Biology subjects

Asthana, S.

Publications and source records attributed to Asthana, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Target-Specific Discovery of BMM_1567 Restores Aminoglycoside Activity Against Multidrug-Resistant Gram-Negative ESKAPE Pathogens

The global emergence of multidrug-resistant (MDR) ESKAPE pathogens has significantly reduced the effectiveness of existing antibiotics, highlighting the urgent need for new strategies to restore antimicrobial susceptibility. Here, we report the discovery and mechanism of BMM_1567, a peptide potentiator that enhances aminoglycoside efficacy against MDR pathogens. A genetically defined reporter-based screen identified BMM_1567 as a potent inhibitor of aminoglycoside resistance, potentiating spectinomycin activity against MDR Gram-negative ESKAPE isolates at low micromolar concentrations. Structural modeling and molecular dynamics simulations indicated that BMM_1567 interacts with residues lining the antibiotic-binding groove of aminoglycoside-modifying enzymes (ANT, APH, AAC), with highest affinity for ANT ({Delta}G_bind = -62.25 kcal/mol), suggesting competitive inhibition of substrate binding. Site-directed mutagenesis of key ANT residues identified critical amino acids involved in BMM_1567 binding, confirming their role in mediating spectinomycin potentiation. In murine abscess model using XDR E. coli, BMM_1567 in combination with spectinomycin significantly reduced bacterial burden and pro-inflammatory cytokine levels, comparable to colistin. Collectively, these findings establish BMM_1567 as a promising aminoglycoside potentiator that restores antibiotic activity against MDR pathogens through direct inhibition of resistance enzymes, while exhibiting in vivo efficacy and a remarkably low propensity for resistance development.

microbiology↗

bioRxiv2026

A human monoclonal antibody from hybrid immunity exhibits exceptional neutralization breadth against antigenically divergent contemporary Omicron JN.1 lineage variants

Hybrid immunity offers stronger and more durable antibody-mediated protection against symptomatic SARS-CoV-2 infection. In the present study, we report the development of a durable antibody response in an individual with hybrid immunity who also received three doses of the prototype COVID-19 vaccine. Polyclonal plasma antibody obtained from this donor also showed extraordinary neutralization breadth against contemporary Omicron variants. One of the functional monoclonal antibodies (ATHS-C30) isolated from this individual, representing the IGHV4-30 lineage-specific B cell with strong binding affinity to JN.1 spike protein, showed extraordinary neutralization breadth, including variants of Omicron lineages that emerged beyond JN.1, such as KP.2, KP.3.1.1, KP.3.2, KP.3.3, LB.1 and XEC. ATHS-C30 was found to bind RBD with high affinity and showed distinct epitope specificity to the other neutralizing mAbs isolated from the same donor through the epitope binning assay. Molecular modelling of the CDHR3 sequence using existing structures indicated that ATHSC-30 belongs to the class 4 antibody, a feature that contributes to breadth, while epitope conservation analysis indicated that the majority of RBD-interacting residues of ATHSC-30 are evolutionarily conserved. Taken together, our study indicate that ATHS-C30 forms the basis of development of a robust and broadly neutralizing antibody response in this individual with hybrid immunity, which overcomes the antigenic variation by targeting highly conserved and cryptic epitopes in destabilizing the spike structure. ImportanceSARS-CoV-2 continues to pose a significant public health threat, particularly to immunocompromised individuals and older adults with underlying comorbidities. Hybrid immunity to SARS-CoV-2 in vaccinated individuals leads to the development of B cells that are qualitatively superior to those that are expected to develop in only vaccinated individuals. In the present study, we found that among individuals with hybrid immunity who developed robust, durable antigen-specific antibody responses, one developed antibody response capable of broadly cross-neutralising contemporary Omicron variants. This was correlated with development of antigen-specific B cell lineage, such as IGHV4-30, that produced antibodies with potent and extraordinary neutralisation breadth against contemporary Omicron lineages, such as KP.2, KP.3.1.1, KP.3.2, KP.3.3, LB.1 and XEC. This is believed to be due to heterologous antigen exposures driving the development of an antigen-specific B cell repertoire that, in turn, facilitates immune imprinting capable of overcoming the ineffectiveness of antibodies to effectively neutralize newly emerging Omicron variants.

immunology↗

bioRxiv2026

Bisphenol-A mediated ubiquitinome alteration triggers PPAR-alpha ubiquitination, affecting trophoblast cell migration

Pregnant women are frequently exposed to various endocrine-disrupting chemicals (EDCs), such as bisphenol A (BPA), causing harm to both the developing placenta and fetus. BPA can promote placental dysfunction by altering key cellular processes such as differentiation, invasion, and migration in trophoblast cells. These cellular processes are also tightly managed by the ubiquitin proteasomal system via maintenance of the ubiquitinated protein pool. However, the BPA-mediated dysregulation of this ubiquitin proteasomal homeostasis is poorly understood. Therefore, we identified 19 deubiquitinases (DUBs) and a dynamic ubiquitinome profile of extravillous trophoblast cells (HTR8/SVneo), which reduced trophoblast cell migration post-BPA exposure. Further investigation using an integrated substrate-ligase-deubiquitinase network shows that BPA binding to PPAR-alpha or indirect regulation of its E3 Ligase MuRF1 and DUB USP5 via BPA resulted in hyper-ubiquitination of PPAR-alpha, triggering its nuclear localization. In the nucleus, the ubiquitinated PPAR-alpha can deregulate its migration-associated target gene expression, causing a reduction in the migration of HTR8/SVneo cells. This physiological alteration of extravillous trophoblast cells (EVTs) through BPA can disrupt placental homeostasis. Hence, we assumed that BPA-induced cellular alteration in EVTs can promote placental defects, which might contribute to adverse pregnancy outcomes.

biochemistry↗

bioRxiv2026

Systematic Screening of a Glycolysis-targeting Small-molecule Library Identifies Novel Inhibitors of Fungal Morphogenesis in Candida albicans

Fungal infections are an increasing global health concern, with Candida albicans emerging as a leading cause of mucosal and life-threatening systemic infections. C. albicans relies on yeast-to-hyphae transition, to establish systemic infections in the host. Our recent work demonstrated that glycolysis is a key regulator of hyphal differentiation in C. albicans. Leveraging this knowledge, we screened a small-molecule compound library containing glycolysis inhibitors for their ability to block fungal morphogenesis and identified multiple inhibitors of glycolysis that robustly block hyphal differentiation without compromising overall growth. While early glycolysis pathway inhibitors showed moderate effects, two compounds (NPD10084 and PKM2-IN-6) targeting pyruvate kinase activity emerged as the most potent inhibitors of fungal morphogenesis, completely blocking hyphal differentiation, in multiple filamentation-inducing conditions. Comparative RNA-Seq analysis revealed that pyruvate kinase inhibition using PKM2-IN-6 resulted in the repression of multiple genes associated with fungal morphogenesis, biofilm formation, and virulence. These transcriptional changes were accompanied by a significant reduction in biofilm formation and increased sensitivity to conventional antifungal drugs, including amphotericin B and fluconazole, in the presence of PKM2-IN-6. Furthermore, administration of PKM2-IN-6 significantly improved the survival of mice in a systemic model of murine candidiasis underscoring the in vivo efficacy of this compound. Molecular docking analysis showed stable binding of both compounds to the ATP-binding pocket of pyruvate kinase, suggesting ATP-competitive inhibition as the mode of action. Collectively, our study has identified novel inhibitors of fungal morphogenesis in C. albicans that target pyruvate kinase activity to increase antifungal susceptibility and attenuate the virulence of C. albicans. ImportanceCandida albicans is a major fungal pathogen that causes fatal infections, particularly in immunocompromised individuals. The increasing prevalence of antifungal resistance and the limited availability of effective antifungal therapies underscore the need for new treatment strategies. In this study, we identify pyruvate kinase, a key glycolytic enzyme, as an important regulator of fungal morphogenesis and virulence. Pharmacological inhibition of pyruvate kinase blocked hyphal differentiation without affecting fungal growth, impaired biofilm formation, and enhanced the activity of clinically relevant antifungal drugs. Transcriptomic analysis further revealed broad repression of genes involved in filamentation, biofilm formation, cell adhesion, antifungal resistance, and virulence, providing a molecular basis for these phenotypes. Importantly, pyruvate kinase inhibition also improved host survival in a murine model of systemic candidiasis. Together, our findings identify pyruvate kinase as a promising therapeutic target and demonstrate the potential of targeting fungal metabolism to effectively combat C. albicans infections.

microbiology↗

bioRxiv2026

Gut microbiota-derived indole-3 acetic acid mitigates key hallmarks of metabolic dysfunction-associated steatotic liver disease (MASLD) in an NLRP3-dependent manner in in vitro and diet-induced mouse models

BackgroundMetabolic-dysfunction associated steatotic liver disease(MASLD) is a global epidemic affecting >30 % global population with no approved therapies till date. Recent reports suggest that activation of NLRP3 inflammasome may be involved in the pathogenesis of MASLD. We, therefore, aimed to identify small molecule inhibitor(s) of NLRP3 inflammasome as a potential therapeutic strategy to manage MASLD and validate their efficacy using in vitro and in vivo models of MASLD. MethodologyWe screened an in-house library of natural products using an in vitro phenotypic assay and identified a gut microbiota derived metabolite of dietary Tryptophan; indole-3 acetic acid (I3A) for its ability to inhibit the levels of IL-1{beta} and IL-18, which are elevated as a result of activation of NLRP3 inflammasome in differentiated THP1 cells. Subsequently, we carried out several in vitro and in vivo studies to confirm the mechanism of action of I3A and its ability to mitigate the key hallmarks of MASLD ResultsOur in vitro data suggest that I3A is an inhibitor of NLRP3 inflammasome. I3A was also found to improve the blood glucose level, plasma lipid profile, hepatic fat, and liver function in high-fat-high-fructose diet induced model of MASLD using C57BL/6 mice. ConclusionOur results show that I3A, which has been previously reported to be a gut microbiota-derived metabolite of dietary tryptophan, mitigates the key hallmarks of MASLD in an NLRP3 dependent manner. A dedicated structure-activity-relationship (SAR) study around the I3A chemotype may be carried out in future to identify novel NLRP3 inhibitors with desirable pharmacological profile.

pharmacology and toxicology↗

bioRxiv2025

Cholesterol-mediated activation of VapC12 toxin modulates growth and drug susceptibility in Mycobacterium tuberculosis

Tuberculosis eradication efforts are severely hampered by antimicrobial resistance (AMR). We have previously reported that VapBC12 TA system in Mycobacterium tuberculosis (M. tuberculosis) regulates both disease and antibiotic persistence in tuberculosis. In this study, we developed a mechanistic understanding of the VapBC12-dependent modulation of growth and antibiotic susceptibility in M. tuberculosis. We identified a unique Cholesterol Recognition Amino acid Consensus (CRAC) motif in VapC12 toxin and demonstrated that its heterologous expression induces toxicity in M. smegmatis. We have solved the crystal structure of VapB12 antitoxin at a 1.6A0 wavelength and using molecular modeling predicted the structure of TA complex. Structure-function analysis revealed specific residues critical for the assembly and activity of the VapBC12 TA system. Our study suggests that cholesterol activates the VapC12 toxin by competitively displacing its cognate antitoxin at the CRAC motif which consequently restricts the growth of M. tuberculosis. Finally, we demonstrated that chemical inhibition of VapC12 prevents growth restriction and potentiates activity of anti-TB drugs.

microbiology↗

bioRxiv2025

Gut bacterial metabolite imidazole propionate potentiates Alzheimer's disease pathology

The gut microbiome modulates metabolic and neurovascular processes implicated in Alzheimers disease and related dementias (ADRD), but the underlying mechanisms remain unclear. Here, we identify the bacterial metabolite imidazole propionate (ImP) as a modifier of ADRD pathology. In a cohort of 1,196 cognitively unimpaired adults, higher plasma ImP levels were associated with lower preclinical cognitive scores and biomarkers of ADRD, both cross-sectionally and longitudinally. Fecal metagenomic analysis linked putative ImP producers to ADRD phenotypes. Genome-wide integrative analysis revealed a locus on chromosome 12 associated with both plasma ImP levels and AD risk in humans, supporting a host genetic contribution to ImP regulation and a causal role of this metabolite in AD. In mice, chronic ImP administration exacerbated AD-like pathology. Mechanistically, ImP impaired brain endothelial barrier and promoted tau hyperphosphorylation in primary neurons, an effect blocked by glycogen synthase kinase-3{beta} inhibition. Together, our study links ImP to hallmarks of neurodegeneration and suggest that targeting ImP may represent a potential strategy to modify ADRD risk.

pathology↗

bioRxiv2025

AGPAT1 is a novel Chikungunya virus receptor on human cells

Chikungunya virus (CHIKV) is a medically important alphavirus whose host receptor is not fully understood. We identified AGPAT1 from the human Huh7 cell plasma membrane binding with CHIKV particles in vitro. The CHIKV binding with AGPAT1 was demonstrated on Huh7, HAP1, and ERMS plasma membrane by confocal microscopy. The AGPAT1 antibody inhibited CHIKV binding to cells, reducing the virus uptake in Huh7 and ERMS cells. CHIKV binding, uptake, and replication were significantly reduced in the AGPAT1 knockout HAP1 cells, and the ectopic expression of AGPAT1 rescued the reduced virus binding, uptake, and replication. AGPAT1 interacted with the E1 surface on the E1-E2 dimer of CHIKV envelope proteins in silico. The E1-AGPAT1 interacting amino acid residues identified in the computational study were experimentally verified. AGPAT1 was also shown to have a role in the binding and uptake of another alphavirus, Ross River virus. These data demonstrate an important role for AGPAT1 as a novel host receptor involved in CHIKV binding and uptake in human cells. AUTHOR SUMMARYChikungunya virus (CHIKV) is a medically important alphavirus. Identifying its receptor on human cells will aid the development of novel antivirals. We have identified AGPAT1 as a novel CHIKV receptor on several human cells. We provide data showing the interaction of AGPAT1 with the E1 protein of CHIKV E1-E2 dimer present on the virion surface and identify the amino acid residues involved in the interaction. AGPAT1 was also shown to have a role in the binding and uptake of Ross River virus, another alphavirus. AGPAT1 is a protein involved in lipid metabolism and has not been reported to have a direct role in any virus infection, or as a virus receptor. Thus, this work identifies AGPAT1 as a novel receptor for CHIKV on human cells, and has implications for understanding the virus pathogenesis and designing the receptor-blocking CHIKV antivirals.

microbiology↗

bioRxiv2025

Sex-Specific Differential DNA Methylation in Mild Cognitive Impairment and Alzheimers Disease

Sex differences in late-onset Alzheimers disease (AD) progression include accelerated decrements in cognitive status and greater amyloid and tau biomarker burdens in females. To identify sex-specific differentially methylated positions (DMPs) and genes in persons with mild cognitive impairment (MCI) and AD, we analyzed whole genome methylation sequencing on blood samples from participants with MCI (N=99, 52% female), AD (N=109, 43% female), and those cognitively unimpaired (CU; N=174, 52% female). Ninety-four percent of DMPs from MCI vs. CU, AD vs. CU, and AD vs. MCI pairwise comparisons were sex-specific. Female-specific DMPs were enriched in neurologic gene sets (e.g., synaptic membrane, ion channel complex), while male-specific DMPs showed limited enrichment. Sex-specific DMPs overlapped blood-specific enhancers, promoters, and transcription factor binding motifs, highlighting divergent epigenetic regulation by sex. These findings identify sex-specific genes and molecular pathways in MCI and AD and support that blood DNA methylation levels can distinguish cognitive status.

genomics↗

bioRxiv2024

Telomere length and cognitive function among middle-aged and older participants from communities underrepresented in aging research: A preliminary study

ObjectiveAccelerated biological aging is a plausible and modifiable determinant of dementia burden facing minoritized communities, but is not well-studied in these historically underrepresented populations. Our objective was to preliminarily characterize relationships between telomere length and cognitive health among American Indian/Alaska Native (AI/AN) and Black/African American (B/AA) middle-aged and older adults. MethodsThis study included data on telomere length and cognitive test performance from 187 participants, enrolled in one of two community-based cognitive aging cohorts and who identified their primary race as AI/AN or B/AA. ResultsNested multivariable regression models revealed preliminary evidence for associations between telomere length and cognitive performance, and these associations were partially independent of chronological age. DiscussionSmall sample size limited estimate precision, however, findings suggest future work on telomere length and cognitive health in underrepresented populations at high risk for dementia is feasible and valuable as a foundation for social and behavioral intervention research.

neuroscience↗

bioRxiv2024

Whole genome methylation sequencing in blood from persons with mild cognitive impairment and dementia due to Alzheimer's disease identifies cognitive status

INTRODUCTIONWhole genome methylation sequencing (WGMS) in blood identifies differential DNA methylation in persons with late-onset dementia due to Alzheimers disease (AD) but has not been tested in persons with mild cognitive impairment (MCI). METHODSWe used WGMS to compare DNA methylation levels at 25,244,219 CpG loci in 382 blood samples from 99 persons with MCI, 109 with AD, and 174 who are cognitively unimpaired (CU). RESULTSWGMS identified 9,756 differentially methylated positions (DMPs) in persons with MCI, including 1,743 differentially methylated genes encoding proteins in biological pathways related to synapse organization, dendrite development, and ion transport. 447 DMPs exhibit progressively increasing or decreasing DNA methylation levels between CU, MCI, and AD that correspond to cognitive status. DISCUSSIONWGMS identifies DMPs in known and newly detected genes in blood from persons with MCI and AD that support blood DNA methylation levels as candidate biomarkers of cognitive status.

genomics↗

bioRxiv2024

pH dependent direct sulfhydrylation pathway is required for pathogenesis of Mycobacterium tuberculosis

Methionine is essential for the survival of Mycobacterium tuberculosis (M. tuberculosis) inside the host. However, the transsulfuration pathway, a major contributor of methionine, is dispensable for the growth of M. tuberculosis suggesting redundancy in the methionine biosynthesis pathway. Orthologues of MetZTB in other bacterial species are known to operate a redundant single-step methionine biosynthesis pathway called direct sulfhydrylation. In this study, we demonstrate that genetic disruption of the metZ-mediated direct sulfhydrylation pathway in M. tuberculosis hinders growth at low pH, an effect mitigated by methionine supplementation. Computational analyses, including in-silico molecular docking and molecular dynamics (MD) simulations, reveal enhanced binding of the MetZ substrate, O-succinyl homoserine (OSH), to the active site of MetZ at acidic pH. Intriguingly, despite increased intracellular ATP levels, a relative decrease in the frequency of Bedaquiline (BDQ)-induced persisters is observed in metZ-deficient strain, suggesting a role of direct sulfhydrylation pathway in modulating BDQ sensitivity. Finally, we demonstrated that the absence of metZ impedes the ability of M. tuberculosis to grow inside the host.

microbiology↗

bioRxiv2024

Oxysterol accumulation in aging cells alters GPCR signalling

Organismal aging is accompanied by the accumulation of senescent cells in the body, which drives tissue dysfunction. Senescent cells have a distinctive profile, including proliferation arrest, resistance to apoptosis, altered gene expression, and high inflammation. Despite global signalling and metabolic dysregulation during senescence, the underlying reasons for changes in signalling remain unclear. GPCRs are pivotal in cellular signalling, dynamically mediating the complex interplay between cells and their surrounding environment to maintain cellular homeostasis. The chemokine receptor CXCR4 plays a crucial role in modulating immune responses and inflammation. It has been shown that expression of CXCR4 increases in cells undergoing senescence, which enhances inflammation post-activation. Here we examine CXCR4 signalling in deeply senescent cells, where cholesterol and its oxidized derivatives, oxysterols, affect receptor function. We report elevated oxysterol levels in senescent cells, which altered classical CXCL12-mediated CXCR4 signalling. Tail-oxidized sterols disrupted signalling more than ring-oxidized counterparts. Molecular dynamics simulations revealed that 27-hydroxycholesterol displaces cholesterol and binds strongly to alter the conformation of critical signalling residues to modify the sterol-CXCR4 interaction landscape. Our study provides a molecular view of the observed mitigated GPCR signalling in the presence of oxysterols, which switched G-protein signalling from Gi/o to Gs class. Overall, we present an altered paradigm of GPCR signalling where cholesterol oxidation alters the signalling outcome in aged cells. Significance StatementOur study brings to light a novel and significant discovery in aged cells: the accumulation of oxysterols, oxidized forms of cholesterol, critically impairs CXCR4-dependent signalling and alters G-protein coupling specificity. This effect of oxysterols is demonstrated for the first time in aged cellular models, providing a molecular basis for a multitude of observed alterations in senescence, such as compromised immune functions and a decline in cellular responsiveness with age. Our research not only fills a crucial gap in understanding the aging process at the molecular level but also identifies potential targets for therapeutic interventions aimed at mitigating age-related cellular dysfunctions and diseases.

cell biology↗

bioRxiv2023

SARS-CoV-2 antibodies cross-react and enhance dengue infection

The presence of non-neutralizing antibodies of any dengue serotype, increase the severity of subsequent infection by other dengue serotypes. During SARS-CoV-2 pandemic, the number of symptomatic dengue cases increased in India. We describe that antibodies isolated from convalescent plasma from COVID-19 patients enhances DENV2 infection in vitro. CR3022, one antibody against SARS-CoV-2 spike protein, also showed elevated DENV2 infection in vitro. In silico protein-protein interactions between the spike antibodies and the DENV2 E-protein revealed significant interactions. Likewise, few monoclonal/polyclonal antibodies against SARS-CoV-2 showed increased dengue infection in vitro. Importantly, the AG129 mice infected with SARS-CoV-2 three-weeks prior to DENV2 infection, showed elevated dengue pathogenesis. Thus, highlighting the possibilities of elevated infection and symptomatic dengue disease in COVID-19 survivors.

immunology↗

bioRxiv2023

Conformational dynamics of a nicotinic receptor neurotransmitter binding site

Agonists increase receptor activity by providing net-favorable binding energy to active versus resting conformations of their target sites. We used molecular dynamics (MD) simulations to explore dynamics of the low-to-high affinity conformational change (L[->]H) at the Torpedo -{delta} nicotinic acetylcholine receptor neurotransmitter site, using 4 agonists. Alternative conformations were identified in trajectories generated from a single starting structure by matching approximate binding energies calculated in silico with exact values measured experimentally in vitro. In all simulations, the L[->]H transition started with a rotation of the agonist about its cationic center (flip), followed by a staged downward displacement of loop C (flop) and the formation of a compact, hydrophobic and stable high-affinity pocket (fix). Agonist rotation and a transient intermediate state are only in simulations but can be confirmed or refuted, for example by time-resolved structures.

biophysics↗

bioRxiv2023

Detergent induced structural perturbations in peanut agglutinin: Insights from spectroscopic and molecular dynamic simulation studies

The three dimensional structure of a protein is very important for its structure. Studies relating to protein structure have been numerous and the effect of denaturants on proteins can help understand the process of protein folding and misfolding. Detergents are important denaturants and play important roles in various fields. Here we explored the effect of sodium dodecyl sulphate (SDS) and cetyltrimethylammonium bromide (CTAB) on the structure of peanut agglutinin (PNA). The protein was purified from its natural source and impact of SDS and CTAB was studied by circular dichroism, intrinsic fluorescence, 8-anilino-1-napthalenesulfonic acid, molecular docking and molecular dynamics simulation. Pure peanut agglutinin showed a trough at 220 nm and positive ellipticity peak at 195 nm, specific for lectins. Results from the experimental and simulation studies suggest how oppositely charged detergents can interact differently and lead to varied structural perturbations in PNA. Both the surfactants induce all protein-like circular dichroism in the protein, above its critical micelle concentrations, with significant change in accessible surface area that became more hydrophobic upon the treatment. Major interactions between the surfactants and protein, resulting in PNA conformational rearrangement, are electrostatic and van der Waals interactions. However, CTAB, like a cationic surfactant, has similar effects as anionic surfactant (SDS) but at significantly very low concentration. Though the effects followed same pattern in both the surfactant treatment, i.e. above respective CMC, the surfactants were inducing -helix/coil like conformation in PNA.

biochemistry↗

bioRxiv2023

Modulated protein-sterol interactions drive oxysterol-induced impaired CXCR4 signalling

CXCR4 is a G-protein coupled receptor which mediates signalling for diverse functions such as cell proliferation and migration, hematopoiesis and plays a role in embryogenesis and development. Signal transduction occurs primarily through transmembrane helices that function in the multicomponent lipid environment of the plasma membrane. Elevated levels of plasma membrane oxysterols occur in cardiovascular and metabolic disorders, physiological stress and inflammatory conditions. We use experimental and simulation approaches to study the impact of oxysterol chemistry and composition on CXCL12-mediated CXCR4 signalling. Experiments on HeLa cells show a pronounced decrease in calcium oscillation response for the tail oxidized sterols in comparison with the ring oxidized sterols with 22(R) hydroxycholesterol showing a near complete loss of signalling followed by 27-hydroxycholesterol and 25-hydroxycholesterol. All-atom molecular dynamics simulations reveal that tail oxidized, 27-hydroxycholesterol, displaces cholesterol and ubiquitously binds to several critical signalling residues, as well as the dimer interface. Enhanced 27-hydroxycholesterol binding alters CXCR4 residue conformations, disrupts the toggle switch and induces secondary structure changes at both N and C termini. Our study provides a molecular view of the observed mitigated CXCR4 signalling in the presence of oxysterols revealing that disruption of cholesterol-protein interactions, important for regulating the active state, is a key factor in the loss of CXCR4 signalling. Additionally, a signalling class switching from Gi to Gs as revealed by increased CREB and ERK phosphorylation is observed in the experiments.

cell biology↗

bioRxiv2022

RNA-protein interactome at the Hepatitis E virus internal ribosome entry site

Multiple processes exist in a cell to ensure continuous production of essential proteins either through cap-dependent or cap-independent translation processes. Viruses depend on the host translation machinery for viral protein synthesis. Therefore, viruses have evolved clever strategies to utilize the host translation machinery. Earlier studies have shown that genotype 1-Hepatitis E virus (g1-HEV) utilizes both cap-dependent and cap-independent translation machineries for its replication and proliferation. Cap-independent translation in g1-HEV is driven by an eighty seven nucleotide-long RNA element which acts as a noncanonical, internal ribosome entry site like (IRESl) element. Here, we have identified the RNA-protein interactome of the HEV IRESl element and characterized the functional significance of some of its components. Our study reveals indispensable roles of host ribosomal protein RPL5 and DHX9 (RNA helicase A) in mediating efficient translation from the IRESl element and establish the function of HEV IRESl as a bonafide internal ribosome entry site. Author summaryProtein synthesis is a fundamental process for survival and proliferation of all living organisms. Majority of cellular proteins are produced through cap-dependent translation. Cells also utilize a variety of cap-independent translation processes to synthesize essential proteins during stress. Viruses depend on the host cell translation machinery to synthesize their own proteins. Hepatitis E virus is a major cause of hepatitis worldwide. The viral genome is a capped positive strand RNA. Viral non-structural and structural proteins are synthesized through a cap-dependent translation process. An earlier study from our laboratory reported the presence of a fourth ORF in genotype 1-HEV, which produced the ORF4 protein using a cap-independent internal ribosome entry site-like (IRESl) element. In the current study, we identified the host proteins that associate with the HEV-IRESl RNA and generated the RNA-protein interactome. Through a variety of experimental approaches, our data proves that HEV-IRESl is a bonafide internal ribosome entry site.

microbiology↗