Biology subjects

Sengupta, N.

Publications and source records attributed to Sengupta, N..

8 recordsLinked to original sources

bioRxiv2026

Multimodal characterization of putative pan-inhibitory viral constructs in macaque monkeys

Transgenic approaches and validated viral vector toolboxes have advanced our understanding of how inhibitory neurons shape cognition and motor control in mice and other rodents. However, such advances have been elusive in non-human primates (NHPs). While many studies demonstrate expression of reporter proteins, few studies have provided clear quantitative evidence of whether these viral constructs can be reliably used for assessment of in vivo and in vitro function in primates, which, in turn, has hindered widespread adoption by NHP researchers. Here, we characterize putative pan-inhibitory cell targeting vectors engineered with regions from the Dlx5-6 intergenic domain. Pan-inhibitory viral vectors are attractive because they enable ground-truth optotagging and modulation of multiple inhibitory subtypes in the same animal and, when combined with machine learning approaches, enable cell-type classifiers for NHPs. We combined in vivo anesthetized electrophysiological recordings, in vitro slice electrophysiology, and immunohistochemistry across seven rhesus macaque monkeys to assess the specificity, completeness and efficacy of three constructs in multiple sensory and higher-order association cortices. The vectors reliably restricted transgene expression to inhibitory neurons (81-97%). However, of the local GABAergic neurons only 32-64% expressed the transgene, depending on the construct. This incompleteness arose in part from uneven coverage across subtypes, with strong transgene expression in parvalbumin-positive interneurons (~60-90%), sparse expression in calbindin-positive interneurons (~1-7%), and near-complete avoidance of calretinin-positive interneurons. A construct pairing DLX2.0 with ChrimsonR was excellent for in vivo optotagging, while h56D paired with bReaChES most effectively suppressed neural activity in vivo. In the slice, activating terminals of transgene-expressing neurons optically evoked inhibitory currents in coupled pyramidal neurons confirming transsynaptic functionality of these constructs and their use for inhibitory circuit-specific synaptic assessments and manipulation. Collectively, the results provide rigorous functional characterization, and quantitative guidance for NHP researchers seeking to deploy GABAergic cell-type-specific tools in their experiments.

neuroscience↗

bioRxiv2026

Surface Binding Sites Orchestrate Long-Range Control of Active-Site Dynamics and Glucose Tolerance in β-glucosidase, BglB

{beta}-Glucosidases are essential for lignocellulosic biomass conversion, yet their industrial utility is limited by inhibition from glucose, the reaction product. Although glucose inhibition has been extensively documented, its molecular basis remains poorly understood. Here, we demonstrate that glucose inhibition in the GH1 {beta}-glucosidase BglB from Paenibacillus polymyxa is mediated by multiple surface-exposed secondary binding sites (SBSs) that function as regulatory elements rather than passive glucose-binding patches. Kinetic analyses revealed a mixed mode of inhibition, with modest activation at low glucose concentrations followed by progressive inhibition at higher concentrations. Glucose association occurred through multiple SBSs in a concentration-dependent manner and induced localized rigidification, particularly at gatekeeper regions surrounding the active-site entrance. These surface interactions propagated through long-range coupling pathways, resulting in structural, energetic, and residue interaction network reorganization that reshaped the catalytic pocket. Functional interrogation of representative SBSs revealed distinct roles for individual sites. Mutation of a gatekeeper-associated SBS reduced local glucose association, increased glucose tolerance by more than 30%, and improved substrate affinity by approximately 38%, whereas disruption of a distal SBS compromised structural integrity and soluble protein production. Together, these findings establish a direct link between surface glucose recognition and active-site regulation, providing, to our knowledge, the first integrated evidence for the existence and functional significance of secondary glucose-binding sites in {beta}-glucosidases. More broadly, this work identifies surface SBSs as promising targets for engineering glucose-tolerant and catalytically robust enzymes for biomass conversion and related biotechnological applications.

biophysics↗

bioRxiv2025

Selective Targeting of Kinesin on Lipid Droplets in the Liver Reduces Serum Lipids

The liver controls plasma lipids by secreting lipid-rich very low density lipoproteins (VLDL) into blood. Inside hepatocytes in the liver, Lipid Droplets (LDs) are transported to the smooth Endoplasmic Reticulum (sER) by kinesin-1 motors, and then catabolized in the sER to supply lipids for VLDL assembly. LDs are the only cellular organelle bounded by a phospholipid monolayer, and are thus distinct from all other (bilayer-bounded) organelles. It is therefore plausible that a given protein can bind to the LD membrane using mechanisms that are completely different from all other organelles. Indeed, here we find that kinesin-1 uses its tail domain to bind LDs, but alternative mechanisms to bind other organelles. A peptide corresponding to kinesins tail domain therefore competes with, and removes kinesin-1 selectively from LDs with minimal effect on other organelles. Delivery of lipids for VLDL assembly is consequently reduced, causing a remarkable reduction of [~]50% of secreted lipids (triglycerides and cholesterol) in cell culture. We further develop Orally fed Egg-liposomes as a method to deliver kinesin tail domain peptide to the liver of Zebrafish. The peptide reverses diet-induced hyperlipidaemia in Zebrafish larvae and brings the larvae back to a normolipidaemic state, thus confirming the effectiveness of our method in a physiologically relevant in-vivo situation. Strikingly, the peptide causes no unwanted accumulation of lipids in the liver, no toxicity and no developmental or behavioural defects in Zebrafish. Using a peptide to displace proteins (e.g. kinesin) selectively from LDs provides a conceptually novel and radically different approach against hyperlipidaemia. This monolayer-versus-bilayer strategy can be potentially extended to target other LD-bound proteins that function as key regulators of Lipid metabolism.

cell biology↗

bioRxiv2025

Repurposing Melatonin in dual-mode for Wilson disease therapy as a Copper Chelator and an antioxidant agent

Loss-of-function mutations in copper-ATPase ATP7B underlie Wilson disease (WD), a disorder characterized by hepatic copper accumulation and severe hepato-neuropathology. Existing chelation therapeutics remove excess copper but lack intrinsic antioxidant capacity and frequently cause systemic toxicity. Here we evaluate melatonin, an FDA-approved indoleamine with antioxidant and putative metal-chelating activity, as a candidate therapeutic for WD. In ATP7B-/- hepatocytes, melatonin restored copper-induced reactive oxygen species (ROS) to basal levels, reduced apoptosis twofold, and attenuated Nrf2 nuclear translocation leading to reduction of Hemoxygenase-1 abundance. Live-cell ratiometric analysis of GSSG/GSH using GRX1-roGFP2 expressed in melatonin-treated ATP7B-/- hepatocytes revealed a significant reduction in intensity-ratio, indicating an effective mitigation of copper-induced glutathione oxidation. Isothermal calorimetric titration revealed moderate Cu2+ affinity (K ITC=4.54 x 103 M-{superscript 1}), rationalized by MD-simulations showing an interaction energy of 18.5 x 10-3 kcal{middle dot}mol-{superscript 1} via amide-Cu{superscript 2} coordination. In-cellulo studies also revealed that copper-induced vesicularized ATP7B reinstates to Golgi in melatonin-treated hepatocytes. In vivo, melatonin treatment reduced copper-induced oxidative stress in zebrafish embryos and lowered copper burden in Caenorhabditis elegans WD model. Our studies revealed that encapsulation of melatonin within an engineered polymeric nanocapsules having dithiol linkers, susceptible to cleavage by GSH, extended melatonins circulatory half-life ten-fold and enhanced its ROS-scavenging efficacy three-fold relative to free melatonin. This work introduces a unique dual-function therapeutic strategy that integrates antioxidant activity with copper chelation, simultaneously addressing copper overload and redox imbalance. Repurposing melatonin, with its established clinical safety, offers rapid and cost-effective translational pathway toward WD-therapy while providing a generalizable platform for redox- and metal-associated disorders.

cell biology↗

bioRxiv2023

Myelin dystrophy in the aging prefrontal cortex leads to impaired signal transmission and working memory decline: a multiscale computational study

Normal aging leads to myelin alterations in the rhesus monkey dorsolateral prefrontal cortex (dlPFC), which are positively correlated with degree of cognitive impairment. It is hypothesized that remyelination with shorter and thinner myelin sheaths partially compensates for myelin degradation, but computational modeling has not yet explored these two phenomena together systematically. Here, we used a two-pronged modeling approach to determine how age-related myelin changes affect a core cognitive function: spatial working memory. First, we built a multicompartment pyramidal neuron model fit to monkey dlPFC empirical data, with an axon including myelinated segments having paranodes, juxtaparanodes, internodes, and tight junctions. This model was used to quantify conduction velocity (CV) changes and action potential (AP) failures after demyelination and subsequent remyelination. Next, we incorporated the single neuron results into a spiking neural network model of working memory. While complete remyelination nearly recovered axonal transmission and network function to unperturbed levels, our models predict that biologically plausible levels of myelin dystrophy, if uncompensated by other factors, can account for substantial working memory impairment with aging. The present computational study unites empirical data from ultrastructure up to behavior during normal aging, and has broader implications for many demyelinating conditions, such as multiple sclerosis or schizophrenia.

neuroscience↗

bioRxiv2022

Cryo-EM reveals the membrane binding phenomenon of EspB, a virulence factor of the Mycobacterial Type VII secretion system

Mycobacterium tuberculosis utilizes sophisticated machinery called the type VII secretion system to translocate virulence factors across its complex lipid membrane. ESX-1 is one of the essential and well-studied secretion systems which transport various virulence factors, including EspB. EspB, a ~36 kDa secreted substrate, has been implicated to play vital role in protecting the bacteria from hostile environment within the host cell phagosome. It is also involved in bacterial pathogenesis and has been shown to bind phospholipids. Recently, two cryo-EM structures of EspB full-length and the secreted isoforms were resolved. Despite the availability of multiple high-resolution structures of EspB, the physiological relevance and mechanism of virulence of this secreted substrate remains poorly characterized. In this current work, we implemented cryo-EM-based structural studies, including various functional assays, TEM imaging, and biophysical approach to demonstrate the interaction of EspB with lipids and bio-membrane. Our findings also indicated that EspB may play a crucial role in binding to and rupturing host mitochondrial membrane. Through cryo-EM studies we were able to show the possible membrane-binding region of EspB. Our study sheds light on host-pathogen interactions and bacterial pathogenesis mediated by EspB.

biophysics↗

bioRxiv2020

Disulfide reduction allosterically destabilizes the β-ladder sub-domain assembly within the NS1 dimer of ZIKV

The Zika virus (ZIKV) was responsible for a recent debilitating epidemic that till date has no cure. A potential way to reduce ZIKV virulence is to limit the action of the non-structural proteins involved in its viral replication. One such protein, NS1, encoded as a monomer by the viral genome, plays a major role via symmetric oligomerization. We examine the homodimeric structure of the dominant {beta}-ladder segment of NS1 with extensive all atom molecular dynamics. We find it stably bounded by two spatially separated interaction clusters (C1 and C2) with significant differences in the nature of their interactions. Four pairs of distal, intra-monomeric disulfide bonds are found to be coupled to the stability, local structure, and wettability of the interfacial region. Symmetric reduction of the intra-monomeric disulfides triggers marked dynamical heterogeneity, interfacial wettability and asymmetric salt bridging propensity. Harnessing the model-free Lipari-Szabo based formalism for estimation of conformational entropy (Sconf), we find clear signatures of heterogeneity in the monomeric conformational entropies. The observed asymmetry, very small in the unperturbed state, expands significantly in the reduced states. This allosteric effect is most noticeable in the electrostatically bound C2 cluster that underlies the greatest stability in the unperturbed state. Allosteric induction of conformational and thermodynamic asymmetry is expected to affect the pathways leading to symmetric higher ordered oligomerization, and thereby affect crucial replication pathways. Statement of significanceControlling viral pathogenesis remains a challenge in the face of modern-day epidemics. Though cumbersome and fraught with misleads, most therapeutic endeavors lean towards the design of drug molecules targeting specific proteins involved in viral pathogenesis. This work demonstrates an alternative approach, namely the usage of allosteric intervention to disrupt the binding integrity of the primary domain of the non-structural NS1 protein dimer crucially important in ZIKV virulence. The intervention, triggered by symmetric reduction of the internal monomeric disulfide bonds, results in weakening and distortion of the distal binding interfaces. It further introduces marked structural and entropic asymmetry within the homooligomeric unit, precluding the formation of higher ordered oligomers of high symmetry. The results have important ramifications for consolidated efforts at limiting ZIKV virulence.

biophysics↗

bioRxiv2019

Defining the Subcellular Distribution and Metabolic Channeling of Phosphatidylinositol

Phosphatidylinositol (PtdIns) is an essential structural component of eukaryotic membranes that also serves as the common precursor for polyphosphoinositide (PPIn) lipids. Despite the recognized importance of PPIn species for signal transduction and membrane homeostasis, there is still a limited understanding of how the dynamic regulation of PtdIns synthesis and transport contributes to the turnover of PPIn pools. To address these shortcomings, we capitalized on the substrate selectivity of a bacterial enzyme, PtdIns-specific PLC, to establish a molecular toolbox for investigations of PtdIns distribution and availability within intact cells. In addition to its presence within the ER, our results reveal low steady-state levels of PtdIns within the plasma membrane (PM) and endosomes as well as a relative enrichment of PtdIns within the cytosolic leaflets of the Golgi complex, peroxisomes, and outer mitochondrial membranes. Kinetic studies also demonstrate the requirement for sustained PtdIns supply from the ER for the maintenance of monophosphorylated PPIn species within the PM, Golgi complex, and endosomal compartments.\n\nSummaryPemberton et al. characterize a molecular toolbox for the visualization and manipulation of phosphatidylinositol (PtdIns) within intact cells. Results using these approaches define the steady-state distribution of PtdIns across subcellular membrane compartments as well as provide new insights into the relationship between PtdIns availability and polyphosphoinositide turnover.

cell biology↗