Biology subjects

Roychowdhury, S.

Publications and source records attributed to Roychowdhury, S..

5 recordsLinked to original sources

bioRxiv2024

Crowder induced conformational fluctuations modulate the phase separation of yeast SUP35 NM domain

Intrinsically disordered proteins (IDPs) like Sup35NM can undergo liquid-liquid phase separation (LLPS) to form biomolecular condensates, a process influenced by their conformational flexibility and the crowded intracellular environment. This study investigates how molecular crowding, specifically the size and shape of crowders like Dextran and Ficoll, modulates the conformational states and phase separation behavior of Sup35NM. Using fluorescence correlation spectroscopy (FCS) and molecular dynamics simulations, we observed that Dextran, depending on its molecular weight, induces both compaction and expansion of Sup35NM, driving phase separation at certain thresholds. Notably, rod-like Dextran crowders promote phase separation, while spherical Ficoll does not, highlighting the impact of crowder geometry on IDP behavior. Computational modelling further revealed that the crowder shape influences Sup35NMs conformational ensemble by modulating intra- and inter-domain interactions. These findings elucidate the role of crowding agents in IDP phase behavior, suggesting that cellular crowding may regulate IDP functionality through conformational control.

biophysics↗

bioRxiv2024

Amyloid Beta Oligomers Accelerate ATP-Dependent Phase Separation of Ago2 to RNA Processing Bodies

Phase separation to insoluble membrane-less organelles is a major way of activity regulation of specific proteins in eukaryotic cells. miRNA-repressed mRNAs and Ago proteins are known to be localized to RNA-processing bodies, the subcellular structures which are formed due to assembly of several RNA binding and regulatory proteins in eukaryotic cells. Ago2 is the most important miRNA binding protein that by forming complex with miRNA binds to mRNAs having cognate miRNA binding sites and represses protein synthesis in mammalian cells. Factors which control compartmentalization of Ago2 and miRNA-repressed mRNAs to RNA processing bodies are largely unknown. We have adopted a detergent permeabilized cell-based assay system to follow the phase separation of exogenously added Ago2 to RNA processing bodies in vitro. The Ago2 phase separation process is ATP dependent and is influenced by osmolarity and salt concentration of the reaction buffer. miRNA binding of Ago2 is essential for its targeting to RNA processing bodies and the compartmentalization process gets retarded by miRNA binding "sponge" protein HuR. This assay system found to be useful in identification of amyloid beta oligomers as miRNA-activity modulators which repress miRNA activity by enhancing Ago2-miRNP targeting to RNA processing bodies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/584939v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1fe06eeorg.highwire.dtl.DTLVardef@7904b1org.highwire.dtl.DTLVardef@1231746org.highwire.dtl.DTLVardef@cd1405_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LImiRNA bound Ago2 gets phase separated in vitro to RNA processing bodies (PBs) in detergent permeabilized mammalian cells. C_LIO_LIPhase separation of Ago2 to PBs is controlled by presence of ATP and RNA. C_LIO_LIAmyloid beta oligomers retard dynamics of Ago2 bodies to inhibit miRNA function and enhance PB targeting of Ago2 miRNPs. C_LIO_LImicroRNA binding protein HuR can rescue Ago2 miRNP from PBs and inverse the effect of amyloid beta oligomers. C_LI

neuroscience↗

bioRxiv2024

Rapid Identification of Genomic Alterations in Tumors affecting lymphocyte Infiltration (RIGATonI)

Tumor genomic alterations have been associated with altered tumor immune microenvironments and therapeutic outcomes. These studies raise a critical question: are there additional genomic variations altering the immune microenvironment in tumors that can provide insight into mechanisms of immune evasion? This question is the backbone of precision immuno-oncology. Current computational approaches to estimate immunity in bulk RNA sequencing (RNAseq) from tumors include gene set enrichment analysis and cellular deconvolution, but these techniques do not consider the spatial organization of lymphocytes or connect immune phenotypes with gene activity. Our new software package, Rapid Identification of Genomic Alterations in Tumors affecting lymphocyte Infiltration (RIGATonI), addresses these two gaps in separate modules: the Immunity Module and the Function Module. Using pathologist-reviewed histology slides and paired bulk RNAseq expression data, we trained a machine learning algorithm to detect high, medium, and low levels of immune infiltration (Immunity Module). We validated this technique using a subset of pathologist-reviewed slides not included in the training data, multiplex immunohistochemistry, flow cytometry, and digital staining of The Cancer Genome Atlas (TCGA). In addition to immune infiltrate classification, RIGATonI leverages another novel machine learning algorithm for the prediction of gain- and loss-of-function genomic alterations (Function Module). We validated this approach using clinically relevant and function-impacting genomic alterations from the OncoKB database. Combining these two modules, we analyzed all genomic alterations present in solid tumors in TCGA for their resulting protein function and immune phenotype. We visualized these results on a publicly available website. To illustrate RIGATonIs potential to identify novel genomic variants with associated altered immune phenotypes, we describe increased anti-tumor immunity in renal cell carcinoma tumors harboring 14q deletions and confirmed these results with previously published single-cell RNA sequencing. Thus, we present our R package and online database, RIGATonI: an innovative software for precision immuno-oncology research.

bioinformatics↗

bioRxiv2022

Pharmacodynamic model of the dynamic response of Pseudomonas aeruginosa biofilms to drug treatments

Chronic infection by gram-negative bacteria such as Pseudomonas aeruginosa is a leading cause of morbidity and mortality in cystic fibrosis patients in whom overabundant mucus and the formation of bacterial biofilms pose barriers to drug delivery and effectiveness. Accurate pharmacokinetic-pharmacodynamic (PK-PD) models of biofilm treatment could be used to guide formulation and administration strategies to better control bacterial lung infections. To this end, we have developed a detailed pharmacodynamic model of P. aeruginosa treatment with the front-line antibiotics, tobramycin and colistin, and validated it on a detailed dataset of killing dynamics. A compartmental model structure was developed in which the key features are diffusion of drug through a boundary layer to the bacteria, concentration dependent interactions with bacteria, and passage of the bacteria through successive transit states before death. The number of transit states employed was greater for tobramycin, which is a ribosomal inhibitor, than for colistin, which disrupts bacterial membranes. For both drugs, the experimentally observed delay in killing of bacteria following drug exposure was replicated and was consistent with the diffusion time, though for tobramycin, there was an additional delay reflected in the model by passage through the transit states. For each drug, the PD model with a single set of parameters described data across a ten-fold range of concentrations and for both continuous and transient exposure protocols. Furthermore, the parameters fit for each drug individually were used to model the response of biofilms to combined treatment with tobramycin and colistin. The ability to predict drug response over a range of administration protocols allows this PD model to be integrated with PK descriptions to describe in vivo antibiotic response dynamics and to predict drug delivery strategies for improved control of bacterial lung infections. Author SummaryBiofilms are self-assembling bacterial communities that adhere to a surface and encase themselves in a protective coating. Biofilm infections are notoriously difficult to treat with conventional antibiotic administrations. To understand better the dynamics of bacterial biofilm killing in response to antibiotic treatment, we developed a mathematical model that integrates several features: drug diffusion through a boundary layer that includes the biofilm casing, concentration dependent cell damage, and passage of the cell through damaged states to eventual death. We validated the model by comparison with an extensive published dataset of biofilm response to treatment with the antibiotics, tobramycin and colistin. The model fits to these datasets were able to capture the observed trends for several antibiotic administration protocols, with model parameters reflecting the differences in mechanism of action between the two drugs. This validated model can be integrated with pharmacokinetic descriptions of drug distribution in the body over time to predict dosing and administration protocols for preclinical and clinical studies.

bioengineering↗

bioRxiv2022

Zn-dependent structural transition of SOD1 modulates its ability to undergo liquid-liquid phase separation.

The toxic gain of function of Cu/Zn superoxide dismutase (SOD1) associated with the neurodegenerative disease - Amyotrophic lateral sclerosis (ALS), is believed to occur via misfolding and/or aggregation. SOD1 is also associated with stress granules (SGs) which are a type of membraneless organelle believed to form via liquid-liquid phase separation (LLPS) of several proteins containing low-complexity, disordered regions. Using a combination of experiments and computer simulations, we report here that structural disorder in two loop regions of SOD1 induced by the absence of metal cofactor - Zn, triggers its LLPS. The phase-separated droplets give rise to aggregates which eventually form toxic amyloids upon prolonged incubation. The addition of exogenous Zn to immature, metal-free SOD1 and the severe ALS mutant - I113T, stabilized the loops and restored the folded structure, thereby inhibiting LLPS and subsequent aggregation. In contrast, the Zn-induced inhibition of LLPS and aggregation was found to be partial in the case of another severe ALS-associated mutant - G85R, which exhibits reduced Zn-binding. Moreover, a less-severe ALS mutant - G37R with perturbed Cu binding does not undergo LLPS. In conclusion, our work establishes a role for Zn-dependent modulation of SOD1 disorder and LLPS as a precursor phenomenon which may lead to the formation of toxic amyloids associated with ALS. Significance StatementThe formation of membraneless organelles such as stress granules (SGs) is believed to occur through the process of liquid-liquid phase separation (LLPS) and involves numerous proteins containing intrinsically disordered regions. Whether SOD1, which is also associated with SGs and whose aggregation is associated with Amyotrophic lateral sclerosis (ALS), can independently undergo LLPS, is not known. SOD1 is a metalloenzyme which is stabilized by the metal co-factor - Zn. In this work, we utilize experimental and simulation techniques to highlight the modulation of SOD1 LLPS propensity in a Zn-dependent manner due to underlying conformational transitions between folded and partially disordered states. Our work establishes a link between SOD1 LLPS and aggregation, which is relevant to ALS pathogenesis.

biophysics↗