Biology subjects

Nanda, A.

Publications and source records attributed to Nanda, A..

3 recordsLinked to original sources

bioRxiv2025

Designing a robust whole-cell biosensor platform for detection of toxic metals using intein splicing inhibition of Mycobacterium tuberculosis SufB protein

Disruption of the natural geochemical cycle by human activities has led to bioaccumulation of metals, posing a global health threat. Hence, there is a pressing need for simple, sensitive, yet eco-friendly biosensor setups to monitor metal contamination in the environment. Existing biosensors are limited by poor efficiency, stability issues, and complex instrumentation requiring skilled operators. To address these caveats, current study explores how intein-mediated protein splicing, a spontaneous post-translational process, can be adapted for metal-biosensing by coupling metal-dependent splicing inhibition to viability loss of native microbial cells. Toxic metal ions like Cd2 and Hg2 attenuated the splicing activity of Mtb SufB precursor protein over a concentration range of 25 {micro}M to 2 mM, while Pb2 and Cr3 failed to do so. An innovative biosensor platform was designed for colorimetric detection of metal ions via simple Alamar Blue assay, where attenuated Mtb strain (H37Ra) served as the indicator cells. Metal-induced SufB splicing inhibition led to loss of viability of H37Ra cells, while addition of metal-specific chelators reversed the effect. Multiplexing ability was evaluated by including known splicing inhibitors like Cu2, Zn2, and Pt4 over various concentration range alongside Cd2 and Hg2. The simple 96-well plate format enables multiplexed qualitative metal detection, while colorimetric absorbance measurement ensures metal quantification. The designed biosensor offers low-cost, user-friendly, and sensitive assay for high-throughput metal detection, utilizing whole-cell native organisms carrying metal-sensing precursor protein. Thus, this approach can be implemented in standard biological laboratories for robust metal screening process in environmental and industrial effluents.

microbiology↗

bioRxiv2022

Emergent dynamics of underlying regulatory network links EMT and androgen receptor-dependent resistance in prostate cancer

Advanced prostate cancer patients initially respond to hormone therapy, be it in the form of androgen deprivation therapy or second-generation hormone therapies, such as abiraterone acetate or enzalutamide. However, most men with prostate cancer eventually develop hormone therapy resistance. This resistance emerges in several ways, such as through genetic mutations, epigenetic mechanisms, or through non-genetic pathways, such as lineage plasticity along epithelial-mesenchymal or neuroendocrine-like axes. These mechanisms of hormone therapy resistance often co-exist within a single patients tumor and can overlap within a single cell. There exists a growing need to better understand how phenotypic heterogeneity and plasticity results from emergent dynamics of the regulatory networks governing androgen independence. Here, we investigated the dynamics of a regulatory network connecting the drivers of androgen receptor (AR) splice variant-mediated androgen independence and those of epithelial-mesenchymal transition. Model simulations for this network revealed four possible phenotypes: epithelial-sensitive (ES), epithelial-resistant (ER), mesenchymal-resistant (MR) and mesenchymal-sensitive (MS), with the latter phenotype occurring rarely. We observed that well-coordinated "teams" of regulators working antagonistically within the network enable these phenotypes. These model predictions are supported by multiple transcriptomic datasets both at single-cell and bulk levels, including in vitro EMT induction models and clinical samples. Further, our simulations reveal spontaneous stochastic switching between the ES and MR states. Addition of the immune checkpoint molecule, PD-L1, to the network was able to capture the interactions between AR, PD-L1, and the mesenchymal marker SNAIL, which was also confirmed through quantitative experiments. This systems-level understanding of the driver of androgen independence and EMT could aid in understanding non-genetic transitions and progression of such cancers and help in identifying novel therapeutic strategies or targets.

cancer biology↗

bioRxiv2021

Concerted dynamics between conserved histidines in the N-extein region regulates Mycobacterium tuberculosis SufB intein cleavage

Inteins are auto-processing domains that implement a multi-step biochemical reaction termed protein splicing, marked by cleavage and formation of peptide bonds. They excise from a precursor protein, generating a functional protein via covalent bonding of flanking exteins. We report the kinetic study of splicing and cleavage reaction in [Fe-S] cluster assembly protein SufB from Mycobacterium tuberculosis. Although it follows a canonical intein splicing pathway, distinct features are added by extein residues present in the active site. Sequence analysis identified two conserved histidines in the N-extein region; His-5 and His-38. Kinetic analyses of His-5Ala and His-38Ala SufB mutants exhibited significant reductions in splicing and cleavage rates relative to the SufB wild-type precursor protein. Structural analysis and molecular dynamics simulations suggested that Mtu SufB displays a unique mechanism where two remote histidines work concurrently to facilitate N-terminal cleavage reaction. His-38 is stabilized by the solvent-exposed His-5, and can impact N-S acyl shift by direct interaction with the catalytic Cys1. Development of inteins as biotechnological tools or as pathogen specific novel antimicrobial targets requires a more complete understanding of such unexpected roles of conserved extein residues in protein splicing.

molecular biology↗