Biology subjects

Sengupta, K.

Publications and source records attributed to Sengupta, K..

29 records · Page 2Linked to original sources

bioRxiv2021

Size-dependent secondary nucleation and amplification of α-synuclein amyloid fibrils

The size of the amyloid seeds is known to modulate their autocatalytic amplification and cellular toxicity. However, the seed size-dependent secondary nucleation mechanism, toxicity, and disease-associated biological processes mediated by -synuclein (-Syn) fibrils are largely unknown. Using the cellular model and in vitro reconstitution, we showed that the size of -Syn fibril seeds not only dictates its cellular internalization and associated cell death; but also the distinct mechanisms of fibril amplification pathways involved in the pathological conformational change of -Syn. Specifically, small-sized fibril seeds showed elongation possibly through monomer addition at the fibril termini; whereas longer fibrils template the fibril amplification by surface-mediated nucleation as demonstrated by super-resolution microscopy. The distinct mechanism of fibril amplification, and cellular uptake along with toxicity suggest that breakage of fibrils into different sizes of seeds determine the underlying pathological outcome of synucleinopathies.

neuroscience↗

bioRxiv2021

Genomic architecture of three newly isolated unclassified Butyrivibrio species elucidate their potential role in the rumen ecosystem

One cellulose-degrading strain CB08 and two xylan-degrading strains XB500-5 and X503 were isolated from buffalo rumen. All the strains were designated as putative novel species of Butyrivibrio based on phylogeny, phylogenomy, digital DNA-DNA hybridization, and average nucleotide identity with their closest type strains. The draft genome length of CB08 was [~]3.54 Mb, while X503 and XB500-5 genome sizes were [~]3.24 Mb and [~]3.27 Mb, respectively. Only 68.28% of total orthologous clusters were shared among three genomes, and 40-44% of genes were identified as hypothetical proteins. The presence of genes encoding diverse carbohydrate-active enzymes (CAZymes) exhibited the lignocellulolytic potential of these strains. Further, the genome annotations revealed the metabolic pathways for monosaccharide fermentation to acetate, butyrate, lactate, ethanol, and hydrogen. The presence of genes for chemotaxis, antibiotic resistance, antimicrobial activity, synthesis of vitamins, and essential fatty acid suggested the versatile metabolic nature of these Butyrivibrio strains in the rumen environment.

genomics↗

bioRxiv2021

PCSK9 affects expression of key surface proteins in human pancreatic beta cells through intra- and extracellular regulatory circuits

Aims/hypothesisProprotein convertase subtilisin/kexin 9 (PCSK9) is involved in the degradation of LDLR. However, PCSK9 can target other proteins in a cell-type specific manner. While PCSK9 has been detected in pancreatic islets, its expression in insulin-producing pancreatic beta cells is debated. Herein, we studied PCSK9 expression, regulation and function in the human pancreatic beta cell line EndoC-{beta}H1. MethodsWe assessed PCSK9 expression in mouse and human pancreatic islets, and in the pancreatic beta cell line EndoC-{beta}H1. We also studied PCSK9 regulation by cholesterol, lipoproteins, Mevastatin, and by SREBPs transcription factors. To evaluate PCSK9 function in pancreatic beta cells, we performed PCSK9 gain-and loss-of-function experiments in EndoC-{beta}H1 using siPCSK9 or recombinant PCSK9 treatments, respectively. ResultsWe demonstrate that PCSK9 is expressed and secreted by pancreatic beta cells. In EndoC-{beta}H1 cells, PCSK9 expression is regulated by cholesterol and by SREBPs transcription factors. Importantly, PCSK9 knockdown results in multiple transcriptome, proteome and secretome deregulations and impaired insulin secretion. By gain- and loss-of-function experiments, we observed that PCSK9 regulates the expression levels of LDLR and VLDLR through an extracellular mechanism while CD36, PD-L1 and HLA-ABC are regulated through an intracellular mechanism. Conclusions/interpretationCollectively, these results highlight PCSK9 as an important regulator of CD36, PD-L1 and HLA-ABC cell surface expression in pancreatic beta cells. Data availabilityRNA-seq data have been deposited to GEO database with accession number GSE182016. Mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the following identifiers: PXD027921, PXD027911 and PXD027913.

cell biology↗

bioRxiv2021

Characterization of the secretome, transcriptome and proteome of human β cell line EndoC-βH1

Early diabetes research is hampered by limited availability, variable quality and instability of human pancreatic islets in culture. Little is known about the human {beta} cell secretome, and recent studies question translatability of rodent {beta} cell secretory profiles. Here, we verify representativeness of EndoC-{beta}H1, one of the most widely used human {beta} cell lines, as a translational human {beta} cell model based on omics and characterize the EndoC-{beta}H1 secretome. We profiled EndoC-{beta}H1 cells using RNA-seq, Data Independent Acquisition (DIA) and Tandem Mass Tag proteomics of cell lysate. Omics profiles of EndoC-{beta}H1 cells were compared to human {beta} cells and insulinomas. Secretome composition was assessed by DIA proteomics. Agreement between EndoC-{beta}H1 cells and primary adult human {beta} cells was ~90% for global omics profiles as well as for {beta} cell markers, transcription factors and enzymes. Discrepancies in expression were due to elevated proliferation rate of EndoC-{beta}H1 cells compared to adult {beta} cells. Consistently, similarity was slightly higher with benign non-metastatic insulinomas. EndoC-{beta}H1 secreted 671 proteins in untreated baseline state and 3,278 proteins when stressed with non-targeting control siRNA, including known {beta} cell hormones INS, IAPP, and IGF2. Further, EndoC-{beta}H1 secreted proteins known to generate bioactive peptides such as granins and enzymes required for production of bioactive peptides. Unexpectedly, exosomes appeared to be a major mode of secretion in EndoC-{beta}H1 cells. We believe that secretion of exosomes and bioactive peptides warrant further investigation with specialized proteomics workflows in future studies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/459582v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1976bc0org.highwire.dtl.DTLVardef@233c4eorg.highwire.dtl.DTLVardef@14c3350org.highwire.dtl.DTLVardef@1bcf0d5_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIWe validate EndoC-{beta}H1 as a translational human {beta} cell model using omics. C_LIO_LIWe present the first unbiased proteomics composition of human {beta} cell line secretome. C_LIO_LIThe secretome of human {beta} cells is more extensive than previously thought. C_LIO_LIUntreated cells secreted 671 proteins and stressed cells secreted 3,278 proteins. C_LIO_LISecretion of exosomes and bioactive peptides constitute directions of future research. C_LI

bioinformatics↗

bioRxiv2021

Active microrheology using pulsed optical tweezers to probe viscoelasticity of Lamin A towards diagnosis of laminopathies

Lamins are nucleoskeletal proteins of mammalian cells that stabilize the structure and maintain the rigidity of the nucleus. These type V intermediate filament proteins which are predominantly of A and B types provide necessary tensile strength to the nucleus. Single amino acid missense mutations occurring all over the lamin A protein form a cluster of human diseases termed as laminopathies, a few of which principally affect the muscle and cardiac tissues responsible for load bearing functionalities of the body. One such mutation is lamin A350P which causes dilated cardiomyopathy in patients. It is likely that a change from alanine to proline in the -helical 2B rod domain of the protein might severely disrupt the propensity of the filaments to polymerise into functional higher order structures required to form a fully functional lamina with its characteristic elasticity. In this study, we validate for the very first time, the application of active microrheology employing oscillating optical tweezers to investigate any alterations in the visco-elastic parameters of the mutant protein meshwork in vitro, which might translate into possible changes in nuclear plasticity. We confirm our findings from this robust yet fast method by imaging both the wild type and mutant lamins using a super resolution microscope, and observe changes in the mesh size which explain our measured changes in the viscoelastic parameters of the lamins. This method could naturally be extended to conduct microrheological measurements on any intermediate filament protein or any protein endowed with elastic behavior, with minor schematic modifications, thus bearing significant implications in laminopathies and other diseases which are associated with changes in structural rigidity of any cellular organelle. SignificanceLamin A mutations produce an array of diseases termed as laminopathies which are primarily characterized by alteration of elastic behavior of the nucleus which in turn leads to defects in mechanotransduction. This is the first report in the lamin arena which shows a fast, accurate and direct quantification of elastic moduli of lamin A using optical tweezers-based microrheology. This has very significant implications and can be registered to be a robust and universal method that could also be suitably used for probing changes in elastic properties of any proteins or surfactants in a disease scenario such as SARS-Cov2 (Covid-19), which is pandemic at this time.

biophysics↗

bioRxiv2020

Principles of organelle membranebridging established using cytosolictether mimics

The interactions between different intra-cellular organelles, including the endoplasmic reticulum, have recently been in focus thanks to the tremendous progress in imaging them using cryogenic transmission electron microscopy. However, they are still difficult to study in cellulo, and reconstituting these systems has been a standing challenge. Here we achieve this task using a giant unilamellar vesicle (GUV) and supported lipid bilayer (SLB) system. The tethers, which may reside in the cytosol when unbound, are mimicked by single (or double) stranded DNA sequences of two different lengths with ends that are self-sticky, and with terminal cholesterol moieties which insert into GUV or SLB membranes. The DNA-tethers, bound by their sticky-end, can exist in two possible states - either with both cholesterols in the same membrane or each cholesterol in a different membrane, the latter conformation leading to adhesion. Exchange of tether-molecules between the membranes occurs through the aqueous phase. By developing theoretical arguments that are supported in our experiments, we show that this possibility of exchange and the relative difference in the projected area between the two states drives the adhesion due to collective entropic considerations, rather than the usually considered enthalpy of binding. The establishment of this fundamentally different interaction between two membranes suggests that in physiological conditions, the regulation of contact formation inside cells may be very different from the case of the much studied ligand-receptor pairing on the external cell membrane.

biophysics↗

bioRxiv2020

Coexistence of long and short DNA constructs within adhesion plaques

Adhesion domains forming at the membrane interfaces between two cells or a cell and the ex-tracellular matrix commonly involve multiple proteins bridges. However, the physical mechanisms governing the domain structures are not yet fully resolved. Here we present a joint experimental and theoretical study of a mimetic model-system, based on giant unilammelar vesicles interacting with supported lipid bilayers, with which the underlying physical effects can be clearly identified. In our case, adhesion is induced by simultaneous action of DNA linkers with two different lengths. We study the organization of bridges into domains as a function of relative fraction of long and short DNA constructs. Irrespective of the composition, we systematically find adhesion domains with coexisting DNA bridge types, despite their relative differences in length of 9 nm. However, at short length scales, below the optical resolution of the microscope, simulations suggest the formation of nanodomains by the minority fraction. The nano-aggregation is more significant for long bridges, which are also more stable, even though the enthalpy of membrane insertion is the same for both species.

biophysics↗

bioRxiv2020

Alteration of epigenetic landscape by lamin A mutations: Hallmark of Dilated Cardiomyopathy

Mutations in lamin A have been reported to be associated with over 16 human diseases including dilated cardiomyopathy (DCM). We have focused on three such DCM causing mutants of lamin A which have to address the contribution of lamins in the pathogenesis of DCM at molecular level. We have elucidated the effect of these mutants for the first time on the epigenetic landscape of a myogenic fibroblast cell line C2C12. C2C12 cells expressing these mutant proteins exhibited alterations in some histone modification marks like H3K4me3, H3K9me3, H3K27me3, H3K36me3 and RNA Polymerase II activity compared to its wild type variants. This report paves the way for further studies involving epigenetic regulation in laminopathies which would be an important step in explaining the molecular mechanism and pathophysiology of the diseases like dilated cardiomyopathy. High LightsO_LIlamin A K97E mutation predominantly alters H3K9me3 histone modifications landscape C_LIO_LIlamin A K97E aggregates within nucleus also sequester the HP1{gamma} C_LIO_LIlamin A K97E mutation affects RNA polymerase II distribution pattern C_LI

cell biology↗

bioRxiv2019

Nup93 modulates spatiotemporal dynamics and function of the HOXA gene cluster during differentiation

Nucleoporins regulate nuclear transport. In addition, nucleoporins also modulate chromatin organization and gene expression. Here we investigated the role of nucleoporin Nup93, in regulating HOXA gene expression during differentiation. ChIP-Seq analysis revealed that Nup93 associates with genes involved in development and differentiation. Furthermore, Nup93 occupancy significantly overlaps with CTCF. Interestingly, Nup93 and CTCF show antagonistic roles in regulating 3 and 5 end HOXA genes in undifferentiated cells. The HOXA gene locus untethered from the nuclear periphery upon Nup93 but not CTCF depletion, consistent with its upregulation. Remarkably, occupancy of Nup93 and CTCF on HOXA gene locus progressively declined during differentiation but was restored in differentiated cells, consistent with the rerepression and re-localization of the HOXA gene locus with the nuclear periphery upon differentiation. In summary, Nup93 is a key modulator of the spatiotemporal dynamics and function of the HOXA gene locus during differentiation.

molecular biology↗

bioRxiv2019

Nuclear deformation and anchorage defect induced by DCM mutants in lamin A

Dilated Cardiomyopathy (DCM) is one of the different types of laminopathies caused by the mutations in A-type lamins in somatic cells. The involuntary cyclic stretching of cardiac muscle cells, as observed in normal physiological conditions is perturbed in DCM which afflict patients globally. As A-type lamins are principal components in nuclear mechanics, we have investigated the effect of the DCM causing mutants- K97E, E161K and R190W on nuclear stretching and deformation by static and dynamic strain inducing experiments. All the mutants exhibited differential nuclear structural aberrations along with a tilt in the nuclear axis compared to the direction of the cell axis and a significant decrease in the lamina thickness which reflected the lower mechanical rigidity. These phenotypes could potentially lead to defects in nuclear anchorage to the actin filaments thereby resulting in the misshapen and misaligned nucleus.

cell biology↗

bioRxiv2017

Biphasic mechanosensitivity of TCR mediated adhesion of T lymphocytes

Force sensitivity of the T cell receptor (TCR) is now believed to be essential for immune recognition, but cellular mechanosensitivity of T cells is still poorly understood. Here we show that T cells adhering via the TCR-complex respond to environmental stiffness in an unusual biphasic fashion. As the stiffness increases, adhesion and spreading first increase, then decrease, attaining their maximal values on an optimally stiff surface, with stiffness comparable to certain antigen presenting cells. Remarkably, in presence of additional ligands for the integrin LFA-1, spreading increases monotonously with stiffness up to a saturation value. Using a mesoscopic semi-analytical model linking spreading to molecular characteristics of bonds, we identify force sensitivity of the off-rate and the effective bond stiffness as the crucial parameters that determine monotonic or biphasic mechanosensitive behavior.

biophysics↗