Biology subjects

Ray, A.

Publications and source records attributed to Ray, A..

At least 73 records · Page 4Linked to original sources

bioRxiv2021

A high content lipidomics method using scheduled MRM with variable retention time window and relative dwell time weightage

Lipid compositions of cells, tissues and bio-fluids are complex, with varying concentrations and structural diversity, which makes their identification challenging. Newer methods for comprehensive analysis of lipids are thus necessary. Herein, we propose a targeted-mass spectrometry based method for large-scale lipidomics using a combination of variable retention time window and relative dwell time weightage. Using this, we detected more than 1000 lipid species, including structural isomers. The limit of detection varied from femtomolar to nanomolar range and the coefficient of variance <30% for 849 lipid species. We used this method to identify lipids altered due to Vitamin B12 deficiency and found that the levels of lipids with {omega}-3 fatty acid chains decreased while those with {omega}-6 increased. This method enables identification of by far the largest number of lipid species with structural isomers in a single experiment and would significantly advance our understanding of the role of lipids in biological processes.

biochemistry↗

bioRxiv2020

CICLOP: A Robust, Faster, and Accurate Computational Frameworkfor Protein Inner Cavity Detection

Internal cavities in proteins are of critical functional importance. They can serve as substrate/ligand binding sites, pave path for movement of bio-molecules and even mediate structural conformations occurring between domain interfaces during structural transitions. Yet, there is a paucity of computational tools that can accurately, and reliably characterize the inner cavities of the proteins, a prerequisite for elucidating their functions. We have developed a novel method, CICLOP, that can accurately identify these regions at an atomic resolution. The method is able to accurately detect residues lining the inner cavity, the diameter and volume occupied by the cavity, as well as physicochemical properties of residues lining the cavity such as their hydrophobicity and secondary structure distribution in detail. Additionally, our method also provides an option for computing conservation scores for the residues detected on the inside, allowing for a thorough functional characterization of the cavity. AvailabilityCICLOP is available at http://ciclop.raylab.iiitd.edu.in/. A compiled Linux executable can be downloaded from https://ciclop.raylab.iiitd.edu.in/standalone/

bioinformatics↗

bioRxiv2020

Global Absence and Targeting of Protective Immune States in Severe COVID-19.

While SARS-CoV-2 infection has pleiotropic and systemic effects in some patients, many others experience milder symptoms. We sought a holistic understanding of the severe/mild distinction in COVID-19 pathology, and its origins. We performed a whole-blood preserving single-cell analysis protocol to integrate contributions from all major cell types including neutrophils, monocytes, platelets, lymphocytes and the contents of serum. Patients with mild COVID-19 disease display a coordinated pattern of interferon-stimulated gene (ISG) expression across every cell population and these cells are systemically absent in patients with severe disease. Severe COVID-19 patients also paradoxically produce very high anti-SARS-CoV-2 antibody titers and have lower viral load as compared to mild disease. Examination of the serum from severe patients demonstrates that they uniquely produce antibodies with multiple patterns of specificity against interferon-stimulated cells and that those antibodies functionally block the production of the mild disease-associated ISG-expressing cells. Overzealous and auto-directed antibody responses pit the immune system against itself in many COVID-19 patients and this defines targets for immunotherapies to allow immune systems to provide viral defense. One Sentence SummaryIn severe COVID-19 patients, the immune system fails to generate cells that define mild disease; antibodies in their serum actively prevents the successful production of those cells.

immunology↗

bioRxiv2020

Cysteine residues in the C-terminal tail of connexin32 regulate its trafficking

Gap junctions (GJ)s are formed by the assembly of constituent transmembrane proteins called connexins (Cxs). Aberrations in this assembly of Cxs are observed in several genetic diseases as well as in cancers. Hence it becomes imperative to understand the molecular mechanisms underlying such assembly defect. The polarized cells in the epithelia express Connexin32 (Cx32). The carboxyl-terminal tail (CT) of Cx32 orchestrates several aspects of GJ dynamics, function and growth. The study here was aimed at determining if post-translational modifications, specifically, palmitoylation of cysteine residues, present in the CT of Cx32, has any effect on GJ assembly. The CT of Cx32 was found to harbor three cysteine residues, which are likely to be modified by palmitoylation. The study here has revealed for the first time that Cx32 is palmitoylated at cysteine 217 (C217). However, it was found that mutating C217 to alanine affected neither the trafficking nor the ability of Cx32 to assemble into GJs. Intriguingly, it was discovered that mutating cysteine 280 and 283 in combination, blocked the transport of Cx32 from the Golgi to the cell surface. Overall, the findings reveal the importance of the two terminal cysteine residues of Cx32 in regulating its trafficking and stability and hence is ability to assemble into GJs, possibly as being part of a CAAX motif in its CT.

cell biology↗

bioRxiv2020

Localized Proteotoxic Stress in Mitochondrial Intermembrane Space and Matrix Elicits Sub-compartment Specific Response Pathways Governed by Unique Modulators

The double-membrane-bound architecture of mitochondria, essential for ATP production, sub-divides the organelle into inter-membrane space (IMS) and matrix. IMS and matrix possess contrasting oxido-reductive environments and distinct protein quality control (PQC) machineries resulting different protein folding environments. To understand the nature of stress response elicited by equivalent proteotoxic stress to sub-mitochondrial compartments, we fused well-described bipartite or matrix-targeting signal sequences to misfolding and aggregation-prone stressor proteins to target and impart stress to yeast mitochondrial IMS or matrix. We show, mitochondrial proteotoxicity leads to growth arrest of yeast cells of varying degrees depending on nature of stressor proteins and the intra-mitochondrial location of stress. Next, using transcriptomics and proteomics, we report a comprehensive stress response elicited by two types of targeting signal-fused stressor proteins. Among global responses by mitochondria-targeted stressors by both types of signal sequences, an adaptive response of abrogated mitochondrial respiration and concomitant upregulation of glycolysis is uncovered. Beyond shared stress responses, specific signatures due to stress within mitochondrial sub-compartments are also revealed. We report that bipartite signal sequence-fused stressor proteins eliciting stress to IMS, leads to specific upregulation of IMS-chaperones and TOM complex components. In contrast, matrix-targeted stressors lead to specific upregulation of matrix-chaperones and cytosolic PQC components. Finally, by systematic genetic interaction using deletion strains of differentially upregulated genes, we found prominent modulatory role of TOM complex components during IMS-stress response. In contrast, VMS1 markedly modulates the stress response originated from matrix.

cell biology↗

bioRxiv2020

Docosahexaenoic acid (DHA), a nutritional supplement, modulates steroid insensitivity in asthma

Asthmatics with poor steroid responsiveness are now found to use health services at higher frequency and contribute to socio-economic burden disproportionately. We have previously shown that a ω-6 fatty acid metabolite leads to a severe and steroid insensitive asthma-like condition in mice. Here, we investigated the role of retinoid-x-receptor gamma (RXRγ) and Docosahexaenoic acid (DHA), a ω3 fatty acid rexinoid ligand of RXR, on the features of steroid insensitivity in asthmatic condition. RXRγ was found to be reduced in the lungs of human asthmatics and mice with steroid insensitive allergic airway inflammation. RXRγ knockdown in naïve mice led to spontaneous asthma like features whereas RXRγ knockdown in allergic mice led to steroid insensitive asthma features. We observed while RXRγ binds to the glucocorticoid receptor (GR) gene and regulates its transcription, DHA increases the GRα expression in human bronchial epithelial cells and reverses the steroid insensitive features in mice with allergic airway inflammation. Docosahexaenoic acid (DHA), a ligand of RXR, was reduced in the sera of steroid-insensitive asthmatics. We conclude that DHA may prove to be a promising steroid sensitizing agent for the treatment of steroid insensitive asthmatics.Summary The molecular regulation of glucocorticoid receptor by retinoid-x-receptor gamma (RXRgama) has an implication in steroid insensitive asthma as we found that Docosahexaenoic acid (DHA), a nutritional supplement and natural ligand of RXRgamma, improves steroid sensitivity in steroid insensitive mice model of asthma and DHA levels are found to be low in steroid insensitive asthmatic patients.Competing Interest StatementThe authors have declared no competing interest.Abbreviations usedAAIAllergic airway inflammationAHRAirway hyper-responsivenessATRAAll-trans retinoic acidAUArbitrary unitsBALBronchoalveolar lavageBEAS-2BHuman bronchial epithelial cellsCECockroach allergen extractChIPChromatin Immunoprecipitation9CRA9-cis retinoic acidDEXDexamethasoneDHADocosahexaenoic acidDMSODimethyl sulfoxideELISAEnzyme-linked immunosorbent assayGAPDHGlyceraldehyde 3-phosphate dehydrogenaseGLucGaussia luciferaseGRαGlucocorticoid receptor alphaGREGlucocorticoid receptor elementH & EHaematoxylin and eosinIFNγInterferon gammaIgEImmunoglobulinE ILInterleukinKCKeratinocyte chemoattractant15-LOX15-lipoxygenaseMCF-7Human breast adenocarcinoma cell lineMCP1-αMonocyte chemoattractant protein1-αNF-κBNuclear Factor kappa-light-chain-enhancer of activated B cellsO.EOverexpressionOVAOvalbuminp38-MAPKp38 mitogen-activated protein kinasePBSPhosphate buffered salineRARRetinoic acid receptorPDTCPyrrolidinedithiocarbamateRXRERexinoid receptor elementRXRγRetinoid-x-receptor gamma13-S-HODE13-hydroxyoctadecadienoic acidSEMStandard error meansiRNASmall interfering RNASTAT-6Signal transducer and activator of transcription 6ThT helperVEHVehicleView Full Text

immunology↗

bioRxiv2020

GroEL/ES Buffers Entropic Traps In Folding Pathway During Evolution Of A Model Substrate

The folding landscape of proteins can change during evolution with the accumulation of mutations that may introduce entropic or enthalpic barriers in the protein folding pathway, making it a possible substrate of molecular chaperones in vivo. Can the nature of such physical barriers of folding dictate the feasibility of chaperone-assistance? To address this, we have simulated the evolutionary step to chaperone-dependence keeping GroEL/ES as the target chaperone and GFP as a model protein in an unbiased screen. We find that the mutation conferring GroEL/ES dependence in vivo and in vitro encode an entropic trap in the folding pathway rescued by the chaperonin. Additionally, GroEL/ES can edit the formation of non-native contacts similar to DnaK/J/E machinery. However, this capability is not utilized by the substrates in vivo. As a consequence, GroEL/ES caters to buffer mutations that predominantly cause entropic traps, despite possessing the capacity to edit both enthalpic and entropic traps in the folding pathway of the substrate protein.

molecular biology↗

bioRxiv2020

FGF signaling regulates development through combinatorial transduction pathways and by modulating cellular adhesion

FGFs are key developmental regulators which engage a signal transduction cascade through receptor tyrosine kinases, typically involving ERK1/2, PI3K/AKT, and other effectors. However, it remains unknown if all FGF activities depend on kinase activity or these canonical signal transduction cascades. To address these questions, we generated allelic series of knock-in Fgfr1 and Fgfr2 mouse strains, carrying point mutations that disrupt binding of signaling effectors to the receptors, alone or in combination. We also produced a kinase dead allele of Fgfr2 which broadly phenocopies the null mutant. When interrogated in cranial neural crest cells, point mutations in either receptor revealed discrete functions for signaling pathways in specific craniofacial contexts, but failed to recapitulate the single or double null mutant phenotypes even in their most extensive combination. Furthermore, we found that together these signaling mutations abrogated the established FGF-induced signal transduction pathways, yet certain FGF functions such as cell-matrix and cell-cell adhesion remained unaffected. Our studies establish combinatorial roles of both Fgfr1 and Fgfr2 in development and identify novel kinase-dependent cell adhesion properties of FGF receptors, independent of well-established roles in intracellular signaling.Competing Interest StatementThe authors have declared no competing interest.View Full Text

developmental biology↗

bioRxiv2020

Western Ghats Myrtaceae are not Gondwana elements but likely dispersed from south-east Asia

Gondwana break-up is one of the key sculptors of the global biogeographic pattern including Indian subcontinent. Myrtaceae, a Gondwana family, demonstrates a high diversity across Indian Western Ghats. A rich paleo-records in Deccan Intertrappean bed in India strive to reconcile two contending hypothesis of origin and diversification of Myrtaceae in India, namely Gondwana elements floated on peninsular India or lineages invading from south-east Asia. In this study, we have reconstructed the biogeographic history of Myrtaceae of Western Ghats of India combining fossil-calibrated phylogeny, ancestral area reconstruction, and comparing various dispersal models. Phylogenetic reconstructions divided lineages into multiple clades at the tribal level that mostly is in agreement with previous descriptions. Dated tree indicated a presumable Gondwanan origin of Myrtaceae (95.7 Ma). The Western Ghats taxa are relatively younger and seem to have arrived either through long-distance oceanic dispersal in middle Eocene (Rhodomyrtus tomentosa), and Miocene (Eugenia) or through major overland dispersals throughout Miocene (Syzygium). However, late Cretaceous fossilsin India lends support to the existence of proto-Myrtaceae on floating peninsular India. Taken together, we conclude that Myrtaceae lineages on the biotic ferry were perhaps extirpated by Deccan volcanism leaving a little or no trace of living Gondwanan link, while the Western Ghats was likely enriched by dispersed elements either from south-east Asia or from South America.

genomics↗

bioRxiv2019

Vibrio deploys Type 2 secreted lipase to esterify cholesterol with host fatty acids and mediate cell egress

Pathogens find diverse niches for survival inside host cells where replication occurs in a relatively protected environment. Vibrio parahaemolyticus, a facultative intracellular pathogen, uses its type 3 secretion system 2 (T3SS2) to invade and replicate inside host cells. However, after extensive analysis, the T3SS2 pathogenicity island appeared to lack a mechanism for egress of this bacterium from the invaded host cell. Using a combination of cell biology, microbial genetics and lipid biochemistry, we found that VPA0226, a constitutively secreted lipase, is required for escape of Vibrio parahaemolyticus from host cells. Remarkably, this lipase must be delivered into the host cytoplasm where it preferentially uses fatty acids associated with innate immune response (i.e. arachidonic acid, 20:4) to esterify cholesterol, weakening the plasma membrane and allowing egress of the bacteria. This study reveals the resourcefulness of microbes and the interplay between virulence systems to evolve an ingenious scheme for survival and escape. Impact StatementConsidering the course of a pathogens evolution, there appears to be interplay between secretion systems, providing unique, synergistic mechanisms to support a successful lifestyle for possibly pathogenesis, symbiosis and/or parasitosis.

microbiology↗

bioRxiv2019

Contributions of the conserved insect carbon dioxide receptor subunits to odor detection

The CO2 receptor in mosquitoes is broadly tuned to detect many diverse odorants. The receptor consists of three 7-TM subunits (Gr1, Gr2, and Gr3) in mosquitoes but only two subunits in Drosophila: Gr21a (Gr1 ortholog) and Gr63a (Gr3 ortholog). We demonstrate that Gr21a is required for CO2 responses in Drosophila as has been shown for Gr63a. Next, we generate a Drosophila double mutant for Gr21a and Gr63a, and in this background we functionally express combinations of Aedes Gr1, 2, and 3 genes in the \"CO2 empty neuron.\" Only two subunits, Gr2 and Gr3, suffice for response to CO2. Addition of Gr1 increases sensitivity to CO2 while it decreases the response to pyridine. The inhibitory effect of the antagonist isobutyric acid is observed upon addition of Gr1. Gr1 therefore increases the diversity of ligands of the receptor, and also modulates the response of the receptor complex.

neuroscience↗

bioRxiv2019

Interactions of Plasmodium falciparum ETRAMP14.1 with PfEMP1, translocon components and other ETRAMP members at the interface of host-parasite

The Early Transcribed Membrane Proteins (ETRAMPs) belong to a multigene family which are conserved, are specific to Plasmodium species, and abundantly present in parasitophorous vacuolar membrane (PVM). The functions of the members of this family are poorly understood. PfETRAMP14.1 (PF3D7_1401400) is a member of this family, present only in Plasmodium falciparum. In this study, we report the potential interacting partners of PfETRAMP14.1 by using immunoprecipitation (IP) LC-MS/MS as well as protein-interaction network reconstructed on in vivo array analyses of severe malaria inflicted patients from malaria endemic Indian regions. We find PfETRAMP14.1 to be the most highly transcribed gene in severe infection. Our studies with western blot analysis and Immuno-flurorescence show that PfETRAMP14.1 is expressed at PVM during all the intraerythrocytic stages of P.falciparum with maximum expression at early trophozoite stage. Further, our results reveal interactions of ETRAMP14.1 with Plasmodium falciparum erythrocyte membrane protein 1(PfEMP1), thioredoxin (TRX2), export protein 2 (EXP2), heat shock protein 70-1 (Hsp70-1) and some of the ETRAMP family members. We propose that ETRAMP14.1 helps trafficking of PfEMP1 to the host RBC surface in conjunction with translocon machinery and the chaperone HSP 70-1.

molecular biology↗

bioRxiv2019

Allergen-induced airway inflammation and Th2 cell development requires Blimp-1

A Th2 immune response is central to allergic airway inflammation, which afflicts millions worldwide. However, the mechanisms that augment GATA3 expression in an antigen-primed developing Th2 cell are not well understood. Here, we describe an unexpected role for Blimp-1, a transcriptional repressor that constrains autoimmunity, as an upstream promoter of GATA3 expression that is critical for Th2 cell development in the lung, but dispensable for TFH function and IgE production. Mechanistically, Blimp-1 acts through Bcl6, which is necessary to drive GATA3 expression. Surprisingly, the anti-inflammatory cytokine IL-10, but not the pro-inflammatory cytokines IL-6 or IL-21, is required via STAT3 activation to upregulate Blimp-1 and promote Th2 cell development. These data reveal a hitherto unappreciated role for an IL-10-STAT3-Blimp-1 circuit as an initiator of an inflammatory Th2 response in the lung to allergens. Thus, Blimp-1 in a context-dependent fashion can drive inflammation by promoting rather than terminating effector T cell responses. SummaryThe transcriptional repressor Blimp-1 acts via a pro-inflammatory IL-10-STAT3 axis as a critical positive regulator of Th2 cells in the lung in response to allergens driving pathophysiology associated with asthma disease.

immunology↗

bioRxiv2019

Circadian regulation of light-evoked attraction/avoidance in day- vs. night-biting mosquitoes

Mosquitoes pose widespread threats to humans and other animals as disease vectors. Day- vs. night-biting mosquitoes occupy distinct time-of-day niches and exhibit very different innate temporal attraction/avoidance behavioral responses to light, yet little is known about their circuit or molecular mechanisms. Day-biting diurnal mosquitoes Aedes aegypti are attracted to light during the day regardless of spectra. In contrast, night-biting nocturnal mosquitoes Anopheles coluzzii avoid short, but not long wavelength light. Attraction/avoidance behavioral responses to light in both species change with time-of-day and show distinct sex and circuit differences. The basis of diurnal versus nocturnal behavior is driven by clock timing, which cycle anti-phase between day-biting versus night-biting mosquito species. Disruption of the circadian molecular clock severely interferes with light-evoked attraction/avoidance behavior in mosquitoes. In summary, attraction/avoidance mosquito behaviors are circadian and light regulated, which may be applied towards species specific control of harmful mosquitoes.

neuroscience↗

bioRxiv2019

Francisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing

Genome editing using the CRISPR Cas9 system has been used to manipulate eukaryotic DNA and make precise heritable changes. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain, regarding their putative off targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target sgRNA are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher HDR rates and very low non-specific genome editing compared to SpCas9. We demonstrate FnCas9 mediated correction of the sickle cell mutation in patient derived iPSCs and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.\n\nSIGNIFICANCE STATEMENTTherapeutic genome editing has been significantly accentuated by the advent of CRISPR based gene correction. However, genomic off-targeting has been a major setback for clinical translation. Although high fidelity versions of Cas9 have been rationally designed, they recognize and bind to off-targets. In this study, we characterize a naturally occurring Cas9 from Francisella novicida (FnCas9) that shows negligible binding affinity to off targets differing by one or more mismatches, rendering it highly specific in target recognition and editing. We show that FnCas9 can direct both HDR and NHEJ mediated DNA repair, generates higher rate of HDR and negligible off-target editing. Finally we show its potential in therapeutic genome editing by correcting the sickle cell anemia mutation in patient derived iPSCs.

molecular biology↗

bioRxiv2019

Exosome-mediated MIR211 modulates tumor microenvironment via the DUSP6-ERK5 axis and contributes to BRAFV600E inhibitor resistance in melanoma.

The microRNA MIR211 is an important regulator of melanoma tumor cell behavior. Previous studies suggested that in certain tumors, MIR211 acted as a tumor suppressor while in others it behaved as an oncogenic regulator. When MIR211 is expressed in BRAFV600E-mutant A375 melanoma cells in mouse xenografts, it promotes aggressive tumor growth accompanied by increased cellular proliferation and angiogenesis. We demonstrate that MIR211 is transferred to adjacent cells in the tumor micro-environment via exosomes. Cross-species genome-wide transcriptomic analysis showed that human tumor-derived MIR211 interacts with the mouse transcriptome in the tumor microenvironment, and activates ERK5 signaling in human tumor cells via the modulation of a feedback loop. Human miR211 directly inhibits human DUSP6 protein phosphatase at the post-transcriptional level. We provide support for the hypothesis that DUSP6 inhibition conferred resistance of the human tumor cells to the BRAF inhibitor vemurafenib and to the MEK inhibitor cobimetinib, with associated increases in ERK5 phosphorylation. These findings are consistent with a model in which MIR211 regulates melanoma tumor proliferation and BRAF inhibitor resistance by inducing ERK5 signaling within the complex tumor microenvironment. We propose that the MIR211-ERK5 axis represents an important and sensitive regulatory arm in melanoma with potential theranostic applications.

cancer biology↗

bioRxiv2018

Taxa-driven functional shifts associated with stormflow in an urban stream microbial community

Urban streams are susceptible to stormwater and sewage inputs that can impact their ecological health and water quality. Microbial communities in streams play important functional roles and their composition and metabolic potential can help assess ecological state and water quality. Although these environments are highly heterogenous, little is known about the influence of isolated perturbations, such as those resulting from rain events on urban stream microbiota. Here, we examined the microbial community composition and diversity in an urban stream during dry and wet weather conditions with both 16S rRNA gene sequencing across multiple years and shotgun metagenomics to more deeply analyze a single stormflow event. Metagenomics was used to assess population-level dynamics as well as shifts in the microbial community taxonomic profile and functional potential before and after a substantial rainfall. Results demonstrated general trends present in the stream under stormflow vs. baseflow conditions across years and seasons and also highlighted the significant influence of increased effluent flow following rain in shifting the stream microbial community from abundant freshwater taxa to those more associated with urban/anthropogenic settings. Shifts in the taxonomic composition were also linked to changes in functional gene content, particularly for transmembrane transport and organic substance biosynthesis. We also observed an increase in relative abundance of genes encoding degradation of organic pollutants and antibiotic resistance after rain. Overall, this study provided evidence of stormflow impacts on an urban stream microbiome from an environmental and public health perspective.\n\nImportanceUrban streams in various parts of the world are facing increased anthropogenic pressure on their water quality, and stormflow events represent one such source of complex physical, chemical and biological perturbations. Microorganisms are important components of these streams from both ecological and public-health perspectives, and analyzing the effect of such perturbations on the stream microbial community can help improve current knowledge on the impact such chronic disturbances can have on these water resources. This study examines microbial community dynamics during rain-induced stormflow conditions in an urban stream of the Chicago Area Waterway System. Additionally, using shotgun metagenomics we identified significant shifts in the microbial community composition and functional gene content following a high rainfall event, with potential environment and public health implications. Previous work in this area has been limited to specific genes/organisms or has not assessed immediate stormflow impact.

microbiology↗

bioRxiv2017

Specific cholesterol binding drives drastic structural alterations in apolipoprotein A1

Protein adopts multitude of flexible and rapidly interconverting conformers, many which are governed by specific protein-interaction domains. ApoA1, a key player involved in high-density lipoprotein (HDL) regulation exists in structurally diverse forms with varying degree of cholesterol association, and each state is associated with different functional properties. While disc-shaped HDL and its oligomeric ApoA1 protein components have been the focus of several investigations, structural properties of monomeric ApoA1 are poorly understood. Here, we undertook large-scale structural analysis of ApoA1 in apo and cholesterol-bound forms using tens of independent simulations with total computing time exceeding 50 s. Examination of multiple lipid-free trajectories of monomeric ApoA1 revealed a common conformation, with distinct spatial proximity between N- and C-terminal domains. With incorporation of physiologically known cholesterol concentration ({approx}100 cholesterol molecules) in ApoA1 simulations, the monomeric protein spontaneously formed an open circular topology. Remarkably, these drastic structural perturbations are driven by specific binding site at C-terminal and a novel cholesterol binding site at the N-terminal. We proposed a mechanism of stage-wise opening of ApoA1 and demonstrated that less cholesterol concentration around interaction sites or mutation within N-terminal binding sites does not lead to open bell-shaped topology. The kinetic barriers between open and closed-states also showed an ensemble of loosely packed helix bundle (H1-H7; H4-H7) that posed as a slow-intermediate step. Lastly we performed complementary experiments, including ITC and CD measurements to confirm that structural changes are induced by ligand association and not driven by random hydrophobic effect. Collectively, our study suggests a previously unknown mechanism of cholesterol sequestering by ApoA1 that could directly aid in developing modulators for cholesterol efflux with chronic cardiovascular diseases.

biophysics↗