Biology subjects

Shakya, A.

Publications and source records attributed to Shakya, A..

4 recordsLinked to original sources

bioRxiv2025

Label-Free Fluorescence Microscopy Reveals Multiphase Organization in Biomolecular Condensates

Phase transitions of proteins and nucleic acids (NA) leading to the formation of biomolecular condensates have been linked to various biological functions. Given the growing number of proteins/NA predicted to undergo liquid-liquid phase separation (LLPS), efficient tools to investigate this behavior are critical to advancing our understanding of biomolecular condensate function. The current standard used to study LLPS involves techniques that utilize exogenous fluorophore labels. The labeling process is often costly and time-consuming and comes with associated complexity that arises from unknown interactions from the bulky fluorescent tags. These aspects limit high throughput analysis of protein/NA phase separation based on external fluorophore labeling. Here, we report the discovery that intrinsic fluorescence, well into the visible spectrum, arises as an emergent property of biomolecular condensates. Leveraging this intrinsic fluorescence, we study condensate formation, directly measure their internal dynamics via Fluorescence Recovery after Photobleaching (FRAP), and examine the 3D morphology and transitions to various multiphase architectures. Through this approach, we find that a variety of G-quadruplex DNA readily form droplets with histone H1 and display dynamic exchange. In addition, we directly demonstrate that the 3D morphology, core-shell architecture, and sub-compartmentalization of condensate droplets are tunable via the charge ratio of components in solution and NA hybridization. Our method utilizes an inherent property of condensates, thus is broadly applicable to any phase-separated systems and can advance our understanding of biological phase transition.

biophysics↗

bioRxiv2023

Protein unfolding thermodynamics predict multicomponent phase behavior

An increasing number of proteins are known to undergo liquid-liquid phase separation (LLPS), with or without nucleic acids or partner proteins, forming dense liquid-like phases termed biomolecular condensates. This physical phenomenon has been implicated in the existence of cellular membraneless organelles as well as in the formation of pathological protein aggregates in several human diseases. While common structural features of proteins with a propensity to undergo LLPS have been well documented, currently there is no thermodynamic framework capable of predicting the phase behavior of native proteins. Here, we show that two fundamental thermodynamic properties associated with the unfolding of a native protein, change in heat capacity ({Delta}Cunfold) and change in Gibbs free energy ({Delta}Gunfold), are sufficient to predict the formation of multicomponent biomolecular condensates. We find that proteins with small{Delta} Cunfold and{Delta} Gunfold values, which indicate a native state that is thermodynamically similar to the fully unfolded state, promote LLPS. In contrast, proteins with large{Delta} Cunfold and{Delta} Gunfold values promote aggregation. We also demonstrate that the stability of the liquid-like condensate can be predicted from the proximity of a proteins thermodynamic variable values to the phase boundary. This work elucidates a deep connection between single-protein thermodynamics and multicomponent phase behavior, and provides an avenue for predicting pathological droplet-aggregate transition. Significance StatementBiomolecular phase transitions, such as LLPS of proteins and nucleic acids, is emerging as an important concept in understanding the link between dysregulation of membraneless compartmentalization in cells and several human diseases. Currently, analysis of structural features such as intrinsic disorder and charged residue content is the "rule-of-thumb" to predict a proteins propensity to undergo LLPS. There are several instances where this qualitative approach fails, potentially due to not accounting for a proteins native structure. We demonstrate an empirical correlation between the unfolding thermodynamics of native proteins and their phase behavior, which enables a quantitative prediction of multicomponent LLPS. This novel approach, based on single-protein thermodynamics, has the potential to quantitatively predict pathological phase transitions of proteins in degenerative human diseases.

biophysics↗

bioRxiv2021

Small molecule allosteric inhibitors of RORγt block Th17-dependent inflammation and associated gene expression in vivo.

Retinoic acid receptor-related orphan nuclear receptor (ROR) {gamma}t is a member of the RORC nuclear hormone receptor family of transcription factors. ROR{gamma}t functions as a critical regulator of thymopoiesis and immune responses. ROR{gamma}t is expressed in multiple immune cell populations including Th17 cells, where its primary function is regulation of immune responses to bacteria and fungi through IL-17A production. However, excessive IL-17A production has been linked to numerous autoimmune diseases. Moreover, Th17 cells have been shown to elicit both pro- and anti-tumor effects. Thus, modulation of the ROR{gamma}t/IL-17A axis may represent an attractive therapeutic target for the treatment of autoimmune disorders and some cancers. Herein we report the design, synthesis and characterization of three selective allosteric ROR{gamma}t inhibitors in preclinical models of inflammation and tumor growth. We demonstrate that these compounds can inhibit Th17 differentiation and maintenance in vitro and Th17-dependent inflammation and associated gene expression in vivo, in a dose-dependent manner. Finally, ROR{gamma}t inhibitors were shown to inhibit tumor formation in pancreatic ductal adenocarcinoma (PDAC) organoids.

immunology↗

bioRxiv2020

Targeting OCA-B/Pou2af1 blocks type-1 diabetes and reduces infiltration of activated, islet-reactive T cells

The transcriptional coregulator OCA-B promotes expression of T cell target genes in cases of repeated antigen exposure, a necessary feature of autoimmunity. We hypothesized that T cell-specific OCA-B deletion and pharmacologic OCA-B inhibition would protect mice from autoimmune diabetes. We developed an Ocab conditional allele and backcrossed it onto a diabetes-prone NOD/ShiLtJ strain background. T cell-specific OCA-B loss protected mice from spontaneous disease. Protection was associated with large reductions in islet CD8+ T cell receptor specificities associated with diabetes pathogenesis. CD4+ clones associated with diabetes were present, but associated with anergic phenotypes. The protective effect of OCA-B loss was recapitulated using autoantigen-specific NY8.3 mice, but diminished in monoclonal models specific to artificial or neoantigens. Rationally-designed membrane-penetrating OCA-B peptide inhibitors normalized glucose levels, and reduced T cell infiltration and proinflammatory cytokine expression in newly-diabetic NOD mice. Together, the results indicate that OCA-B is a potent autoimmune regulator and a promising target for pharmacologic inhibition. ~40-word summary statement for the online JEM table of contents and alertsKim and colleagues show that OCA-B in T cells is essential for the generation of type-1 diabetes. OCA-B loss leaves the pancreatic lymph nodes largely undisturbed, but associates autoreactive CD4+ T cells in the pancreas with anergy while deleting potentially autoreactive CD8+ T cells. SummaryKim et al. show that loss or inhibition of OCA-B in T cells protects mice from type-1 diabetes.

immunology↗