Biology subjects

Sen, A.

Publications and source records attributed to Sen, A..

51 records · Page 3Linked to original sources

bioRxiv2021

Mitochondrial membrane proteins and VPS35 orchestrate selective removal of mtDNA

Integrity of mitochondrial DNA (mtDNA), encoding several subunits of the respiratory chain, is essential to maintain mitochondrial fitness. Mitochondria, as a central hub for metabolism, are affected in a wide variety of human diseases but also during normal ageing, where mtDNA integrity is compromised. Mitochondrial quality control mechanisms work at different levels, and mitophagy and its variants are critical to remove dysfunctional mitochondria together with mtDNA to maintain cellular homeostasis. Understanding the mechanisms governing a selective turnover of mutation-bearing mtDNA without affecting the entire mitochondrial pool is fundamental to design therapeutic strategies against mtDNA diseases and ageing. Here we show that mtDNA depletion after expressing a dominant negative version of the mitochondrial helicase Twinkle, or by chemical means, is due to an exacerbated mtDNA turnover. Targeting of nucleoids is controlled by Twinkle which, together with the mitochondrial transmembrane proteins ATAD3 and SAMM50, orchestrate mitochondrial membrane remodeling to form extrusions. mtDNA removal depends on autophagy and requires the vesicular trafficking protein VPS35 which binds to Twinkle-enriched mitochondrial subcompartments upon mtDNA damage. Stimulation of autophagy by rapamycin selectively removes mtDNA deletions which accumulated during muscle regeneration in vivo, but without affecting mtDNA copy number. With these results we unveil a new complex mechanism specifically targeting and removing mutant mtDNA which occurs outside the mitochondrial network. We reveal the molecular targets involved in a process with multiple potential benefits against human mtDNA related diseases, either inherited, acquired or due to normal ageing.

cell biology↗

bioRxiv2021

Allele-specific expression links diverse cis-regulatory mutations to recurrent gene dysregulation in high-risk neuroblastoma

Neuroblastoma is a pediatric malignancy with a high frequency of metastatic disease at initial diagnosis. Neuroblastoma tumors have few protein-coding mutations but contain extensive somatic copy number alterations (SCNAs) suggesting that mutations that alter gene dosage are important drivers of tumorigenesis. Here we analyze allele-specific expression (ASE) in 96 high-risk neuroblastoma tumors to discover genes with cis-acting mutations that alter dosage. We identify 1,049 genes with recurrent, neuroblastoma-specific ASE, 68% of which lie within common SCNA regions. However, many genes exhibit ASE in copy neutral samples and are enriched for mutations that cause nonsense-mediated decay, indicating that neuroblastoma tumors select for multiple types of mutations that alter gene expression. We also find 24 genes with reduced expression in stage 4 disease that have neuroblastoma-specific ASE that is independent of SCNAs. At least two of these genes have evidence for tumor suppressor activity including the transcription factor TFAP2B and the protein tyrosine phosphatase PTPRH. In summary, our ASE analysis discovers genes that are recurrently dysregulated by both large SCNAs and other cis-acting mutations in high-risk neuroblastoma.

cancer biology↗

bioRxiv2021

Leveraging Allele-Specific Expression for Therapeutic Response Gene Discovery in Glioblastoma.

Glioblastoma is the most prevalent primary malignant brain tumor in adults and is characterized by poor prognosis and universal tumor recurrence. Effective glioblastoma treatments are lacking, in part due to somatic mutations and epigenetic reprogramming that alter gene expression and confer drug resistance. Here, we interrogated allele-specific expression (ASE) in 43 patient-derived glioblastoma stem cells (GSCs) to identify recurrently dysregulated genes in glioblastoma. We identified 118 genes with recurrent ASE preferentially found in GSCs compared to normal tissues. These genes were enriched for apoptotic regulators, including Schlafen Family Member 11 (SLFN11). Loss of SLFN11 gene expression was associated with aberrant promoter methylation and conferred resistance to chemotherapy and poly ADP ribose polymerase inhibition. Conversely, low SLFN11 expression rendered GSCs susceptible to the oncolytic flavivirus Zika, which suggests a potential alternative treatment strategy for chemotherapy resistant GBMs.

cancer biology↗

bioRxiv2021

Discovering single nucleotide variants and indels from bulk and single-cell ATAC-seq

Genetic variants and de novo mutations in regulatory regions of the genome are typically discovered by whole-genome sequencing (WGS), however WGS is expensive and most WGS reads come from non-regulatory regions. The Assay for Transposase-Accessible Chromatin (ATAC-seq) generates reads from regulatory sequences and could potentially be used as a low-cost capture method for regulatory variant discovery, but its use for this purpose has not been systematically evaluated. Here we apply seven variant callers to bulk and single-cell ATAC-seq data and evaluate their ability to identify single nucleotide variants (SNVs) and insertions/deletions (indels). In addition, we develop an ensemble classifier, VarCA, which combines features from individual variant callers to predict variants. The Genome Analysis Toolkit (GATK) is the best-performing individual caller with precision/recall on a bulk ATAC test dataset of 0.92/0.97 for SNVs and 0.87/0.82 for indels. On bulk ATAC-seq reads, VarCA achieves superior performance with precision/recall of 0.99/0.95 for SNVs and 0.93/0.80 for indels. On single-cell ATAC-seq reads, VarCA attains precision/recall of 0.98/0.94 for SNVs and 0.82/0.82 for indels. In summary, ATAC-seq reads can be used to accurately discover non-coding regulatory variants in the absence of whole-genome sequencing data and our ensemble method, VarCA, has the best overall performance.

bioinformatics↗

bioRxiv2020

Estrogen receptors in human bladder cells regulate innate cytokine responses to differentially modulate uropathogenic E. coli colonization

The bladder epithelial cells elicit robust innate immune responses against urinary tract infections (UTIs) for preventing the bacterial colonization. Physiological fluctuations in circulating estrogen levels in women increase the susceptibility to UTI pathogenesis, often resulting in adverse health outcomes. Dr adhesin bearing Escherichia coli (Dr E. coli) cause recurrent UTIs in menopausal women and acute pyelonephritis in pregnant women. Dr E. coli bind to epithelial cells via host innate immune receptor CD55, under hormonal influence. The role of estrogens or estrogen receptors (ERs) in regulating the innate immune responses in the bladder are poorly understood. In the current study, we investigated the role of ER, ER{beta} and GPR30 in modulating the innate immune responses against Dr E. coli induced UTI using human bladder epithelial carcinoma 5637 cells (HBEC). Both ER and ER{beta} agonist treatment in bladder cells induced a protection against Dr E. coli invasion via upregulation of TNF and downregulation of CD55 and IL10, and these effects were reversed by action of ER and ER{beta} antagoinsts. In contrast, the agonist-mediated activation of GPR30 led to an increased bacterial colonization due to suppression of innate immune factors in the bladder cells, and these effects were reversed by the antagonist-mediated suppression of GPR30. Further, siRNA-mediated ER knockdown in the bladder cells reversed the protection against bacterial invasion observed in the ER positive bladder cells, by modulating the gene expression of TNF, CD55 and IL10, thus confirming the protective role of ER. We demonstrate for the first time a protective role of nuclear ERs, ER and ER{beta} but not of membrane ER, GPR30 against Dr E. coli invasion in HBEC 5637 cells. These findings have many clinical implications and suggest that ERs may serve as potential drug targets towards developing novel therapeutics for regulating local innate immunity and treating UTIs. HighlightsO_LIEstrogen receptor (ER) subtypes regulate the gene expression of innate immune molecules, CD55, TNF and IL10 in human bladder epithelial cells impacting the bacterial colonization by Dr E. coli. C_LIO_LIActivation of nuclear ER subtypes, ER and ER{beta}, upregulate the gene expression of proinflammatory cytokine, TNF, but downregulate the gene expression of anti-inflammatory cytokine, IL10, and Dr E. coli colonization receptor, CD55, thus leading to efficient bacterial clearance in human bladder cells. C_LIO_LIIn contrast, activation of the membrane ER subtype, GPR30, shows opposite effects to ER and ER{beta} that were mediated on TNF, IL10 and CD55 gene expression, thus leading to impaired bacterial clearance of Dr E. coli in human bladder cells. C_LIO_LIER subtypes can serve as potential drug candidates for designing new therapies to boost or modulate the local immunity in the human bladder preventing the establishment of E. coli infections. C_LI

immunology↗

bioRxiv2020

Scaffolding protein IQ motif containing GTPase activating protein 2 (IQGAP2) regulates glycogen metabolism

The liver is critical in maintaining metabolic homeostasis, regulating both anabolic and catabolic processes of fats, proteins, and carbohydrates. The IQ motif containing GTPase activating protein 2 (IQGAP2) is a member of the IQGAP family. Of the three homologous isoforms, the IQGAP2 scaffolding protein is predominantly found in the liver. To characterize its role in regulating metabolism, Iqgap2-/- female and male mice, and their WT controls, were fed ad libitum or fasted for 24 hours. Hepatic gene expression, protein levels, and the metabolic response were compared between WT and Iqgap2-/- mice, using RT-qPCR, western blot analysis, and histological stains. We found that loss of IQGAP2 alters the phosphorylation of active glycogen synthase kinase 3 (GSK3) expression, a known regulator of glycogen synthesis and lipogenesis. Consistent with this result, Iqgap2-/- female mice displayed depletion of periportal glycogen even in the fed state. We also observed the blunted expression of genes involved in glycogenesis and lipogenesis when IQGAP2 was deleted. Since GSK3 is known to regulate the activity of {beta}-catenin, we examined and found it to be reduced in Iqgap2-/- mice. Our findings demonstrate that IQGAP2 plays an important role in regulating glycogen synthesis.

molecular biology↗

bioRxiv2020

Ubiquitination and Phosphorylation are Independently Required for Epsin-Mediated Internalization of Cargo in S. cerevisiae

It is well-known that in addition to its classical role in protein turnover, ubiquitination is required for a variety of membrane protein sorting events. However, and despite substantial progress in the field, a long-standing question remains: given that all ubiquitin (Ub) units are identical, how do different elements of the sorting machinery recognize their specific cargoes? Here we provide an answer to this question as we discovered a mechanism based on the coincidence detection of lysine ubiquitination and Ser/Thr phosphorylation for the endocytic adaptor epsin to mediate the internalization of the yeast Na+ pump Ena1. Internalization of Ena1-GFP was abolished in double epsin knock-out in S. cerevisiae and was rescued by re-introducing either one of the 2 yeast epsins, Ent1 or Ent2 in an UIM (Ub Interacting Motif)-dependent manner. Further, our results indicate that ubiquitination of its C-terminal Lys1090 is needed for internalization of Ena1 and requires the arrestin-related-trafficking adaptor, Art3. We determined that in addition to ubiquitination of K1090, the presence of a Ser/Thr-rich patch (S1076TST1079) within Ena1 was also essential for its internalization. Our results suggest that this ST motif is targeted for phosphorylation by casein kinases. Nevertheless, phosphorylation of this S/T patch was not required for ubiquitination. Instead, ubiquitination of K1090 and phosphorylation of the ST motif were independently needed for epsin-mediated internalization of Ena1. We propose a model in which a dual detection mechanism is used by Ub-binding elements of the sorting machinery to differentiate among multiple Ub-cargoes.

cell biology↗

bioRxiv2020

A comprehensive and comparative study on the action of pentacyclic triterpenoids on Vibrio cholerae biofilms.

While serving as environmental reservoir for V. cholerae infection, biofilms are also crucial for intestinal colonization of the pathogen. Triterpenoids, a group of bioactive phytochemicals, have been tested for antibiofilm activity against model biofilm forming bacteria in recent times. In this context, glycyrrhetinic acid (GRA), ursolic acid (UA) and betulinic acid (BA), representing three categorically distinct groups of pentacyclic triterpenoids, are targeted for profiling their impact on Vibrio cholerae C6709 biofilms. The triterpenoids substantially affected biofilm associated attributes like formation, substratum adherence and dispersion from preformed biofilms. Though at variable degree, the compounds decreased cell surface hydrophobicity and composition in terms of macromolecular content. Not only EPS-associated extracellular enzyme activities were estimated to be reduced by triterpenoid exposure, ultra structural analysis also revealed that GRA, UA and BA can affect extracellular polymeric substance (EPS) content. Albeit total extracellular proteolytic activity remained unaffected by the triterpenoids, GRA treatment resulted in considerable reduction of extracellular gelatinase activity. Molecular docking analysis indicated potential interaction with cyclic di-GMP sensor VpsT, autoinducer-2 sensor kinase LuxP-LuxQ and transcriptional activator HapR, component of complex quorum sensing networks modulating biofilm formation. Comprehensive analysis of antibiotic action revealed accentuation of cephalosporin antibiotics with GRA and UA while BA potentiated action of fluoroquinolones, widening the scope of combinatorial therapeutic strategy.

microbiology↗

bioRxiv2019

Rotavirus infection induces both interferon (IFN) and inflammatory resistance by re-programming intestinal IFN receptor signaling

Rotaviruses (RV) cause acute severe diarrhea in the absence of substantial intestinal inflammation. They are also highly infectious in their homologous host species. The efficient replication capacity of RV in the small bowel is substantially linked to its ability to inhibit different types of interferons (IFNs). Here, we find that during RV infection in vitro, both virus-infected and uninfected bystander cells resist STAT1 phosphorylation and IRF7 induction in response to exogenous IFN. Functionally, cellular transcription in response to exogenous stimulation with IFN, but not intracellular dsRNA, was inhibited by RV. Further, IFNAR1 stimulation during RV infection significantly repressed a set of virus-induced transcripts. Regulation of IFN signaling in vivo was studied in suckling mice using the highly infectious homologous murine EW RV strain. Kinetic studies indicated that whereas sustained EW RV replication and IFN induction occurred in the small intestine, IFN-stimulated transcripts significantly decreased over time. In addition, LPS-mediated intestinal damage, driven by STAT1-induced inflammation, was prevented in EW RV-infected mice. Remarkably, ectopic stimulation of either IFNAR1 or IFNGR1 in murine RV-infected mice eliminated several intestinal antiviral and inflammatory transcriptional responses to RV. In contrast to homologous murine RV, infection with a STAT1-sensitive heterologous simian RV strain induced multiple IFN-stimulated transcripts, inflammatory cytokines, and intestinal expression of STAT1-pY701. Finally, RV strain-specific STAT1 regulation in the gut plays a prominent role in the activation of multiple intestinal caspases. On the other hand, the simian RRV strain, but not murine EW RV, uniquely triggers the cleavage of both extrinsic and intrinsic caspases (-8, -9, and -3) in a STAT1-mediated manner. Collectively, these findings reveal efficient re-programing of multiple IFN receptors in the gut towards a negative feedback mode of signaling, accompanied by suppression of IFN-mediated antiviral, apoptotic, and inflammatory functions, during natural RV intestinal infection.

immunology↗

bioRxiv2019

Motility of Enzyme-Powered Vesicles

Autonomous nanovehicles powered by energy derived from chemical catalysis have potential applications as active delivery agents. For in vivo applications, it is necessary that the engine and its fuel, as well as the chassis itself, be biocompatible. Enzyme molecules have been shown to generate mechanical force through substrate turnover and are attractive candidates as engines; phospholipid vesicles are biocompatible and can serve as cargo containers. Herein, we describe the autonomous movement of vesicles with membrane-bound enzymes in the presence of the substrate. We find that the motility of the vesicles increases with increasing enzymatic turnover rate. The enhanced diffusion of these enzyme-powered systems was further substantiated in real time by tracking the motion of the vesicles using optical microscopy. The membrane-bound protocells that move by transducing chemical energy into mechanical motion serve as models for motile living cells and are key to the elucidation of the fundamental mechanisms governing active membrane dynamics and cellular movement.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC=\"FIGDIR/small/645986v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (30K):\norg.highwire.dtl.DTLVardef@49a48org.highwire.dtl.DTLVardef@2d0bc0org.highwire.dtl.DTLVardef@15e9466org.highwire.dtl.DTLVardef@60ba5e_HPS_FORMAT_FIGEXP M_FIG \"For Table of Contents Only\"\n\nC_FIG

biochemistry↗

bioRxiv2019

BreakCA, a method to discover indels using ChIP-seq and ATAC-seq reads, finds recurrent indels in regulatory regions of neuroblastoma genomes

Most known cancer driver mutations are within protein coding regions of the genome, however, there are several important examples of oncogenic non-coding regulatory mutations. We developed a method to identify insertions and deletions (indels) in regulatory regions using aligned reads from chromatin immunoprecipitation followed by sequencing (ChIP-seq) or the assay for transposase-accessible chromatin (ATAC-seq). Our method, which we call BreakCA for Breaks in Chromatin Accessible regions, allows non-coding indels to be discovered in the absence of whole genome sequencing data, out-performs popular variant callers such as the GATK-HaplotypeCaller and VarScan2, and detects known oncogenic regulatory mutations in T-cell acute lymphoblastic leukemia cell lines. We apply BreakCA to identify indels in H3K27ac ChIP-seq peaks in 23 neuroblastoma cell lines and, after removing common germline variants, we identify 23 rare germline or somatic indels that occur in multiple neuroblastoma cell lines. Among them, 4 indels are candidate oncogenic drivers that are present in 4 or 5 cell lines, absent from the genome aggregation database of over 15,000 whole genome sequences, and within the promoters or first introns of known genes (PHF21A, ADAMTS19, GPR85 and RALGDS). In addition, we observe a rare 7bp germline deletion in two cell lines, which is associated with high expression of the histone demethylase KDM5B. Overexpression of KDM5B is prognostic for many cancers and further characterization of this indel as a potential oncogenic risk factor is therefore warranted.

bioinformatics↗

bioRxiv2019

Non-uniform Crowding Enhances Transport: Relevance to Biological Environments

The cellular cytoplasm is crowded with macromolecules and other species that occupy up to 40% of the available volume. Previous studies have reported that for high crowder molecule concentrations, colloidal tracer particles have a dampened diffusion due to the higher solution viscosity. However, these studies employed uniform distributions of crowder molecules. We report a scenario, previously unexplored experimentally, of increased tracer transport driven by a non-uniform concentration of crowder macromolecules. In gradients of polymeric crowder, tracer particles undergo transport several times higher than that of their bulk diffusion rate. The direction of the transport is toward regions of lower crowder concentration. Mechanistically, hard-sphere interactions and the resulting volume exclusion between the tracer and crowder increases the effective diffusion by inducing a convective motion of tracers. Strikingly, soft deformable particles show even greater enhancement in transport in crowder gradients compared to similarly sized hard particles. We propose a model that supports the data and quantifies a \"diffusiophoretic buoyancy force\" when a tracer is in a gradient of crowder concentration. Overall, this demonstration of enhanced transport in non-uniform distributions of crowder is anticipated to clarify aspects of multi-component intracellular transport.

biophysics↗

bioRxiv2019

Clueless forms dynamic, insulin-responsive bliss particles sensitive to stress

Mitochondria perform a myriad of biochemical functions in the cell that integrate ATP production and metabolism. While mitochondria contain their own genome, mtDNA, it only encodes thirteen proteins required for oxidative phosphorylation, thus well over one thousand proteins required for all mitochondrial functions are encoded in the nucleus. One such protein is Drosophila Clueless (Clu), whose vertebrate homolog is Clustered mitochondria homolog (Cluh). Clu/Cluh is a ribonucleoprotein that regulates mRNAs destined for import into mitochondria and is an essential protein that regulates cellular metabolism. Clu forms large particles in the cytoplasm, although how these particles relate to nutrition and metabolic stress is unknown. Using live-imaging, we show Clu particles are highly dynamic. Clu particles appear to be unique as they do not colocalize with many known cytoplasmic bodies. In addition, Clu particle formation is highly dependent on diet as ovaries from starved females no longer contain Clu particles although Clu protein levels remain the same and insulin is necessary and sufficient for Clu particle formation. Oxidative stress also disperses particle. Since Clu particles are only present under optimal conditions we are naming them bliss particles. These observations identify Clu particles as unique, stress-sensitive cytoplasmic ribonucleoprotein particles whose absence corresponds with altered mitochondrial function and localization.

cell biology↗

bioRxiv2018

Functional Balance between TCF21-Slug defines phenotypic plasticity and sub-classes in high-grade serous ovarian cancer

Cellular plasticity and transitional phenotypes add to complexities of cancer metastasis initiated by single cell epithelial to mesenchymal transition or cooperative cell migration (CCM). We identified novel regulatory cross-talks between Tcf21 and Slug in mediating phenotypic and migration plasticity in high-grade serous ovarian adenocarcinoma. Live imaging discerned CCM as being achieved either through rapid cell proliferation or sheet migration. Transitional states were enriched over the rigid epithelial or mesenchymal phenotypes under conditions of environmental stresses. The Tcf21-Slug interplay identified in HGSC tumors through effective stratification of subtypes also contributed to class-switching in response to disease progression or therapy. Our study effectively provides a framework for understanding the relevance of cellular plasticity in situ as a function of two transcription factors.

cancer biology↗

bioRxiv2017

Proximity labeling reveals an extensive steady-state stress granule interactome and insights to neurodegeneration

Stress granules (SGs) are transient ribonucleoprotein (RNP) aggregates that form in response to proteotoxic stress. Although SGs are distinct from aggregates observed in neurodegenerative disorders, they share protein components. We used APEX-mediated proximity labeling combined with quantitative mass spectrometry and high-throughput imaging to identify >100 previously unknown SG proteins in human cells, about 10% of which localize to SGs in a cell type- or stress type-dependent manner. Supporting a link between SG proteins and neurodegeneration, we demonstrate aberrant SG composition and subcellular distribution in iPSC-derived motor neurons from ALS patients, and identify several known and previously unidentified SG proteins that modify toxicity of mutant FUS and TDP-43 overexpression in Drosophila. We show that even in an unstressed steady-state, SG proteins form a densely-connected protein interaction network (PIN) and propose a model in which existing RNPs coalesce rapidly into microscopically visible granules that can act as gateways to pathological protein aggregation.\n\nHighlights O_LIAPEX proximity labeling of dynamic RNP granules identifies over 100 novel SG proteins\nC_LIO_LISG proteins form a densely-connected protein interaction network in unstressed cells\nC_LIO_LISystematic immunofluorescence analysis reveals stress- and cell type-specific SG composition\nC_LIO_LIALS motor neurons contain SGs with distinct content and subcellular distribution\nC_LI

cell biology↗