receptor (AR-NTD), resulting in a combination of toxic gain- and loss-of-function mechanisms. The structural basis of these processes has not been resolved due to the disordered nature of the NTD, which hinders experimental analyses of its detailed conformations. Here, using extensive computational modelling, we show that AR-NTD forms dynamic compact regions, which upon pQe re-organize dynamically, mediated partly by direct pQ interaction with the Androgen N-Terminal Signature (ANTS) motif. The altered dynamics of the NTD result in a perturbation of interdomain interactions, with potential implications for binding of the receptor protein to its response element. Oligomeric aggregation of the dynamic misfolded NTD exposes pQe, but blocks tau-5 and the FQNLF motif, which could lead to aberrant receptor transcriptional activity. These observations suggest a structural mechanism for AR dysfunction in SBMA.

pathology↗

bioRxiv2024

Rapid prediction of thermodynamically destabilizing tyrosine phosphorylations in cancers

Tyrosine phosphorylations are a prominent characteristic of numerous cancers, necessitating the use of computational tools to comprehensively analyze phosphoproteomes and identify potentially (dys)functional phosphorylations. Here we propose a machine learning-based method to predict the thermodynamic stability change resulting from tyrosine phosphorylation. Our approach, based on prediction of phosphomimetic delta-delta-G from structural features, strongly correlates with experimental mutational scanning cDNA proteolysis data (R = 0.71). We predicted the destabilizing effects of all 384,857 tyrosine residues from the Alphafold2 database. We then applied our approach to a pan-cancer phosphoproteomics dataset, comprising over 600 unique tyrosine phosphorylations across 11 cancer subtypes. We predict destabilizing phosphorylations in both oncogenes and tumor suppressors, where the former likely reflects a generalized relief of auto-inhibition or activating conformational change. We find that the number of circuit topological parallel relations with respect to residues contacting the phosphorylated site is greater for autoinhibited oncogenes than for other proteins (Wilcoxon p = 0.03). Utilizing an extreme gradient-boosting machine learning approach, we obtain an AUC of 0.85 for the prediction of autoinhibited phosphorylation states from circuit topological features. The top destabilized proteins from the pan-cancer data are enriched for chemical and oxidative stress pathways. Among metabolic proteins, highly destabilizing phosphorylations tend to occur in more peripheral proteins with lower network centrality measures (Wilcoxon p = 0.005). We predict 58% of recurrent tyrosine cancer phosphorylations to be destabilizing at the 1 kcal/mol threshold. Our approach can enable rapid screening of destabilizing phosphorylations and phosphomimetic mutations.

systems biology↗

bioRxiv2024

Phosphorylation regulated conformational diversity and topological dynamics of an intrinsically disordered nuclear receptor

Site-specific phosphorylation of disordered proteins is often considered as a marker of protein activity, yet it is unclear how phosphorylation alters conformational dynamics of disordered protein chains, such as those in the nuclear receptor superfamily. In the case of disordered human glucocorticoid receptor N-terminal domain (GR NTD), a negatively charged region known as core activation function 1 (AF1c) features three phosphorylation sites, regulating its function and intracellular localization. Deletion of this sequence reduces GR transcriptional activation ability dramatically in cell experiments. By developing a circuit topology-based fold analysis approach, combined with atomistic simulations, we reveal that site-specific phosphorylation facilitates formation of non-local contacts, leading to the emergence of disordered compact topologies with significant entanglement, which are distinct from solvent exposed topologies. While we observe that the topological buildup of solvent-exposed states is similar in different phosphovariants, it depends on the exact phosphorylation site for the disordered compact states. This study thus reveals the complex regulatory role of the GR phosphorylation and introduces a unique analysis framework that can be broadly applied to studying topological dynamics of disordered proteins.

biophysics↗

bioRxiv2024

Scanning probe microscopy elucidates gelation and rejuvenation of biomolecular condensates

Comprehensive understanding of dynamics and disease-associated solidification of biomolecular condensates is closely tied to analysis of their mechanical characteristics. Despite recent technical advances in rheological studies of condensates, these still vastly rely on methods restricted to small forces, rendering measurements of droplets with higher elasticities and after transition to solid challenging. Here, we develop assays for in-depth mechanical characterization of biomolecular condensates by scanning probe microscopy. We demonstrate this technique by measuring the rheological behavior of heterotypic poly-L-lysine heparin condensates, showcasing their multi-route liquid to gel transition, as well as their rejuvenation by chemical alterations to the medium. Due to the wide-spread application of scanning probe microscopy in biological fields, its capability for rapid, high throughput, high force range studies, and integration with nanoscale morphological measurements, our probe-based method is a significant breakthrough in investigating condensate behavior, leading to accelerated development of therapies.

biophysics↗

bioRxiv2021

Single cell micro-pillar-based characterization of endothelial and fibroblast cell mechanics

Mechanotransduction, the ability of cells to sense and respond to the mechanical cues from their microenvironment, plays an important role in numerous cellular processes, ranging from cell migration to differentiation. Several techniques have been developed to investigate the underlying mechanisms of mechanotransduction, in particular, force measurement-based techniques. However, we still lack basic single cell quantitative comparison on the mechanical properties of commonly used cell types, such as endothelial and fibroblast cells. Such information is critical to provide a precedent for studying complex tissues and organs that consist of various cell types. In this short communication, we report on the mechanical characterization of the commonly used endothelial and fibroblast cells at the single cell level. Using a micropillar-based assay, we measured the traction force profiles of these cells. Our study showcases differences between the two cell types in their traction force distribution and morphology. The results reported can be used as a reference and to lay the groundwork for future analysis of numerous disease models involving these cells.

biophysics↗

bioRxiv2021

Direct observation of Hsp90-induced compaction in a protein chain

The chaperone Hsp90 is well known to undergo important conformational changes, which depend on nucleotide, co-chaperones, substrate interactions and post-translational modifications. Conversely, how the conformations of its unstable and disordered substrates are affected by Hsp90 is difficult to address experimentally, yet central to its function. Here, using optical tweezers and luciferase and glucocorticoid receptor substrates, we find that Hsp90 promotes local contractions in unfolded chains that drive their global compaction down to dimensions of folded states. This compaction has a gradual nature while showing small steps, is stimulated by ATP, and performs mechanical work against counteracting forces that expand the chain dimensions. The Hsp90 interactions suppress the formation of larger-scale folded, misfolded and aggregated structures. The observations support a model in which Hsp90 alters client conformations directly by promoting local intra-chain interactions while suppressing distant ones. We conjecture that chain compaction may be central to how Hsp90 protects unstable kinases and receptor clients, regulates their activity, and how Hsp90 cooperates with Hsp70.

biophysics↗