Biology subjects

Kumari, P.

Publications and source records attributed to Kumari, P..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Structure of the human Duffy antigen receptor

The Duffy antigen receptor, also known as FY glycoprotein or CD234, is a seven transmembrane protein expressed primarily at the surface of red blood cells, which displays promiscuous binding to multiple chemokines. Not only does it serve as the basis of the Duffy blood group system but it also acts as the primary attachment site for malarial parasite Plasmodium vivax on erythrocytes and as one of the nucleating receptors for the pore forming toxins secreted by Staphylococcus aureus. Despite a predicted 7TM architecture and efficient binding to a spectrum of chemokines, it fails to exhibit canonical second messenger response such as calcium release, likely due to a lack of G protein coupling. Unlike prototypical GPCRs and {beta}-arrestin-biased atypical chemokine receptors, the Duffy antigen receptor also appears to lack {beta}-arrestin binding, making it an enigmatic 7TM chemokine receptor. In order to decipher the molecular mechanism of this intriguing functional divergence exhibited by the Duffy antigen receptor, we have determined its cryo-EM structure in complex with a C-C type chemokine, CCL7. The structure reveals a relatively superficial binding mode of CCL7, with the N-terminus of the receptor serving as the key interaction interface, and a partially formed orthosteric binding pocket lacking the second site for chemokine recognition compared to prototypical chemokine receptors. The structural framework allows us to employ HDX-MS approach to uncover ligand-induced structural changes in the receptor and draw important insights into the promiscuous nature of chemokine binding. Interestingly, we also observe a dramatic shortening of TM5 and 6 on the intracellular side, compared to prototypical GPCRs, which precludes the coupling of canonical signal-transducers namely G proteins, GRKs and {beta}-arrestins, as demonstrated through extensive cellular assays. Taken together, our study uncovers a previously unknown structural mechanism that imparts unique functional divergence on the 7TM fold encoded in the Duffy antigen receptor while maintaining its scavenging function and should facilitate the designing of novel therapeutics targeting this receptor.

biochemistry↗

bioRxiv2023

Supramolecular catch-and-release strategy for bioorthogonal fluorogenic imaging across the visible spectrum

Fluorogenic probes that unmask fluorescence signals in response to a bioorthogonal reaction are a powerful new addition to biological imaging. They can provide significantly reduced background fluorescence and minimize non-specific signals, potentially allowing real-time high-contrast imaging without washing out excess fluorophores. While diverse classes of highly refined synthetic fluorophores are readily available now, their integration into a bioorthogonal fluorogenic scheme still necessitates another level of extensive design efforts and customized structural alterations to optimize quenching mechanisms for each given fluorophore scaffold. Herein, we present an easy-to-implement and highly generalizable supramolecular catch-and-release strategy for generating an efficient bioorthogonal fluorogenic response from essentially any readily available fluorophores without further structural alterations. We designed this distinct strategy based on the macrocyclic cucurbit[7]uril (CB7) host, where a fluorogenic response is achieved by programming a guest displacement reaction from the macrocycle cavity. We used this strategy to rapidly generate fluorogenic probes across the visible spectrum from structurally diverse classes of fluorophore scaffolds, including coumarin, bodipy, rhodamine, and cyanine. These probes were applied to no-wash fluorogenic imaging of various target molecules in live cells and tissue with minimal background and no appreciable non-specific signal. Notably, the orthogonal reactivity profile of the system allowed us to pair this host-guest fluorogenic probe with the covalently clickable fluorogenic probe to achieve high-contrast super-resolution and multiplexed fluorogenic imaging in cells and tissue.

bioengineering↗

bioRxiv2022

Mapping the unicellular transcriptome of the ascending thoracic aorta to changes in mechanosensing and mechanoadaptation during aging

Aortic stiffening is an inevitable manifestation of chronological aging, yet the mechano-molecular programs that orchestrate region- and layer-specific adaptations along the length and through the wall of the aorta are incompletely defined. Here, we show that the decline in passive cyclic distensibility is more pronounced in the ascending thoracic (ATA) compared to distal segments of the aorta and that tissues in both the medial and adventitial compartments of the ATA stiffen during aging. Single-cell RNA sequencing of aged ATA tissues reveals altered cellular senescence, remodeling, and inflammatory responses accompanied by enrichment of T-lymphocytes and rarefaction of vascular smooth muscle cells, compared to young samples. T-lymphocytes accumulate in the adventitia and likely promote fibrosis, while activation of mechanosensitive piezo-1 enhances medial vasoconstriction. These results portray the immuno-mechanical aging of the ATA as a process that culminates in a stiffer conduit permissive to the accrual of multi-gerogenic signals priming to disease development.

bioengineering↗

bioRxiv2022

Molecular insights into intrinsic transducer-coupling bias in the CXCR4-CXCR7 system

Chemokine receptors constitute an important subfamily of G protein-coupled receptors (GPCRs), and they are critically involved in a broad range of immune response mechanisms. Ligand promiscuity among these receptors makes them an interesting target to explore novel aspects of biased agonism. Here, we comprehensively characterize two chemokine receptors namely, CXCR4 and CXCR7, which share a common chemokine agonist (CXCL12), in terms of their G-protein coupling, {beta}-arrestin ({beta}arr) recruitment, contribution of GRKs, and ERK1/2 MAP kinase activation. We observe that CXCR7 lacks G-protein coupling while maintaining robust {beta}arr recruitment with a major contribution of GRK5/6. On the other hand, CXCR4 displays robust G-protein activation as expected, however, it exhibits significantly reduced {beta}arr-coupling compared to CXCR7 in response to their shared natural agonist, CXCL12. These two receptors induce distinct {beta}arr conformations even when activated by the same agonist, and CXCR7, unlike CXCR4, fails to activate ERK1/2 MAP kinase. We further determine the crystal structure of {beta}arr2 in complex with a carboxyl-terminal phosphopeptide derived from CXCR7, which reveals a smaller interdomain rotation than observed previously for activated {beta}arrs. Importantly, structure-guided cellular experiments reveal a key contribution of a single phosphorylation site in CXCR7 on {beta}arr recruitment and endosomal trafficking. Taken together, our study provides molecular insights into intrinsic bias encoded in the CXCR4-CXCR7 system, and it has broad implications for therapeutically important framework of biased agonism.

biochemistry↗

bioRxiv2022

Differential pathogenesis of SARS-CoV-2 variants of concern in human ACE2-expressing mice

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic resulting in millions of deaths worldwide. Increasingly contagious variants of concern (VoC) have fueled recurring global infection waves. A major question is the relative severity of disease caused by the previous and currently circulating variants of SARS-CoV-2. In this study, we evaluated the pathogenesis of SARS-CoV-2 variants in human ACE-2-expressing (K18-hACE2) mice. Eight-week-old K18-hACE2 mice were inoculated intranasally with a representative virus from the original B.1 lineage, or the emerging B.1.1.7 (alpha), B.1.351 (beta), B.1.617.2 (delta) or B.1.1.529 (omicron) lineages. We also infected a group of mice with the mouse-adapted SARS-CoV-2 (MA10). Our results demonstrate that B.1.1.7, B.1.351 and B.1.617.2 viruses are significantly more lethal than B.1 strain in K18-hACE2 mice. Infection with B.1.1.7, B.1.351 and B.1.617.2 variants resulted in significantly higher virus titers in the lungs and brain of mice compared to the B.1 virus. Interestingly, mice infected with the B.1.1.529 variant exhibited less severe clinical signs and high survival rate. We found that B.1.1.529 replication was significantly lower in the lungs and brain of infected mice in comparison to other VoC. Transcription levels of cytokines and chemokines in the lungs of the B.1.1.529-infected mice were significantly less when compared to those challenged with the B.1.1.7 virus. Together, our data provide insights into the pathogenesis of the previous and circulating SARS-CoV-2 VoC in mice.

microbiology↗

bioRxiv2022

Commensal fungi Candida albicans modulates dietary high-fat induced alterations in metabolism, immunity, and gut microbiota

Obesity and its associated diseases represent a growing health concern. Despite a strong correlation between gut microbiota and obesity, a limited study is available to suggest the direct involvement of fungus in host physiology. Candida albicans survives as a commensal fungus in the gastrointestinal tract, and its excessive growth causing infections in immune-suppressed individuals is widely accepted. However, any mutualistic relationship that may exist between C. albicans and the host remains outstanding. Here we show that the dietary C. albicans do not cause any noticeable infections, and our metagenomics analyses suggest that it colonizes in the gut and modulates microbiome dynamics which in turn negates the high-fat diet induced uncontrolled body weight gain, metabolic hormonal imbalances, and inflammatory response. Interestingly, adding C. albicans to a non-obesogenic diet stimulates the appetite regulated hormones and helped the mice gain healthy body weight. In concert, our results suggest a mutualism between C. albicans and the host, therefore, contrary to the notion, C. albicans is not always an adversary rather a bonafide admirable companion of the host. Finally, we discuss its potential translational implication as a probiotic especially in obese people or people dependent on high fat calorie intakes to manage obesity and associated complications.

microbiology↗

bioRxiv2022

Influenza virus-like particle-based hybrid vaccine containing RBD induces immunity against influenza and SARS-CoV-2 viruses

Several approaches have produced an effective vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the influence of immune responses induced by other vaccinations on the durability and efficacy of the immune response to SARS-CoV-2 vaccine is still unknown. We have developed a hybrid vaccine for SARS-CoV-2 and influenza viruses using influenza virus-like particles (VLP) incorporated by protein transfer with glycosylphosphatidylinositol (GPI)-anchored SARS-CoV-2 S1 RBD fused to GM-CSF as an adjuvant. GPI-RBD-GM-CSF fusion protein was expressed in CHO-S cells, purified and incorporated onto influenza VLPs to develop the hybrid vaccine. Our results show that the hybrid vaccine induced a strong antibody response and protected mice from both influenza virus and mouse-adapted SARS-CoV-2 challenges, with vaccinated mice having significantly lower lung viral titers compared to naive mice. These results suggest that the hybrid vaccine strategy is a promising approach for developing multivalent vaccines to prevent influenza A and SARS-CoV-2 infections.

immunology↗

bioRxiv2021

An mRNA silencing mechanism reliant on the cooperation between REGE-1/Regnase-1 and RLE-1/Roquin-1

Regnase-1 is an evolutionarily conserved endoribonuclease, degrading diverse mRNAs important, among others, for immune homeostasis, development, and cancer. There are two competing models of Regnase-1 mediated mRNA silencing. One model postulates that Regnase-1 works together with another RNA-binding protein, Roquin-1. The other model proposes that the two proteins function separately. Studying the C. elegans Regnase-1 ortholog, REGE-1, we have uncovered a functional relationship between REGE-1 and the nematode counterpart of Roquin-1, RLE-1. While REGE-1 and RLE-1 associate with mRNA independently of each other, both proteins are essential for mRNA silencing. Intriguingly, the functional interdependence between REGE-1 and RLE-1 is reminiscent of the proposed cooperation between mammalian Regnase-1 and Roquin-1, which may underlie a prototypic silencing mechanism involving both proteins.

molecular biology↗

bioRxiv2021

Atomic resolution Protein Allostery from the multi-state Structure of a PDZ Domain

Recent methodological advances in solution NMR allow the determination of multi-state protein structures and provide insights into correlated motion at atomic resolution as demonstrated here for the well-studied PDZ2 domain of protein human tyrosine phosphatase 1E for which protein allostery was predicted. Two-state protein structures were calculated for both the free form and in complex with the RA-GEF2 peptide using the exact nuclear Overhauser effect (eNOE) method. In the apo protein states an allosteric conformational preselection step comprising almost 60% of the domain was detected with an "open" ligand welcoming state and a "closed" state that obstructs the binding site by the distance between the {beta}-sheet, -helix 2 and sidechains of residues Lys38 and Lys72. Observed apo-holo structural rearrangements of induced fit-type are in line with previously published evolution-based analysis covering ~25% of the domain with only a partial overlap with the protein allostery of the open form. These presented structural studies highlight the presence of a dedicated highly optimized dynamic interplay of the complexity of the PDZ2 domain owed by the structure-dynamics landscape.

biophysics↗

bioRxiv2021

SARS-CoV-2 variants of concern infect the respiratory tract and induce inflammatory response in wild-type laboratory mice

The emergence of new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern poses a major threat to the public health due to possible enhanced virulence, transmissibility and immune escape. These variants may also adapt to new hosts in part through mutations in the spike protein. In this study, we evaluated the infectivity and pathogenicity of SARS-CoV-2 variants of concern in wild-type C57BL/6 mice. Six-week-old mice were inoculated intranasally with a representative virus from the original B.1 lineage or emerging B.1.1.7 and B.1.351 lineages. We also infected a group of mice with a mouse-adapted SARS-CoV-2 (MA10). Viral load and mRNA levels of multiple cytokines and chemokines were analyzed in the lung tissues on day 3 after infection. Our data show that unlike the B.1 virus, the B.1.1.7 and B.1.351 viruses are capable of infecting C57BL/6 mice and replicating at high concentrations in the lungs. The B.1.351 virus replicated to higher titers in the lungs compared to the B.1.1.7 and MA10 viruses. The levels of cytokines (IL-6, TNF-, IL-1{beta}) and chemokine (CCL2) were upregulated in response to the B.1.1.7 and B.1.351 infection in the lungs. Overall, these data indicate a greater potential for infectivity and adaptation to new hosts by emerging SARS-CoV-2 variants.

microbiology↗

bioRxiv2021

Temporal resolution of melanogenesis determine fatty acid metabolism as key skin pigment regulator

Therapeutic methods to modulate skin pigmentation has important implications for skin cancer prevention and for treating meta-inflammatory-triggered cutaneous conditions. Modulators of cAMP signalling of melanocyte have met with minimal clinical efficacy. Towards defining new potential targets, we followed temporal dynamics orchestrating melanocyte differentiation by using a cell-autonomous pigmentation model. Our study elucidates three dominant phases of synchronized metabolic and transcriptional reprogramming. The induction phase is concomitant with a paradoxical decrease in MITF levels, reduced proliferation, and increased anabolic metabolism mediated by AKT phosphorylation. The melanogenic phase shows rapid uptake of glucose and fatty acid, transiently forming lipid droplets through SREBF1-mediated regulation of fatty acid metabolism. This heightened bioenergetic activity impairs mitochondria and the recovery phase is marked by a shift to aerobic glycolysis and activation of the NRF2 detoxication pathway. Finally, we show that inhibitors of lipid metabolism indeed resolve hyper-pigmentary conditions in a guinea pig UV-tanning model. Our studies reveal metabolic control mechanisms of melanocytes that could govern the balance between differentiation and proliferation in a variety of cutaneous diseases.

cell biology↗

bioRxiv2021

Innate Immune sensing of Influenza A viral RNA through IFI16 promotes pyroptotic cell death

Programmed cell death pathways are triggered by various stresses or stimuli, including viral infections. The mechanism underlying the regulation of these pathways upon Influenza A virus IAV infection is not well characterized. We report that a cytosolic DNA sensor IFI16 is essential for the activation of programmed cell death pathways in IAV infected cells. We have identified that IFI16 functions as an RNA sensor for influenza A virus by binding to genomic RNA. The activation of IFI16 triggers the production of type I, III interferons, and also other pro-inflammatory cytokines via the STING-TBK1 and Pro-caspase-1 signaling axis, thereby promoting cell death (apoptosis and pyroptosis in IAV infected cells). Whereas, IFI16 knockdown cells showed reduced inflammatory responses and also prevented cell mortality during IAV infection. These results demonstrate the pivotal role of IFI16-mediated IAV sensing and its essential role in activating programmed cell death pathways.

immunology↗

bioRxiv2021

Intrinsic bias at non-canonical, β-arrestin-coupled seven transmembrane receptors

G protein-coupled receptors (GPCRs) are typically characterized by their seven transmembrane (7TM) architecture, and interaction with two universal signal-transducers namely, the heterotrimeric G-proteins and {beta}-arrestins ({beta}arrs). Synthetic ligands and receptor mutants have been designed to elicit transducer-coupling preferences and distinct downstream signaling outcomes for many GPCRs. This raises the question if some naturally-occurring 7TMRs may selectively engage one of these two signal-transducers, even in response to their endogenous agonists. Although there are scattered hints in the literature that some 7TMRs lack G-protein coupling but interact with {beta}arrs, an in-depth understanding of their transducer-coupling preference, GRK-engagement, downstream signaling and structural mechanism remains elusive. Here, we use an array of cellular, biochemical and structural approaches to comprehensively characterize two non-canonical 7TMRs namely, the human decoy D6 receptor (D6R) and the human complement C5a receptor (C5aR2), in parallel with their canonical GPCR counterparts, CCR2 and C5aR1, respectively. We discover that D6R and C5aR2 couple exclusively to {beta}arrs, exhibit distinct GRK-preference, and activate non-canonical downstream signaling partners. We also observe that {beta}arrs, in complex with these receptors, adopt distinct conformations compared to their canonical GPCR counterparts despite being activated by a common natural agonist. Our study therefore establishes D6R and C5aR2 as bona-fide arrestin-coupled receptors (ACRs), and provides important insights into their regulation by GRKs and downstream signaling with direct implications for biased agonism.

biochemistry↗

bioRxiv2020

Neuroinvasion and encephalitis following intranasal inoculation of SARS-CoV-2 in K18-hACE2 mice

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection can cause neurological disease in humans, but little is known about the pathogenesis of SARS-CoV-2 infection in the central nervous system. Herein, using K18-hACE2 mice, we demonstrate that SARS-CoV-2 neuroinvasion and encephalitis is associated with mortality in these mice. Intranasal infection of K18-hACE2 mice with 105 plaque-forming units of SARS-CoV-2 resulted in 100% mortality by day 6 after infection. The highest virus titers in the lungs were observed at day 3 and declined at days 5 and 6 after infection. In contrast, very high levels of infectious virus were uniformly detected in the brains of all the animals at days 5 and 6. Onset of severe disease in infected mice correlated with peak viral levels in the brain. SARS-CoV-2-infected mice exhibited encephalitis hallmarks characterized by production of cytokines and chemokines, leukocyte infiltration, hemorrhage and neuronal cell death. SARS-CoV-2 was also found to productively infect cells within the nasal turbinate, eye and olfactory bulb, suggesting SARS-CoV-2 entry into the brain by this route after intranasal infection. Our data indicate that direct infection of CNS cells together with the induced inflammatory response in the brain resulted in the severe disease observed in SARS-CoV-2-infected K18-hACE2 mice.

microbiology↗

bioRxiv2020

Stagewise identification of significantly mutated gene clusters in major molecular classes of breast invasive carcinoma

Breast invasive carcinoma (BRCA) is most malignant and leading cause of death in women. The efforts are ongoing for improvement in early detection, prevention and treatment. Therefore, identification of biomarkers/candidate genes has become very important. The current work includes comprehensive analysis of RNA-sequencing data of 1097 BRCA samples and 114 normal adjacent tissues to identify dysregulated genes in major molecular classes of BRCA in various clinical stages. Huge number of dysregulated genes were found, some were stage-specific, and others were common. The pathways as interferon signaling, tryptophan degradation III, granulocyte adhesion & diapedesis and catecholamine biosynthesis were found in ER/PR+/HER-2- (p-value<0.010), pathways as RAR activation, adipogenesis, role of JAK1, JAK2 in interferon signaling, TGF-{beta} and STAT3 signaling (p-value<0.014) intricated in ER/PR-/HER-2+ and pathways as IL-1/IL-8 signaling, TNFR1/TNFR2 signaling, TWEAK and relaxin signaling (p-value<0.005) were found in triple negative breast cancer. The genes were clustered based on their mutation profile which revealed nine mutated clusters, some of which were known to be involved in well-known cancer signalling pathways while others were less characterized. Each cluster was analyzed in detail which led us to discover genes viz. NLGN3, MAML2, TTN, SYNE1, ANK2 as candidates in BRCA. The genes were found to be involved in important processes as chemotaxis, axon guidance, notch binding, cell-adhesion-molecule binding etc. They are central genes in the protein-protein-interaction network indicating they can have important regulatory roles. The qRT-PCR and western blot confirmed our findings in breast cancer cell-lines. Further, immunohistochemistry corroborated the results in ~100 tissue samples. The genes can be used as biomarker in BRCA.

bioinformatics↗

bioRxiv2020

Downy Mildew effector HaRxL21 interacts with the transcriptional repressor TOPLESS to promote pathogen susceptibility

Hyaloperonospora arabidopsidis (Hpa) is an oomycete pathogen causing Arabidopsis downy mildew. Effector proteins secreted from the pathogen into the plant play key roles in promoting infection by suppressing plant immunity and manipulating the host to the pathogens advantage. One class of oomycete effectors share a conserved RxLR motif critical for their translocation into the host cell. Here we characterize the interaction between an RxLR effector, HaRxL21 (RxL21), and the Arabidopsis transcriptional co-repressor Topless (TPL). We establish that RxL21 and TPL interact via an EAR motif at the C-terminus of the effector, mimicking the host plant mechanism for recruiting TPL to sites of transcriptional repression. We show that this motif, and hence interaction with TPL, is necessary for the virulence function of the effector. Furthermore, we provide evidence that RxL21 uses the interaction with TPL, and its close relative TPL-related 1, to repress plant immunity and enhance host susceptibility to both biotrophic and necrotrophic pathogens.

plant biology↗

bioRxiv2020

The FDA-approved gold drug Auranofin inhibits novel coronavirus (SARS-COV-2) replication and attenuates inflammation in human cells

SARS-COV-2 has recently emerged as a new public health threat. Herein, we report that the FDA-approved gold drug, auranofin, inhibits SARS-COV-2 replication in human cells at low micro molar concentration. Treatment of cells with auranofin resulted in a 95% reduction in the viral RNA at 48 hours after infection. Auranofin treatment dramatically reduced the expression of SARS-COV-2-induced cytokines in human cells. These data indicate that auranofin could be a useful drug to limit SARS-CoV-2 infection and associated lung injury due to its anti-viral, anti-inflammatory and anti-ROS properties. Auranofin has a well-known toxicity profile and is considered safe for human use.

microbiology↗

bioRxiv2020

Human DND1-RRM2 forms a non-canonical domain-swapped dimer

Human DND1 (Dead end protein homolog1) is an RNA binding protein. DND1 plays pivotal role in animal development and has been implicated in cancer. DND1 consists of two RNA recognition motifs (RRMs) in tandem and a double stranded RNA binding domain at the C terminal separated by 40 residues flexible linker. The conserved RNP site in the RRM1 domain helps in specific RNA recognition while the RNP sites in RRM2 are not well conserved. DND1 has been reported to be involved in inhibition of microRNA access to target mRNA and it also associate with CCR4-NOT complex that targets mRNA. In order to understand this intriguing contrasting molecular function, we have determined the 2.3 [A] resolution crystal structure of the human DND1 RRM2 domain. The structure revealed an interesting non-canonical RRM fold that is maintained by the formation of a domain swapped dimer between {beta}1 and {beta}4 strands across two chains. The domain swapping is attributed by a hinge loop between 2 and {beta}4 that helps in mediating a domain swap forming anti-parallel {beta} sheets. We have delineated the structural basis of stable dimer formation using the residue level dynamics of protein explored by NMR spectroscopy and MD simulations. Our structural and dynamics studies demonstrate the molecular basis for the dimerization of the RRM2 domain and shed light on the possibility for this motif for interaction with other proteins which helps in transcription regulation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/978023v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@4975d2org.highwire.dtl.DTLVardef@d65418org.highwire.dtl.DTLVardef@12b0272org.highwire.dtl.DTLVardef@ada8b7_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFirst report of a domain swapped dimer formation by an RRM identified through crystal structure determination of the human DND1 RRM2 domain. C_LIO_LIRRM2 exhibit domain swapped dimerization attributed by hinge loop and disulfide bond formation. C_LIO_LIDimer formation is under redox regulation. C_LIO_LIMajor determinants of swapping were identified. C_LIO_LIDND1 RRM2 is not involved in RNA recognition. C_LI

biophysics↗