Biology subjects

Bhattacherjee, A.

Publications and source records attributed to Bhattacherjee, A..

7 recordsLinked to original sources

bioRxiv2025

A molecularly defined basalo-prefrontal-thalamic circuit regulates sensory and affective dimensions of pain

Both the medial prefrontal cortex (mPFC) and thalamus have been implicated in pain regulation. However, the roles of the mPFC-thalamus connection in pain and how the mPFC modulates nociceptive processing remain unclear. Here, we show that the mPFC neurons projecting to thalamus, marked by Foxp2 expression, are deactivated in both acute and chronic pain in male mice. Persistent inactivation of the mPFC Foxp2+ neurons enhances nociceptive sensitivity, while their activation alleviates multiple aspects of pain. Circuit-specific manipulations revealed that the projections to parataenial nucleus, mediodorsal and ventromedial thalamus differentially modulate sensory and affective pain. Additionally, the mPFC Foxp2+ neurons receive cholinergic input from the basal forebrain, particularly the horizonal diagonal band (HDB). Notably, activation of the 4{beta}2-containing nicotinic acetylcholine receptor in mPFC exerts antinociceptive effects in Foxp2+ neuron-dependent manner. Together, our study defines an HDB[->]mPFCFoxp2[->]thalamus circuit essential for sensory and affective pain modulation and underscores the therapeutic potential of targeting mPFC cholinergic signaling in chronic pain management.

neuroscience↗

bioRxiv2025

Mechanism of phosphorylation dependent interactions of complete Retinoblastoma-AB pocket domain with its linker

Retinoblastoma protein (pRb) is a critical tumor suppressor, whose activity is regulated through phosphorylation of Serine/Threonine residues within unstructured linker regions. Due to the absence of high-resolution structures encompassing these linkers, many studies have indirectly examined the phosphorylation effects on pRb-protein interactions. Here, we present computational models of the full pRb pocket domain (pRbAB), incorporating the flexible linker (pRbPL) that connects boxes A and B. Three models, generated by ROBETTA and AlphaFold, were used to explore the mechanisms by which phosphorylation at Ser608 and Ser612 influence the interaction of pRbAB with E2F transactivation domain (E2FTA). All models revealed a short helix (residues 602-607), similar to that observed in a previously published crystal structure of pRbAB with shortened pRbPL linker and phosphomimetic mutation at Ser608, with E2FTA (PDB:4ELL), and proposed to competitively inhibit E2FTA binding. Interestingly, this helix formed irrespective of phosphorylation, indicating that additional factors influencing the interaction upon phosphorylation. In our models, the pRbPL linker displayed significant motion, especially near Ser608 and Ser612, facilitating phosphorylation, but without inducing large conformational changes in the pocket domains. Phosphorylation at Ser608 and Ser612 resulted in changes in multiple parameters including electrostatic properties, compactness and energy landscape of pRbAB constituting important mechanisms that promote E2F release. Additionally, recapitulating known R661W mutation in these models correctly showed reduced E2FTA binding, demonstrating the predictive power of these models to study the effects of missense mutations on the stability, conformation and interactions of pRbAB with E2FTA.

cell biology↗

bioRxiv2022

Understanding prefrontal cortex functions by decoding its molecular, cellular and circuit organization

The prefrontal cortex (PFC) is functionally one of the most complex regions of mammalian brain. Unlike other cortical areas that process single sensory modalities (like vision, touch, smell, etc.), the PFC integrates information across brain regions to regulate diverse functions ranging from cognition, emotion, executive action to even pain sensitivity. However, it is unclear how such diverse functions are organized at the cellular and circuit levels within the anatomical modules of the PFC. Here we employed spatially resolved single-cell transcriptome profiling to decode PFCs organizational heterogeneity. The results revealed that PFC has very distinct cell type composition relative to all neighboring cortical areas. Interestingly, PFC also adopts specialized transcriptional features, different from all neighbors, with differentially expressed genes regulating neuronal excitability. The projections to major subcortical targets of PFC emerge from combinations of neuron subclusters determined in a target-intrinsic fashion. These cellular and molecular features further segregated within subregions of PFC, alluding to the subregion-specific specialization of several PFC functions. Finally, using these unique cellular, molecular and projection properties, we identified distinct cell types and circuits in PFC that engage in pathogenesis of chronic pain. Collectively, we not only present a comprehensive organizational map of the PFC, critical for supporting its diverse functions, but also reveal the cluster and circuit identity of a pathway underlying chronic pain, a rapidly escalating healthcare challenge limited by molecular understanding of maladaptive PFC circuits. Major pointsO_LIPFC adopts unique cellular composition, distinct from other cortical areas C_LIO_LISelective transcriptomic features emerge in PFC to support its divergent functional portfolio C_LIO_LISubcortical projections of PFC assume target-intrinsic specification for innervating clusters C_LIO_LIA molecularly defined L5 projection neuron cluster (to PAG) potentially mediates chronic pain pathogenesis C_LI

neuroscience↗

bioRxiv2021

Decoding molecular and cellular heterogeneity of nucleus accumbens with high-throughput scRNA-seq and MERFISH

The nucleus accumbens (NAc) plays an important role in regulating multiple behaviors and its dysfunction has been linked to many neural disorders. However, the molecular, cellular and anatomic heterogeneity underlying its functional diversity remains incompletely understood. Here, we generate a cell census of the mouse NAc using high-throughput single cell RNA sequencing and multiplexed error-robust FISH, revealing a high level of cell heterogeneity in this brain region. We show that the transcriptional and spatial diversity of neuron subtypes underlie NAcs anatomic and functional heterogeneity, and possibly contribute to the pathogenesis of different neurological disorders. These findings explain how the seemingly simple neuronal composition of the NAc achieves its highly heterogenous structure and diverse functions. Collectively, our study generates a spatially resolved cell taxonomy for understanding the NAc structure and function, which demonstrates the importance of combining molecular and spatial information in revealing the fundamental features of the nervous system.

neuroscience↗

bioRxiv2021

Carbohydrate sulfation as a mechanism for fine-tuning Siglec ligands

The immunomodulatory family of Siglecs recognize sialic acid-containing glycans as self, which is exploited in cancer for immune-evasion. The biochemical nature of Siglec ligands remains incompletely understood with emerging evidence suggesting the importance of carbohydrate sulfation. Here, we investigate how specific sulfate modifications affect Siglec ligands by overexpressing eight carbohydrate sulfotransferases (CHSTs) in five cell lines. Overexpression of three CHSTs (CHST1, CHST2, or CHST4) significantly enhances the binding of numerous Siglecs. Unexpectedly, two other CHSTs (Gal3ST2 and Gal3ST3) diminish Siglec binding, suggesting a new mode to modulate Siglec ligands via sulfation. Results are cell type dependent, indicating that the context in which sulfated glycans are presented is important. Moreover, pharmacological blockade of N- and O-glycan maturation reveals a cell type-specific pattern of importance for either class of glycan. Production of a highly homogenous CD33 (Siglec-3) fragment enabled a mass spectrometry-based binding assay to determine 10-fold and 3-fold enhanced affinity for Neu5Ac2-3(6-O-sulfo)Gal{beta}1-4GlcNAc and Neu5Ac2-3Gal{beta}1-4(6-O- sulfo)GlcNAc, respectively, over Neu5Ac2-3Gal{beta}1-4GlcNAc. CD33 showed significant additivity in affinity (36-fold) for the disulfated ligand, Neu5Ac2-3(6-O-sulfo)Gal{beta}1-4(6-O-sulfo)GlcNAc. Moreover, overexpression of both CHST1 and CHST2 in cells greatly enhanced the binding of several Siglecs, including CD33. Finally, we reveal that CHST1 is upregulated in numerous cancers, correlating with poorer survival rates and sodium chlorate sensitivity for the binding of Siglecs to cancer cell lines. These results provide new insights into carbohydrate sulfation as a modification that is a general mechanism for tuning Siglec ligands on cells, including in cancer.

immunology↗

bioRxiv2021

Increasing phagocytosis of microglia through targeting CD33 with liposomes displaying glycan ligands

CD33 is an immunomodulatory receptor expressed on microglia and genetically linked to Alzheimers disease (AD) susceptibility. While antibodies targeting CD33 have entered clinical trials to treat neurodegeneration, it is unknown whether the glycan-binding properties of CD33 can be exploited to modulate microglia. Here, we use liposomes that multivalently display glycan ligands of CD33 (CD33L liposomes) to engage CD33. We find that CD33L liposomes increase phagocytosis of cultured monocytic cells and microglia in a CD33-dependent manner. Enhanced phagocytosis strongly correlates with loss of CD33 from the cell surface and internalization of liposomes. Increased phagocytosis by treatment with CD33L liposomes is dependent on a key intracellular signaling motif on CD33 as well as the glycan-binding ability of CD33. These effects are specific to trans engagement of CD33 by CD33L liposomes, as cis engagement through insertion of lipid-linked CD33L into cells produces the opposite effect on phagocytosis. Moreover, intracerebroventricular injection of CD33L liposomes in mice enhances phagocytosis of microglia in a CD33-dependent manner. These results demonstrate that multivalent engagement of CD33 with glycan ligands can modulate microglial cell function.

immunology↗

bioRxiv2020

Interaction of 14-3-3I and CDPK1 mediates the growth of human malaria parasite

Scaffold proteins play pivotal role as modulators of cellular processes by operating as multipurpose conformation clamps. 14-3-3 proteins are gold-standard scaffold modules that recognize phosphoSer/Thr (pS/pT) containing conserved motifs of target proteins and confer conformational changes leading to modulation of their functional parameters. Modulation in functional activity of kinases has been attributed to their interaction with 14-3-3 proteins. Herein, we have characterized Plasmodium falciparum 14-3-3 and its interaction with key kinase of the parasite, Calcium-Dependent Protein Kinase 1 (CDPK1) by performing various analytical biochemistry and biophysical assays. Towards this, we annotated PF3D7_0818200 as 14-3-3 isoform I through extensive phylogenetic and comparative sequence analysis. Molecular dynamics simulation studies indicated that phosphoSer64 present in CDPK1 polypeptide sequence (61KLGpS64) behaves as canonical Mode I-type (RXXpS/pT) consensus 14-3-3 binding motif, mediating the interaction. The protein-protein interaction was validated in vitro with ELISA and SPR, which confirmed that CDPK1 interacts with 14-3-3I in a phosphorylation dependent manner, with binding affinity constant of 670 {+/-} 3.6 nM. The interaction of 14-3-3I with CDPK1 was validated with well characterized optimal 14-3-3 recognition motifs: ARSHpSYPA and RLYHpSLPA as CDPK1 mimetics, by simulation studies and ITC. Further, interaction antagonizing peptidomimetics showed growth inhibitory impact on the parasite indicating crucial physiological role of 14-3-3/CDPK1 interaction. Overall, this study characterizes 14-3-3I as a scaffold protein in the malaria parasite and unveils CDPK1 as its previously unidentified target. This sets a precedent for the rational design of 14-3-3 based PPI inhibitors by utilizing 14-3-3 recognition motif peptides, as a potential antimalarial strategy.

cell biology↗