Biology subjects

Srinivasan, N.

Publications and source records attributed to Srinivasan, N..

11 recordsLinked to original sources

bioRxiv2026

High-throughput platforms for genetic perturbation screening using CRISPR/Cas9 in human iPSC-derived macrophages for drug discovery

Human induced Pluripotent Stem Cell (hiPSC) models have revolutionised drug discovery, offering high translational relevance for recapitulating disease biology and thereby the potential to help reduce drug attrition. Macrophages are pivotal for maintaining tissue homeostasis and orchestrating immune responses; their dysregulation underpins several diseases, including autoinflammatory disorders, neurodegeneration, and cancer. Therapeutically targeting this cell type presents an attractive strategy to simultaneously influence multiple cellular mediators and functions. We have established a scalable, semi-automated, and physiologically relevant hiPSC-derived macrophage model, rigorously characterised through deep comparative multi-omics. We have also integrated our hiPSC-derived macrophage platform with large-scale CRISPR screening workflows designed for parallel genetic interrogation of thousands of gene targets, in both arrayed and pooled formats. In this manuscript, we apply those genetic screening methods to hiPSC-derived macrophages and showcase how genetic perturbations alter pro- and anti-inflammatory transcriptional signatures and significantly impact functional phenotypes in this cell model. This integrated approach allows for the exploration of novel genetic insights linked to causal disease biology, advancing myeloid cell-associated target discovery across a broad spectrum of therapeutic areas.

immunology↗

bioRxiv2026

Cromolyn inhibits PGE2-mediated sensitisation of TRPV1 in a GPR35-dependent manner in sensory neurons

There is a pressing need for effective alternatives to opioid analgesics, the development of which requires the identification of novel anti-nociceptive drug targets. Here, we have further investigated the anti-nociceptive properties of a GPR35 agonist, cromolyn, in an in vitro model of inflammatory sensitisation. We used ratiometric Ca2+ imaging of cultured sensory neurons to examine the effect of cromolyn on prostaglandin E2 (PGE2)-mediated sensitisation of the pro-nociceptive ion channel, transient receptor potential cation channel, subfamily V, member 1 (TRPV1). The sensitisation of TRPV1 by PGE2 was inhibited by cromolyn in a GPR35-dependent manner. These observations provide further evidence in support of an anti-nociceptive role for GPR35, highlighting the potential use of GPR35 agonists as analgesics.

neuroscience↗

bioRxiv2025

Structural network comparison of domains across remote homologues arising from different domain architectures

The homology of protein domains indicates an evolutionary relationship between them. Remote homologues, on the other hand, share very low sequence similarity while maintaining a highly similar structural topology. These similar structures can be compared using several metrices, but a comparison of networks built from amino acid residues to mimic non-covalent interactions provides a better understanding of their similarities or minute differences. Comparison of structurally related domains are studied to some extent, while the impact of domain combinations is poorly studied. In this study, several types of pairs of domains, arising from different multi-domain proteins were compared to understand the effect of cognate domain on the structural architecture of remote homologues. Several network parameters were compared among these domains. We found that an interacting domain of unrelated structure exerts higher structural and evolutionary pressure on the remote homologues, leading to structural embellishments and functional modifications. This exerted change is mediated by local rearrangements of the amino acid residues in the structure. Case studies were conducted to understand these effects. These results will assist in re-defining the homology among domains, which would rather help in better functional annotations and structural modelling.

bioinformatics↗

bioRxiv2025

SIRT2 attenuates stress-induced skeletal muscle atrophy by inhibiting glucocorticoid receptor signaling

Skeletal muscle atrophy occurs in several diseases and is associated with chronic stress. Studies indicate that glucocorticoid receptor signalling is the major signalling pathway that mediates stress-induced muscle degeneration. Although the glucocorticoid signalling pathway is relatively well characterized, there is a need to identify modulators of this pathway that may be useful for drug targeting to ameliorate muscle atrophy. SIRT2 is a mammalian Sirtuin isoform known to mediate the longevity benefits of calorie restriction and exercise. Currently, the role of SIRT2 in regulating stress-induced skeletal muscle atrophy is unclear. Our study found that SIRT2 is a critical regulator of muscle homeostasis and is required to protect against stress-induced muscle atrophy. Interestingly, SIRT2 levels are reduced during glucocorticoid-induced muscle atrophy in mice. SIRT2 depletion exacerbates glucocorticoid-induced reduction in myotube diameter and atrophy gene expression. In contrast, SIRT2 overexpression ameliorates myotube atrophy in primary myotubes. Our findings indicate that SIRT2 knockout mice are susceptible to glucocorticoid-induced muscle atrophy, while muscle-specific SIRT2-transgenic mice exhibit improved muscle function and are protected from glucocorticoid-induced atrophy. Mechanistically, SIRT2 binds to the glucocorticoid receptor to negatively regulate its activity, possibly via deacetylation of critical residues in its DNA-binding domain. Our findings suggest that SIRT2 activation may protect against glucocorticoid-induced skeletal muscle atrophy and serve as a potential therapeutic target for treating muscle atrophy.

pathology↗

bioRxiv2024

Understanding the roles of secondary shell hotspots in protein-protein complexes

Hotspots are interfacial residues in protein-protein complexes that contribute significantly to complex stability. Methods for identifying interfacial residues in protein-protein complexes are based on two approaches, namely, (a) distance-based methods, which identify residues that form direct interactions with the partner protein and (b) Accessibility Surface Area (ASA)-based methods, which identify those residues which are solvent-exposed in the isolated form of the protein and become buried upon complex formation. In this study, we introduce the concept of secondary shell hotspots, which are hotspots uniquely identified by the distance-based approach, staying buried in both the bound and isolated forms of the protein and yet forming direct interactions with the partner protein. From the analysis of the dataset curated from Docking Benchmark 5.5, comprising of 94 protein-protein complexes, we find that secondary shell hotspots are more evolutionarily conserved and have distinct Chou-Fasman propensities and interaction patterns compared to other hotspots. Finally, we present detailed case studies to show that the interaction network formed by the secondary shell hotspots is crucial for complex stability and activity. Further, they act as potentially allosteric propagators and bridge interfacial and non-interfacial sites in the protein. Their mutations to any other amino acid types cause significant destabilization. Overall, this study sheds light on the uniqueness and importance of secondary shell hotspots in protein-protein complexes.

bioinformatics↗

bioRxiv2024

Computational analysis of the effect of a binding protein (RbpA) on the dynamics of Mycobacterium tuberculosis RNA polymerase assembly

RNA polymerase-binding protein A (RbpA) is an actinomycetes-specific protein crucial for the growth and survival of the pathogen Mycobacterium tuberculosis. Its role is essential and influences the transcription and antibiotic responses. However, the regulatory mechanisms underlying RbpA-mediated transcription remain unknown. In this study, we employed various computational techniques to investigate the role of RbpA in the formation and dynamics of the RNA polymerase complex. Our analysis reveals significant structural rearrangements in RNA polymerase happen upon interaction with RbpA. Hotspot residues, crucial amino acids in the RbpA-mediated transcriptional regulation, were identified through our examination. The study elucidates the dynamic behavior within the complex, providing insights into the flexibility and functional dynamics of the RbpA-RNA polymerase interaction. Notably, potential allosteric mechanisms, involving the interface of subunits 1 and 2 were uncovered, shedding light on how RbpA modulates transcriptional activity. Finally, potential ligands meant for the 1-2 binding site were identified through virtual screening. The outcomes of our computational study serve as a foundation for experimental investigations into inhibitors targeting the RbpA-regulated dynamics in RNA polymerase. Overall, this research contributes valuable information for understanding the intricate regulatory networks of RbpA in the context of transcription and suggests potential avenues for the development of RbpA-targeted therapeutics. Author SummaryInfection studies by Mycobacterium tuberculosis (Mtb) acquires primary importance due to its severe infection and antibiotic resistance. There is an open need for highly effective drugs and one needs to employ novel approaches such as detailed structural analysis and the possibility to focus on allosteric inhibitors. We have exploited the availability of cryo-EM structures of RNA polymerase of Mtb, with and without its transcription-activator protein namely RNA polymerase-binding protein A (RbpA). In this study, we employed various computational techniques to investigate the role of RbpA in the formation and dynamics of the RNA polymerase complex. The assemblies were subject to molecular dynamics and perturbation scanning, followed by structural comparisons and measurement of subunit interface strength. These analyses could clearly show that subunits, which are far away from the RbpA binding site, undergo differential structural changes. Hence, we focused on the site to recognize potential small molecule inhibitors using virtual screening. These analyses demonstrate that it is possible to perform comparative structural analysis of different forms of assemblies, which can be useful towards drug design.

bioinformatics↗

bioRxiv2023

Single-Site Phosphorylation Elicits Structural, Dynamic, and Accessibility Changes in Proteins at both Proximal and Distal Regions to the Phosphosite.

Phosphorylation, a fundamental cellular mechanism, intricately regulates protein function and signaling pathways. Our study employs extensive computational analyses on a curated dataset of phosphorylated and unphosphorylated protein structures to explore the multifaceted impact of phosphorylation on protein conformation. Our findings reveal that phosphorylation induces not only local changes at the phosphorylation site but also extensive alterations in distant regions, showcasing its far-reaching influence on protein structure-dynamics. Using Normal Mode Analysis (NMA), we investigate changes in protein flexibility post-phosphorylation, highlighting an enhanced level of structural dynamism. Through in-depth case studies on Polyubiquitin-B and Glycogen Synthase Kinase-3 Beta, we elucidate how phosphorylation at distinct sites leads to variable structural and dynamic modifications, potentially dictating functional outcomes. While phosphorylation largely preserves residue motion correlation, it significantly disrupts low-frequency global modes, presenting a dualistic impact on protein dynamics. We also explore alterations in the total accessible surface area (ASA), emphasizing region-specific changes around phosphorylation sites. This study sheds light on phosphorylation-induced conformational changes, dynamic modulation, and surface accessibility alterations, contributing to a comprehensive understanding of cellular regulation and suggesting promising avenues for therapeutic interventions.

biophysics↗

bioRxiv2023

Interfacial residues in protein-protein complexes are in the eyes of the beholder

Interactions between proteins are vital in almost all biological processes. The characterization of protein-protein interactions helps us understand the mechanistic basis of biological processes, thereby enabling the manipulation of proteins for biotechnological and clinical purposes. The interface residues of a protein-protein complex are assumed to have the following two properties: a) they always interact with a residue of a partner protein, which forms the basis for distance-based interface residue identification methods, and b) they are solvent-exposed in the isolated form of the protein and become buried in the complex form, which forms the basis for Accessible Surface Area (ASA)-based methods. The study interrogates this popular assumption by recognizing interface residues in protein-protein complexes through these two methods. The study shows that a few residues are identified uniquely by each method, and the extent of conservation, propensities and their contribution to the stability of protein-protein interaction varies substantially between these residues. The case study analyses showed that interface residues, unique to distance, participate in crucial interactions that hold the proteins together, whereas the interface residues unique to the ASA method have a potential role in the recognition, dynamics and specificity of the complex and can also be a hotspot. Overall, the study recommends applying both distance and ASA methods so that some interface residues missed by either method but crucial to the stability, recognition, dynamics and function of protein-protein complexes are identified in a complementary manner.

bioinformatics↗

bioRxiv2020

D614G substitution enhances the stability of trimeric SARS-CoV-2 spike protein

SARS-CoV-2 spike protein with D614G substitution has become the dominant variant in the ongoing COVID-19 pandemic. Several studies to characterize the new virus expressing G614 variant show that it exhibits increased infectivity compared to the ancestral virus having D614 spike protein. Here, using in-silico mutagenesis and energy calculations, we analyzed inter-residue interaction energies and thermodynamic stability of the dominant (G614) and the ancestral (D614) variants of spike protein trimer in closed and partially open conformations. We find that the local interactions mediated by aspartate at the 614th position are energetically frustrated and create unfavourable environment. Whereas, glycine at the same position confers energetically favourable environment and strengthens intra-as well as inter-protomer association. Such changes in the local interaction energies enhance the thermodynamic stability of the spike protein trimer as free energy difference ({Delta}{Delta}G) upon glycine substitution is -2.6 kcal/mol for closed conformation and -2.0 kcal/mol for open conformation. Our results on the structural and energetic basis of enhanced stability hint that G614 may confer increased availability of functional form of spike protein trimer and consequent in higher infectivity than the D614 variant.

bioinformatics↗

bioRxiv2017

PB-kPRED: Knowledge-Based Prediction Of Protein Backbone Conformation Using A Structural Alphabet

Libraries of structural prototypes that abstract protein local structures are known as structural alphabets and have proven to be very useful in various aspects of protein structure analyses and predictions. One such library, Protein Blocks (PBs), is composed of 16 standard 5-residues long structural prototypes. This form of analyzing proteins involves drafting its structure as a string of PBs. Thus, predicting the local structure of a protein in terms of protein blocks is a step towards the objective of predicting its 3-D structure. Here a new approach, kPred, is proposed towards this aim that is independent of the evolutionary information available. It involves (i) organizing the structural knowledge in the form of a database of pentapeptide fragments extracted from all protein structures in the PDB and (ii) apply a purely knowledge-based algorithm, not relying on secondary structure predictions or sequence alignment profiles, to scan this database and predict most probable backbone conformations for the protein local structures.\n\nBased on the strategy used for scanning the database, the method was able to achieve efficient mean Q16 accuracies between 40.8% and 66.3% for a non-redundant subset of the PDB filtered at 30% sequence identity cut-off. The impact of these scanning strategies on the prediction was evaluated and is discussed. A scoring function that gives a good estimate of the accuracy of prediction was further developed. This score estimates very well the accuracy of the algorithm (R2 of 0.82). An online version of the tool is provided freely for non-commercial usage at http://www.bo-protscience.fr/kpred/.

bioinformatics↗

bioRxiv2017

Novel Repolarisation Metric Predicts Arrhythmia Origin And Clinical Events In ARVC And Brugada Syndrome

Structured abstractO_ST_ABSBackgroundC_ST_ABSInitiation of re-entrant ventricular tachycardia (VT) involves complex interactions between activation (AT) and repolarization times (RT). The re-entry vulnerability index (RVI) is a recently proposed activation-repolarization metric designed to quantify tissue susceptibility to re-entry.\n\nObjectivesThe study aimed to test the feasibility of an RVI-based algorithm to predict the exit site of VT and occurrence of clinical events.\n\nMethodsPatients with Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) (n=11), Brugada Syndrome (BrS) (n=13) and focal RV outflow tract VT (n=9) underwent programmed stimulation with unipolar electrograms recorded from a non-contact array. The distance between region of lowest RVI and site of VT breakout (Dmin), and global minimum RVI (RVIG) were computed to assess prediction of site of VT breakout and occurrence of clinical events, respectively.\n\nResultsLowest values of RVI, representing sites of highest susceptibility to re-entry, co-localised with site of VT breakout in ARVC/BrS but not in focal VT and Dmin values were lower in ARVC/BrS. ARVC/BrS patients with inducible VT had lower RVIG than those who were non-inducible or those with focal VT. Patients were followed up for 112 {+/-} 19 months; those with clinical VT events had lower RVIg than those without VT or those with focal VT.\n\nConclusionsThe proposed methodology based on RVI localises the origin of re-entrant but not focal ventricular arrhythmias and predicts clinical events. This index could be applied to target ablation for arrhythmias which are difficult to induce or are haemodynamically unstable and also risk stratify patients for ICD prophylaxis.\n\nAbbreviations list

physiology↗