Biology subjects

Ray, A.

Publications and source records attributed to Ray, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

An algorithm-based investigation reveals the differential dynamics of water inside protein cavity as a function of distance from its wall

Hydration forces exerted by water in the form of hydrogen bonding networks or electrostatic interactions play an essential role in protein structure and function. These interactions often govern chemical catalysis, ion transport, protein stability, and folding. While waters role as a biological solvent and on the protein surface is widely studied, its function inside protein cavities is often neglected due to the existing challenges in its detection using experimental and computational approaches. The importance of studying these special protein-water interactions is further underscored by the fact that water spatially confined within cavities exhibits deviations from bulk behavior, directly impacting processes occurring inside protein cavities. With these challenges in mind and building upon our method that accurately identifies the protein inner cavity surface (CICLOP), we have developed a tool that can accurately distinguish water occurring within cavities from the bulk solvent around the protein. Our tool can characterize the dynamic properties of water within protein cavities, such as diffusion, residence time, and rotational and orientational relaxation, using molecular dynamics (MD) simulation trajectories as input. We demonstrate the robustness of our tool on several cavity-containing proteins and describe its applicability in characterizing the biological function of water confined within the cavity of an archaeal group II chaperonin.

bioinformatics↗

bioRxiv2024

The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19F-NMR

Endogenous phospholipids influence the conformational equilibria of G protein-coupled receptors, regulating their ability to bind drugs and form signaling complexes. However, most studies of GPCR-lipid interactions have been carried out in mixed micelles or lipid nanodiscs. Though useful, these membrane mimetics do not fully replicate the physical properties of native cellular membranes associated with large assemblies of lipids. We investigated the conformational equilibria of the human A2A adenosine receptor (A2AAR) in phospholipid vesicles using 19F solid-state magic angle spinning NMR (SSNMR). By applying an optimized sample preparation workflow and experimental conditions, we were able to obtain 19F-SSNMR spectra for both antagonist- and agonist-bound complexes with sensitivity and linewidths closely comparable to those achieved using solution NMR. This facilitated a direct comparison of the A2AAR conformational equilibria across detergent micelle, lipid nanodisc, and lipid vesicle preparations. While antagonist-bound A2AAR showed a similar conformational equilibria across all membrane and membrane mimetic systems, the conformational equilibria of agonist-bound A2AAR exhibited differences among different environments. This suggests that the conformational equilibria of GPCRs may be influenced not only by specific receptor-lipid interactions but also by the membrane properties found in larger lipid assemblies.

biophysics↗

bioRxiv2024

Reactive Oxygen Detoxification Contributes to Mycobacterium abscessus Antibiotic Survival

When a population of bacteria is exposed to a bactericidal antibiotic, most cells die rapidly. However, a sub-population of antibiotic-tolerant cells known as "persister cells" can survive for prolonged periods. In addition, antibiotic tolerance can be broadly induced throughout the population by stresses such as nutrient deprivation. However, the pathways required to maintain viability in this setting, and how stress induces antibiotic tolerance are both poorly understood. To identify genetic determinants of antibiotic tolerance in mycobacteria, we carried out transposon mutagenesis insertion sequencing (Tn-Seq) screens in Mycobacterium abscessus (Mabs) exposed to bactericidal translation-inhibiting antibiotics. This analysis identified genes essential for the survival of both spontaneous persister cells as well as for stress-induced tolerance, allowing the first genetic comparison of these states in mycobacteria. Pathway analysis identified multiple genes involved in the detoxification of reactive oxygen species (ROS), including the catalase-peroxidase katG, which contributed to survival in both unstressed and nutrient-starved cells. In addition, we found that endogenous ROS were generated by translation-inhibiting antibiotics, and that hypoxia impaired bacterial killing. KatG specifically contributed to survival following exposure to transcription or translation inhibitors, but not other antibiotic classes tested. Thus, the lethality of some antibiotics is amplified by toxic ROS accumulation, and antibiotic-tolerant cells require detoxification systems to remain viable. These findings further demonstrate that antibiotic-induced ROS plays a broad role in mediating antibiotic lethality across diverse organisms.

microbiology↗

bioRxiv2024

The binary protein interactome mapping of the Giardia lamblia proteasome lid reveals extra proteasomal functions of GlRpn11

Giardia lamblia does not encode Rpn12 and Sem1, two proteins crucial for assembling the proteasome lid. To understand how the interactions between the giardial proteasome lid subunits may have changed to compensate for their absence, we used the yeast two-hybrid assay to generate a binary protein interaction map of the Giardia lid subunits. Most interactions within the Giardia proteasome lid are stronger than those within the Saccharomyces cerevisiae lid. These may compensate for the absence of Rpn12 and Sem1. A notable exception was the weaker interaction between GlRpn11 and GlRpn8, compared to the strong interaction between Rpn11-Rpn8 of yeast. The Rpn11-Rpn8 dimer provides a platform for lid assembly and their interaction involves the insertion of a methionine residue of Rpn11 into a hydrophobic pocket of Rpn8. Molecular modeling indicates that GlRpn8s pocket is wider, reconciling the experimental observation of its weak interaction with GlRpn11. This weaker interaction may have evolved to support extra proteasomal functions of GlRpn11, which localizes to multiple subcellular regions where other proteasome subunits have not been detected. One such location is the mitosome. Functional complementation in yeast shows that GlRpn11 can influence mitochondrial function and distribution. This, together with its mitosomal localization, indicates that GlRpn11 functions at the mitosome. Thus, this parasites proteasome lid has a simpler subunit architecture and structural attributes that may support dual functionalities for GlRpn11. Such parasite-specific proteasome features could provide new avenues for controlling the transmission of Giardia. 1 Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/613619v1_ufigs1.gif" ALT="Figure 0"> View larger version (33K): org.highwire.dtl.DTLVardef@989e69org.highwire.dtl.DTLVardef@1c8134aorg.highwire.dtl.DTLVardef@6a9794org.highwire.dtl.DTLVardef@5ac20_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIGiardia genome does not encode two proteasomal lid subunits: Rpn12 and Sem1 C_LIO_LIUnique interactions within the lid may compensate for the absence of these two C_LIO_LIGlRpn8:GlRpn11 weakly interacts to support GlRpn11s extra-proteasomal distribution C_LIO_LIGlRpn11 localizes at mitosomes, OZ of VD, and to the VFP C_LIO_LIThe 182-218 fragment of GlRpn11 may regulate mitosomal function C_LI

cell biology↗

bioRxiv2024

Prior exposure to malaria decreases SARS-CoV-2 mediated mortality in K18-hACE2 mice without influencing viral load in lungs

BackgroundEpidemiological evidence for decreased prevalence and/or mortality due to SARS-CoV-2 infections in countries endemic for malaria have been reported. However, such associational studies in human population are limited by known and several unknown confounding factors. The current study, the first of its kind, was designed to seek experimental evidence to test the hypothesis if prior exposure to Plasmodial infections cross-protect against SARS-CoV-2 challenge infection in a murine model, K-18 human ACE2 transgenic mice. MethodsMice that had recovered from Plasmodium chabaudi infection 40 days earlier were challenged with a virulent strain of SARS-CoV-2 and viral load in lungs as well as mortality were scored and compared with K18 hACE2 mice that had not experienced prior malaria. ResultsThe viral load in lungs 6 days post challenge were comparable in malaria recovered mice and controls suggesting no significant generation of anti-viral immunity. However, mice with prior malaria exposure were significantly protected against SARS-CoV-2 induced mortality. Significant differences were observed in several host immune responses between the two groups when cytokines, chemokines and transcription factors were quantified in lungs. The plasma levels of several cytokines and chemokines were also significantly different between the two groups. ConclusionThe results of the study suggest that prior exposure to malaria protects mice against viral induced mortality in K18 hACE2 transgenic mice challenged with a virulent isolate of SARS- CoV-2 in the absence of demonstrable host immunity inhibiting viral growth in lungs.

immunology↗

bioRxiv2023

Machine Learning Based Modelling of Human and Insect Olfaction Screens Millions of compounds to Identify Pleasant Smelling Insect Repellents

The rational discovery of behaviorally active odorants is impeded by limited information on how the olfactory system generates percept or valence for a volatile chemical. In previous studies, we showed that chemical informatics could be used to predict ligands for a large repertoire of odorant receptors in Drosophila (Boyle et al., 2013). However, it remained difficult to predict behavioral valence of volatiles since the activities of a large ensembles of odor receptors encode odor information, and little is known about the complex information processing circuitry. This is a systems-level challenge well-suited for Machine-learning approaches which we have used to model olfaction in two organisms with completely unrelated olfactory receptor proteins: humans ([~]400 GPCRs) and insects ([~]100 ion-channels). We use chemical structure-based Machine Learning models for prediction of valence in insects and for 146 human odor characters. Using these predictive models, we evaluate a vast chemical space of >10 million compounds in silico. Validations of human and insect behaviors yield very high success rates. The discovery of desirable fragrances for humans that are highly repulsive to insects offers a powerful integrated approach to discover new insect repellents.

neuroscience↗

bioRxiv2023

DIGEST: An online tool for designing of multiple reaction monitoring assays

Targeted proteomics using multiple reaction monitoring (MRM) assays enables fast and sensitive detection of a preselected set of target peptides. This technique utilizes the specificity of precursors to product transitions for quantitative analysis of multiple proteins in a single sample. The success of an MRM experiment depends on the selection of transitions however, given the existing resources, accurately predicting signal intensity of peptides and their fragmentation patterns ab initio is challenging task. We present an alternative for rapid design of MRM transitions for proteomics research: DIGEST. Our method predicts the b and y ions with +1 and +2 charge produced in a collision cell of a mass spectrometer from peptides of multiple proteotypically digested proteins. Additionally, by using the existing knowledge of the fundamental rules for designing transitions, the tool provides optimal MRM transitions, negating the need to undertake prior "discovery" MS studies. We demonstrate that our algorithm is directed toward the selection of MRM precursor and product-ions pairs, and can avoid the pitfalls of interference due to cross-contamination of samples by selecting ion combinations that uniquely map to target peptides. Comparison with SRMAtlas showed that DIGEST successfully predicted the peptide and production pairs in the majority of cases. We believe that DIGEST will facilitate rapid design of MRM assays with increased specificity, reducing the overall time required to design an MRM assay for routine mass-spectrometry. DIGEST is available as a web-based tool at https://digest.raylab.iiitd.edu.in/

bioinformatics↗

bioRxiv2023

An explainable model using Graph-Wavelet for predicting biophysical properties of proteins and measuring mutational effects

Proteins hold multispectral patterns of different kinds of physicochemical features of amino acids in their structures, which can help understand proteins behavior. Here, we propose a method based on the graph-wavelet transform of signals of features of amino acids in protein residue networks derived from their structures to achieve their abstract numerical representations. Such abstract representations of protein structures hand in hand with amino-acid features can be used for different purposes, such as modelling the biophysical property of proteins. Our method outperformed graph-Fourier and convolutional neural-network-based methods in predicting the biophysical properties of proteins. Even though our method does not predict deleterious mutations, it can summarize the effect of an amino acid based on its location and neighbourhood in protein-structure using graph-wavelet to estimate its influence on the biophysical property of proteins. Such an estimate of the influence of amino-acid has the potential to explain the mechanism of the effect of deleterious non-synonymous mutations. Thus, our approach can reveal patterns of distribution of amino-acid properties in the structure of the protein in the context of a biophysical property for better classification and more insightful understanding.

bioinformatics↗

bioRxiv2023

Critical role of CD206+ macrophages in organizing anti-tumor immunity

Tumor-associated macrophages (TAMs) are frequently categorized as being M1 or M2 polarized, even as substantial data challenges this binary modeling of macrophage cell state. One molecule consistently referenced as a delineator of a putative immunosuppressive M2 state is the surface protein CD206. We thus made a novel conditional CD206 (Mrc1) knock-in mouse to specifically visualize and/or deplete CD206+ M2-like TAMs and assess their correspondence with pro-tumoral immunity. Early, but not late depletion of CD206+ macrophages and monocytes (here, Mono/Macs) led to an indirect loss of a key anti-tumor network of NK cells, conventional type I dendritic cells (cDC1) and CD8 T cells. Among myeloid cells, we found that the CD206+ TAMs are the primary producers of CXCL9, and able to differentially attract activated CD8 T cells. In contrast, a population of stress-responsive TAMs ("Hypoxic" or Spp1+) and immature monocytes, which lack CD206 expression and become prominent following early depletion, expressed markedly diminished levels of CXCL9. Those NK and CD8 T cells which enter CD206- depleted tumors express vastly reduced levels of the corresponding receptor Cxcr3, the cDC1- attracting chemokine Xcl1 and cDC1 growth factor Flt3l transcripts. Consistent with the loss of this critical network, early CD206+ TAM depletion decreased tumor control by antigen specific CD8 T cells in mice. Likewise, in humans, the CD206Replete, but not the CD206Depleted Mono/Mac gene signature correlated robustly with CD8 T cell, NK cell and stimulatory cDC1 gene signatures and transcriptomic signatures skewed towards CD206Replete Mono/Macs associated with better survival. Together, these findings negate the unqualified classification of CD206+ M2-like macrophages as immunosuppressive by illuminating contexts for their role in organizing a critical tumor-reactive archetype of immunity.

immunology↗

bioRxiv2023

Membrane Mimetic-Dependence of GPCR Energy Landscapes

Protein function strongly depends on temperature, which is related to temperature-dependent changes in the equilibria of protein conformational states. We leveraged variable-temperature 19F-NMR spectroscopy to interrogate the temperature dependence of the conformational landscape of the human A2A adenosine receptor (A2AAR), a class A GPCR. Temperature-induced changes in the conformational equilibria of A2AAR in lipid nanodiscs were markedly dependent on the efficacy of bound drugs. While antagonist complexes displayed only modest changes as the temperature rose, both full and partial agonist complexes exhibited substantial increases in the active state population. Importantly, the temperature-dependent response of complexes with both full and partial agonists exhibited a pronounced sensitivity to the specific membrane mimetic employed. In striking contrast to observations within lipid nanodiscs, in detergent micelles the active state population exhibited different behavior for A2AAR complexes with both full and partial agonists. This underscores the importance of the protein environment in understanding the thermodynamics of GPCR activation. HighlightsO_LIActive A2AAR population increases with increasing temperature in lipid nanodiscs C_LIO_LIActive A2AAR population exhibits different temperature dependence in detergents C_LIO_LIPartial agonist complexes present a unique conformational state in nanodiscs C_LIO_LITemperature dependence of partial agonist equilibria depends on membrane mimetic C_LI

biophysics↗

bioRxiv2023

Increased epithelial mTORC1 activity in chronic rhinosinusitis with nasal polyps

BackgroundThe airway epithelium plays a central role in the pathogenesis of chronic respiratory diseases such as asthma and chronic rhinosinusitis with nasal polyps (CRSwNP), but the mechanisms by which airway epithelial cells (EpCs) maintain inflammation are poorly understood. ObjectiveWe hypothesized that transcriptomic assessment of sorted airway EpCs across the spectrum of differentiation would allow us to define mechanisms by which EpCs perpetuate airway inflammation. MethodsEthmoid sinus EpCs from adult patients with CRS were sorted into 3 subsets, bulk RNA sequenced, and analyzed for differentially expressed genes and pathways. Single cell RNA-seq (scRNA-seq) datasets from eosinophilic and non-eosinophilic CRSwNP and bulk RNA-seq of EpCs from mild/moderate and severe asthma were assessed. Immunofluorescent staining and ex vivo functional analysis of sinus EpCs were used to validate our findings. ResultsAnalysis within and across purified EpC subsets revealed an enrichment in glycolytic programming in CRSwNP vs CRSsNP. Correlation analysis identified mammalian target of rapamycin complex 1 (mTORC1) as a potential regulator of the glycolytic program and identified EpC expression of cytokines and wound healing genes as potential sequelae. mTORC1 activity was upregulated in CRSwNP, and ex vivo inhibition demonstrated that mTOR is critical for EpC generation of CXCL8, IL-33, and CXCL2. Across patient samples, the degree of glycolytic activity was associated with T2 inflammation in CRSwNP, and with both T2 and non-T2 inflammation in severe asthma. ConclusionsTogether, these findings highlight a metabolic axis required to support epithelial generation of cytokines critical to both chronic T2 and non-T2 inflammation in CRSwNP and asthma. KEY MESSAGESO_LIEpithelial mTORC1 activity is upregulated in CRSwNP. C_LIO_LImTOR regulates EpC cytokine generation. C_LIO_LIEpithelial metabolic reprograming correlates with T2 inflammation in CRSwNP, and with both T2 and non-T2 inflammation in asthma. C_LI CAPSULE SUMMARYmTORC1 mediates EpC cytokine generation in CRSwNP.

immunology↗

bioRxiv2023

Multimodal identification of rare potent effector CD8 T cells in solid tumors

The anti-tumor function of CD8 T cells is limited through well-established pathways of T cell exhaustion (TEX). Strategies to capture emergent functional states amongst this dominant trajectory of dysfunction are necessary to find pathways to durable anti-tumor immunity. By leveraging transcriptional reporting (by the fluorescent protein TFP) of the T cell activation marker Cd69, related to upstream AP-1 transcription factors, we define a classifier for potent versus sub-optimal CD69+ activation states arising from T cell stimulation. In tumors, this delineation acts an additional functional readout along the TEX differentiation trajectory, within and across TEX subsets, marked by enhanced effector cytokine and granzyme B production. The more potent state remains differentially prominent in a T cell-mediated tumor clearance model, where they also show increased engagement in the microenvironment and are superior in tumor cell killing. Employing multimodal CITE-Seq in human head and neck tumors enables a similar strategy to identify Cd69RNAhiCD69+ cells that also have enhanced functional features in comparison to Cd69RNAloCD69+ cells, again within and across intratumoral CD8 T cell subsets. Refining the contours of the T cell functional landscape in tumors in this way paves the way for the identification of rare exceptional effectors, with imminent relevance to cancer treatment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/559470v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@4a9429org.highwire.dtl.DTLVardef@4535d9org.highwire.dtl.DTLVardef@1d26e03org.highwire.dtl.DTLVardef@34acd2_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Metagenomics untangles metabolic adaptations of Antarctic endolithic 1 bacteria at the fringe of habitability

BackgroundEndolithic niches offer an ultimate refuge, supplying buffered conditions for microorganisms that dwell inside rock airspaces. Yet, survival and growth strategies of Antarctic endolithic microbes residing in Earths driest and coldest desert remains virtually unknown. ResultsFrom 109 endolithic microbiomes, 4,539 metagenome-assembled genomes were generated, 49.3% of which were novel candidate bacterial species. We present evidence that trace gas oxidation and atmospheric chemosynthesis may be the prevalent strategies supporting metabolic activity and persistence of these ecosystems at the fringe of life and the limits of habitability. ConclusionsThese results represent the foundation to untangle adaptability at the edge of sustainability on Earth and on other dry Earth-like planetary bodies such as Mars.

microbiology↗

bioRxiv2023

GLUT4 dynamic subcellular localization is controlled by AMP kinase activation as revealed by proximal proteome mapping in human muscle cells

Regulation of glucose transport into muscle and adipocytes, central for control of whole-body metabolism, is determined by the amount of GLUT4 glucose transporter in the plasma membrane (PM). Physiologic signals (activated insulin receptor or AMP kinase [AMPK]), acutely increase PM GLUT4 to enhance glucose uptake. Here we show in kinetic studies that intracellular GLUT4 is in equilibrium with the PM in unstimulated cultured human skeletal muscle cells, and that AMPK promotes GLUT4 redistribution to the PM by regulating both exocytosis and endocytosis. AMPK-stimulation of exocytosis requires Rab10 and Rab GTPase activating protein TBC1D4, requirements shared with insulin control of GLUT4 in adipocytes. Using APEX2 proximity mapping, we identify, at high-density and high-resolution, the GLUT4 proximal proteome, revealing GLUT4 traverses both PM proximal and distal compartments in unstimulated muscle cells. These data support intracellular retention of GLUT4 in unstimulated muscle cells by a dynamic mechanism dependent on the rates of internalization and recycling. AMPK promoted GLUT4 translocation to the PM involves redistribution of GLUT4 among the same compartments traversed in unstimulated cells, with a significant redistribution of GLUT4 from the PM distal Trans Golgi Network Golgi compartments. The comprehensive proximal protein mapping provides an integrated, whole cell accounting of GLUT4s localization at a resolution of [~]20 nm, a structural framework for understanding the molecular mechanisms regulating GLUT4 trafficking downstream of different signaling inputs in physiologically relevant cell type and as such, sheds new light on novel key pathways and molecular components as potential therapeutic approaches to modulate muscle glucose uptake.

cell biology↗

bioRxiv2023

Plasticity of gene expression in the nervous system by exposure to environmental odorants that inhibit HDACs

Eukaryotes are often exposed to microbes and respond to their secreted metabolites, such as the microbiome in animals or commensal bacteria in roots. Little is known about the effects of long-term exposure to volatile chemicals emitted by microbes, or other volatiles that we are exposed to over a long duration. Using the model system Drosophila melanogaster, we evaluate a yeast emitted volatile, diacetyl, found in high levels around fermenting fruits where they spend long periods of time. We find that exposure to just the headspace containing the volatile molecules can alter gene expression in the antenna. Experiments showed that diacetyl and structurally related volatile compounds inhibited human histone-deacetylases (HDACs), increased histone-H3K9 acetylation in human cells, and caused wide changes in gene expression in both Drosophila and mice. Diacetyl crosses the blood-brain barrier and exposure causes modulation of gene expression in the brain, therefore has potential as a therapeutic. Using two separate disease models known to be responsive to HDAC-inhibitors, we evaluated physiological effects of volatile exposure. First, we find that the HDAC inhibitor also halts proliferation of a neuroblastoma cell line in culture as predicted. Next, exposure to vapors slows progression of neurodegeneration in a Drosophila model for Huntingtons disease. These changes strongly suggest that unbeknown to us, certain volatiles in the surroundings can have profound effects on histone acetylation, gene expression and physiology in animals.

neuroscience↗

bioRxiv2023

DksA homologs, ppGpp and RegAB regulate dissimilatory nitrogen metabolism in Paracoccus denitrificans

The periplasmic (NAP) and membrane-associated (Nar) nitrate reductases of Paracoccus denitrificans are responsible for nitrate reduction under aerobic and anaerobic conditions, respectively. Expression of NAP is elevated in cells grown on a relatively reduced carbon and energy source (such as butyrate); it is believed that NAP contributes to redox homeostasis by coupling nitrate reduction to the disposal of excess reducing equivalents. Here we show that deletion of either dksA1 (encoding one of two dksA homologs in the P. denitrificans genome) or relA/spoT (encoding a bifunctional ppGpp synthetase and hydrolase) eliminates the butyrate-dependent increase in nap promoter and NAP enzyme activity. We conclude that ppGpp likely signals growth on a reduced substrate and, together with DksA1, mediates increased expression of the genes encoding NAP. Support for this model comes from the observation that nap promoter activity is increased in cultures exposed to a protein synthesis inhibitor that is known to trigger ppGpp synthesis in other organisms. We also show that, under anaerobic growth conditions, the redox-sensing RegAB two-component pair acts as a negative regulator of NAP expression and as a positive regulator of expression of the membrane-associated nitrate reductase Nar. The dksA1 and relA/spoT genes are conditionally synthetically lethal; the double mutant has a null phenotype for growth on butyrate and other reduced substrates while growing normally on succinate and citrate. We also show that the second dksA homolog (dksA2) and relA/spoT have roles in regulation of expression of the flavohemoglobin Hmp and in biofilm formation. IMPORTANCEParacoccus denitrificans is a metabolically versatile Gram-negative bacterium that is used as a model for studies of respiratory metabolism. The organism can utilize nitrate as an electron acceptor for anaerobic respiration, reducing it to dinitrogen via nitrite, nitric oxide, and nitrous oxide. This pathway (known as denitrification) is important as a route for loss of fixed nitrogen from soil and as a source of the greenhouse gas nitrous oxide. Thus, it is important to understand those environmental and genetic factors that govern flux through the denitrification pathway. Here we identify four proteins and a small molecule (ppGpp) which function as previously unknown regulators of expression of enzymes that reduce nitrate and oxidize nitric oxide.

microbiology↗

bioRxiv2023

Anionic Phospholipids Control Mechanisms of GPCR-G Protein Recognition

G protein-coupled receptors (GPCRs) are embedded in phospholipids that strongly influence drug-stimulated signaling. Anionic lipids are particularly important for GPCR signaling complex formation, but a mechanism for this role is not understood. Using NMR spectroscopy, we visualized the impact of anionic lipids on the function-related conformational equilibria of the human A2A adenosine receptor (A2AAR) in bilayers containing defined mixtures of zwitterionic and anionic phospholipids. Anionic lipids primed the receptor to form complexes with G proteins through a conformational selection process. Without anionic lipids, signaling complex formation proceeded through a less favorable induced fit mechanism. In computational models, anionic lipids mimicked interactions between a G protein and positively charged residues in A2AAR at the receptor intracellular surface, stabilizing a pre-activated receptor conformation. Replacing these residues strikingly altered the receptor response to anionic lipids in experiments. High sequence conservation of the same residues among all GPCRs supports a general role for lipid-receptor charge complementarity in signaling.

biophysics↗

bioRxiv2022

Mapping phenotypic heterogeneity in melanoma onto the epithelial-hybrid-mesenchymal axis

Epithelial to mesenchymal transition (EMT) is a well-studied hallmark of epithelial-like cancers that is characterized by loss of epithelial markers and gain of mesenchymal markers. Interestingly, melanoma, which is derived from melanocytes of the skin, also undergo phenotypic plasticity toward mesenchymal-like phenotypes under the influence of various micro-environmental cues. Our study connects EMT to the phenomenon of de-differentiation (i.e., transition from proliferative to more invasive phenotypes) observed in melanoma cells during drug treatment. By analyzing 78 publicly available transcriptomic melanoma datasets, we found that de-differentiation in melanoma is accompanied by upregulation of mesenchymal genes, but not necessarily a concomitant loss of an epithelial program, suggesting a more "one-dimensional" EMT that leads to a hybrid epithelial/ mesenchymal phenotype. Samples lying in the hybrid epithelial/mesenchymal phenotype also correspond to the intermediate phenotypes in melanoma along the proliferative-invasive axis - neural crest and transitory ones. Interestingly, as melanoma cells progress along the invasive axis, the mesenchymal signature does not increase monotonically. Instead, we observe a peak in mesenchymal scores followed by a decline, as cells further de-differentiate. This biphasic response recapitulates the dynamics of melanocyte development, suggesting close interactions among genes controlling differentiation and mesenchymal programs in melanocytes. Similar trends were noted for metabolic changes often associated with EMT in carcinomas in which progression along mesenchymal axis correlates with the downregulation of oxidative phosphorylation, while largely maintaining glycolytic capacity. Overall, these results provide an explanation for how EMT and dedifferentiation axes overlap with respect to their transcriptional and metabolic programs in melanoma.

cancer biology↗