Biology subjects

Agarwal, T.

Publications and source records attributed to Agarwal, T..

10 recordsLinked to original sources

bioRxiv2026

Opposing Regulation of Tau Condensation by Phosphorylation Levels and Molecular Chaperone Interactions

Aggregation of the microtubule-stabilizing protein Tau into insoluble deposits is characteristic for several neurodegenerative diseases, including Alzheimer's disease. While phosphorylated Tau (pTau) appears as a normal post-translational modification, the accumulation of hyperphosphorylated Tau aggregates represents a hallmark of these disorders. The molecular mechanisms involved in the conversion from functional Tau into pathological assemblies are still not elucidated. Here, we identified the role of phosphorylation levels on the phase separation and aggregation behavior of Tau, and how these processes can be counteracted by the molecular chaperone-like Bri2 BRICHOS domain. We found that the phosphorylation pattern modulates Tau fibrillation and enhances its droplet-formation propensity. Remarkably, Bri2 BRICHOS generally shifts Tau phase boundaries, counteracting the dense state, and inhibits Tau fibril formation, irrespectively of the degree of phosphorylation. Solution NMR results pinpointed the binding site of Bri2 BRICHOS on pTau, both in the dilute phase and droplet state, revealing an effect on specific regions and phosphorylated residues, which appear to be crucial for the formation and maintenance of the droplet state. Taken together, this study uncovered the opposing regulatory roles of phosphorylation and the anti-amyloid chaperone Bri2 BRICHOS on biomolecular condensation and fibrillation of Tau, providing detailed insights into the molecular mechanisms involved in tauopathies, which may facilitate future treatment strategies using chaperone-based inhibitors.

biophysics↗

bioRxiv2026

Genotypic Responses of Rice to Ammonium and Nitrate under Contrasting Water Regimes: Agronomic, Biochemical, and Gene-Expression Analyses

Considering the increasing water and labor constraints in rice production, direct-seeded rice is becoming increasingly important. However, direct-seeded rice is vulnerable to water stress and inefficient nutrient use. Nitrogen use efficiency in rice is generally low, and improving nitrogen acquisition and assimilation is important for sustainable productivity. In this study, 48 recombinant inbred lines were evaluated under ammonium and nitrate nutrition in irrigated and rainfed environments across two cropping seasons. Agronomic, physiological, biochemical and molecular responses were assessed. Significant effects of genotype, nitrogen form, environments and their interactions were observed for several traits. Rainfed conditions reduced grain and biological yield compared with irrigated conditions. Ammonium produced a higher mean grain yield than nitrate by approximately 26.2 g m-2 under irrigated conditions and 154.2 g m-2 under rainfed conditions, although the responses varied among genotypes. Biological yield and SPAD values were generally higher with ammonium, whereas total chlorophyll showed season and environment-dependent responses. Glutamine synthetase activity was highest under ammonium treatment, while glutamate synthase activity was higher under nitrate treatment. Grain yield showed strong positive correlations with biological yield, but correlations between grain yield and GS or GOGAT activity were not significant. Expression of OsGln1.2, OsGln2 and OsGlt1 varied according to genotype, tissue and nitrogen treatment. Overall, the results demonstrate that responses to nitrogen forms are genotype- and environment-dependent, and that ammonium may provide a yield advantage under rainfed conditions. Integrating field performance with biochemical and molecular traits may help identify rice genotypes suited to water-limited systems.

genomics↗

bioRxiv2026

Systematic identification of pH-sensing amyloid core motifs reveals a widespread mechanism for reversible protein assembly upon stress

Unlike irreversible pathological amyloids, reversible fibrils can be regulated via pH-sensing core motifs, characterized by amyloid properties unleashed upon stress-induced protonation of critical residues. Here, we combined bioinformatic predictions and an in vitro validation pipeline to search for pH-responsive, reversible amyloid core peptides in yeast and human proteomes. This approach uncovered biophysical properties distinguishing pH-sensing and constitutive amyloid cores and established reliable criteria to identify novel reversible assemblies based on sequence data. Selected full-length candidate proteins with evolutionarily conserved pH-sensing motifs were analyzed in Saccharomyces cerevisiae using fluorescence microscopy and SDS-resistance assays, revealing multiple proteins forming reversible assemblies in stationary phase. We found that protonation of a specific histidine in the amyloid core motif of the asparagine synthase Asn1 is necessary and sufficient for assembling catalytically inactive, reversible structures called cytoophidia. Interestingly, mutant cells that fail to assemble Asn1-cytoophidia show defects to recover from stationary phase, demonstrating functional relevance of pH-sensing amyloid core motifs in vivo. Taken together, we uncovered a widespread and conserved pH-sensing mechanism that regulates the reversible assembly and function of structurally diverse fibrils upon stress.

biochemistry↗

bioRxiv2026

Lipoengineering of Biomolecular Condensates Controls Material Properties and Multiphase Hierarchy to Guide Organoid Morphogenesis

Cells use post-translational modifications (PTMs) to reconfigure biomolecular condensates across length scales, space, and time.1,2 While charged PTMs are well-known electrostatic switches,3,4 how ubiquitous neutral PTMs shape condensate plasticity and hierarchy remains unclear. Here, we establish a set of design principles for using site-specific lipidation, a class of neutral hydrophobic PTMs, to rationally control properties and interactions of engineered biomolecular condensates. Through systematic analysis of over 80 lipidated synthetic intrinsically disordered proteins (IDPs), we uncovered two distinct axes of control. First, the interplay between the lipid and the local three-residue sequence of its attachment site acts as a programmable switch for cohesion--the homotypic interactions that define the material state of the condensed phase-- directing assemblies toward dynamic liquids, arrested gels, or ordered fibrillar solids. Second, the lipid, together with the global properties of the IDP scaffold, tunes adhesion--the heterotypic interactions that govern condensate miscibility and hierarchical organization. We harnessed these principles to rationally engineer complex, multi-phase architectures and create hybrid hydrogels with programmed microstructure and material properties that guide the morphogenesis of functional intestinal organoids. These findings establish a new framework for lipoengineering advanced biomaterials and provide a blueprint for dissecting structure-property relationships across diverse classes of PTMs.

bioengineering↗

bioRxiv2025

Mapping high resolution, multidimensional phase diagrams of physiological protein condensates

Biomolecular condensates are membraneless compartments, crucial for organising and regulating diverse cellular processes. Current approaches to study condensate biology either use simplified recombinant protein systems with limited physiological relevance, or complex live-cell models with restricted experimental control and scalability. Here, we present ExVivo PhaseScan, a droplet microfluidics platform that couples mammalian lysate-based reconstitution with scalable analysis to generate high-resolution phase diagrams of physiological protein condensates. We apply this approach to study two complex multicomponent condensate systems, stress granules and nucleoli, and dissect the physicochemical interactions that influence their stability. We further developed a machine learning pipeline to analyse condensate morphology which we use to reveal how mutations in the ALS-linked protein FUS remodels condensate phase landscapes. We identify liquid-to-solid transitions of mutant FUS within stress granules and nucleoli, and show that these transitions can be reversed by RNA aptamer-based interventions. Together, these findings establish ExVivo PhaseScan as a versatile tool for dissecting the physicochemical and pathological regulation of condensates, with potential to inform therapeutic strategies for diseases driven by aberrant phase transitions.

biophysics↗

bioRxiv2025

In vitro liquid-liquid phase separation induced by respiratory syncytial virus proteins and RNA

Respiratory syncytial virus (RSV) causes severe respiratory infections, with viral replication occurring in cytoplasmic membraneless viral factories formed by liquid-liquid phase separation (LLPS). The interactions between the RSV nucleoprotein N, phosphoprotein P, transcription factor M2-1, and RNA drive these condensates. Here we employed a microfluidic Phase Scan platform, together with biochemical and cellular assays to systematically characterise LLPS involving RSV proteins and RNA. We identified optimal stoichiometric ratios of N oligomers and P tetramers for condensate formation, demonstrated that monomeric N inhibits LLPS, and revealed that M2-1 enhances condensate formation by increasing multivalency. Notably, we discovered that M2-1 preferentially binds 5 capped RNA, distinguishing it from N, which binds uncapped RNA. These findings elucidate molecular determinants of RSV viral factory assembly and subcompartmentalisation, providing insights into viral replication mechanisms and informing potential antiviral strategies targeting LLPS processes.

microbiology↗

bioRxiv2025

The role of N-terminal acetylation on biomolecular condensation

N-terminal acetylation (Nt-acetylation) is one of the most prevalent co-translational modifications in eukaryotes, affecting nearly 80% of the human proteome. Despite its ubiquity, the potential impact of this phenomenon on biomolecular condensation has been largely overlooked. Here, we uncover how this chemically subtle modification can exert broad and multifaceted control over phase behaviour, using Grh1, a Golgi-associated protein involved in stress-induced secretion in yeast, as a model system. We show that Nt-acetylation increases the saturation concentration for condensation, reduces droplet size and number, dampens pH sensitivity, weakens electrostatic contributions, and suppresses water dipolar relaxation within condensates, indicating reduced internal hydration and environmental responsiveness. These effects are accompanied by acetylation-dependent dimerisation and local structural changes, including a concentration-dependent gain in -helicity. Remarkably, co- condensation assays reveal that acetylated and non-acetylated forms of the same protein are only partially miscible, giving rise to core-shell architectures driven by differences in interfacial tension. Together, our findings highlight Nt-acetylation as a potent, generalizable regulator of condensate material properties, linking primary sequence chemistry to mesoscale organisation. Given its evolutionary conservation and prevalence across eukaryotic proteomes, Nt-acetylation may represent a widespread mechanism for modulating protein condensation in health and disease.

biophysics↗

bioRxiv2025

Phosphorylation enables allosteric control of a viral condensate

In many viruses, intrinsically disordered proteins (IDPs) drive the formation of replicative organelles essential for viral production. In species A rotaviruses, the disordered protein NSP5 forms condensates in cells via liquid-liquid phase separation (LLPS). Yet the sequence diversity of NSP5 raises the question of whether condensate formation is conserved across all strains and if distinct variants employ alternative mechanisms for nucleating phase separation. Using a machine learning approach, we demonstrate that NSP5 variants differ significantly in their propensity to phase-separate. We engineered a variant incorporating amino acid signatures from strains with low LLPS tendency, which failed to phase separate in vitro yet supported the formation of replicative condensates in recombinant viruses in cells. Low-tendency LLPS strains require phosphorylation of NSP5 to nucleate phase separation, whereas high-tendency strains do not, suggesting distinct nucleation mechanisms. Furthermore, hydrogen-deuterium exchange mass spectrometry revealed a phosphorylation-driven allosteric switch between binding sites on the high-propensity variant. These findings establish that phosphorylation plays a context-dependent role in the formation of replicative organelles across diverse rotaviruses.

biophysics↗

bioRxiv2023

Genome-wide, Organ-delimited gene regulatory networks (OD-GRNs) provide high accuracy in candidate TF selection across diverse processes.

Construction of organ-specific gene expression datasets that include hundreds to thousands of experiments would greatly aid reconstruction of gene regulatory networks with organ-level spatial resolution. However, creating such datasets is greatly hampered by the requirements of extensive and tedious manual curation. Here we trained a supervised classification model that can accurately classify the organ-of-origin for a plant transcriptome. This K-Nearest Neighbor-based multiclass classifier was used to create organ-specific gene expression datasets for the leaf, root, shoot, flower, seed, seedling, silique, and stem in the model plant Arabidopsis thaliana. In the leaf, root, flower, seed and, a gene regulatory network (GRN) inference approach was used to determine: i. influential transcription factors (TFs) in that organ and, ii. the most influential TFs for specific biological processes in the organ. These genome-wide, organ-delimited GRNs (OD-GRNs), identified de novo many known regulators of organ development and processes operating in those organs. Moreover, many previously unknown TF regulators were highly ranked as potential master regulators of organ development or organ-specific processes. As a proof-of-concept, we focused on experimentally validating the predicted TF regulators of lipid biosynthesis in seeds, with relevance to food and biofuel production. Of the top twenty candidate TFs, eight (e.g., WRI1, LEC1, and FUS3) are known regulators of seed oil content. Importantly, we validated that seven more candidate TFs, whose role was previously unknown in seed lipid biosynthesis, indeed affect this process by genetics and physiological approaches, thus yielding a net accuracy rate of >75% for the de novo TF predictions. The general approach developed here could be extended to any species with sufficiently large gene expression datasets to speed up hypothesis generation and testing for constructing gene regulatory networks at a high spatial resolution. Significance StatementOur study develops a machine-learning framework for building extremely large gene expression datasets for each organ, and to infer organ-delimited gene regulatory networks. We show that this approach is very successful at predicting which transcription factors are going to regulate processes at an organ level. We validated the accuracy of the predictions for transcription factor regulators using the seed lipid synthesis pathway as a case study. We demonstrated a very high success rate for uncovering both known and novel transcription factor regulators for the seed lipid biosynthesis pathway. The approach described in this study is broadly applicable across any organism (plant or animal) that has a large body of public gene expression data.

plant biology↗

bioRxiv2019

Gibbs Process Determines Survival and Reveals Contact-Inhibition Genes in Glioblastoma Multiforme

Tumor growth is a spatiotemporal birth-and-death process with loss of heterotypic contact-inhibition of locomotion (CIL) of tumor cells promoting invasion and metastasis. Therefore, representing tumor cells as two-dimensional points, we can expect the tumor tissues in histology slides to reflect realizations of spatial birth-and-death process which can be mathematically modeled to reveal molecular mechanisms of CIL, provided the mathematics models the inhibitory interactions. Gibbs process as an inhibitory point process is a natural choice since it is an equilibrium process of the spatial birth-and-death process. That is if the tumor cells maintain homotypic contact inhibition, the spatial distributions of tumor cells will result in Gibbs hard core process over long time scales. In order to verify if this is the case, we applied the Gibbs process to 411 TCGA Glioblastoma multiforme patient images. Our imaging dataset included all cases for which diagnostic slide images were available. The model revealed two clusters, one of which - the "Gibbs cluster," showed the convergence of the Gibbs process with significant survival difference. Further smoothing the discretized (and noisy) inhibition metric, for both increasing and randomized survival time, we found a significant association of the patients in the Gibbs cluster with increasing survival time. The mean inhibition metric also revealed the point at which the homotypic CIL establishes in tumor cells. Besides, RNAseq analysis between patients with loss of heterotypic CIL and intact homotypic CIL in the Gibbs cluster unveiled cell movement gene signatures and differences in Actin cytoskeleton and RhoA signaling pathways as key molecular alterations. These genes and pathways have established roles in CIL. Taken together, our integrated analysis of patient images and RNAseq data provides for the first time a mathematical basis for CIL in tumors, explains survival as well as uncovers the underlying molecular landscape for this key tumor invasion and metastatic phenomenon.

cancer biology↗