Biology subjects

Anand, P. K.

Publications and source records attributed to Anand, P. K..

6 recordsLinked to original sources

bioRxiv2026

Lipid droplet accumulation drives glutamine-dependent NLRP3 inflammasome activation

The NLRP3 inflammasome is implicated in the pathogenesis of many inflammatory and metabolic diseases. Although metabolic cues can shape NLRP3 inflammasome activation, the role of lipid metabolism in this response is poorly understood. Lipids are essential for energy and redox homeostasis, membrane biogenesis, and diverse cellular processes. However, excessive accumulation of lipids such as free fatty acids can be highly cytotoxic. Consequently, surplus free fatty acids are esterified into triglycerides and cholesteryl esters, which are then stored within lipid droplets (LDs). Interestingly, transcriptomic data analyses revealed that macrophages exposed to inflammasome-activating stimulus undergo a broad metabolic rewiring favouring lipid storage, suggesting a link between LDs and inflammasome response. Here, we show that LD accumulation actively promotes NLRP3 inflammasome activation. Increasing LD abundance, either by fatty acid supplementation or by inhibition of lipolysis, led to robust caspase-1 activation and IL-1{beta} release both in vitro and in vivo. Mechanistically, LD expansion promoted cellular reorganisation resulting in the formation of peri-droplet mitochondria, a distinct mitochondrial subpopulation which exhibited elevated membrane potential and increased ATP-linked respiration. Furthermore, LD-rich macrophages underwent metabolic reconfiguration whereby they exhibited a pronounced reliance on pyruvate and glutamine metabolism, but not fatty acid oxidation, to sustain mitochondrial bioenergetics. Surprisingly, inflammasome activation was found dependent predominantly on glutaminolysis, highlighting a key role for glutamine catabolism in LD-rich cells. Collectively, these findings identify a glutamine-dependent pathway as a key driver linking excess lipid storage to NLRP3 inflammasome, with profound implications in metabolic diseases.

immunology↗

bioRxiv2026

Distinct lipid transport proteins are regulated by innate immune stimuli

Lipid transport plays a critical role in the distribution of lipids across subcellular compartments. This is pivotal during infection and other stress stimuli that increase metabolic demands. While lipid biosynthesis is regulated by immune stimuli, whether immune signalling also influences lipid transport mechanisms remains unexplored. Here, we demonstrate that TLR signalling impacts the gene expression of lipid transport proteins in human monocytic THP-1 cell line and compare it to the expression in primary bone marrow-derived mouse macrophages. Our data demonstrates that TLR4 signalling selectively modulates the expression of oxysterol-binding protein-related proteins (ORPs), a key family of proteins that transport lipids between organelles. Remarkably, TLR4 activation led to the downregulation of several ORP family members in human THP1-derived macrophages. However, this response was less profound in mouse macrophages. In contrast, the expression of steroidogenic acute regulatory domain (STARD) proteins, many of which transport lipids between mitochondria and other compartments, remained broadly unaffected. Moreover, IFNG, a cytokine that plays a key role in the immune response, did not considerably impact human ORP or STARD expression levels, either alone or in combination with LPS. Together, these results reveal that TLR signalling exerts selective and critical control over lipid trafficking pathways with important species-specific differences. These findings provide new insights into the crosstalk between immune signalling and lipid metabolism, which may offer novel targets to treat diseases characterized by dysregulated lipid pathways.

immunology↗

bioRxiv2025

Molecular docking of Pyocyanin from Pseudomonas aeruginosa with NADPH oxidase and Toll-like receptor 4 : Exploring the Link Between Oral Microbiome and Oral Cancer Pathogenesis

Oral cancer is among the leading cancers in India, with its progression influenced not only by genetic and environmental factors but also by dysbiosis in the oral microbiome. Pseudomonas aeruginosa, an opportunistic Gram-negative bacterium, has been implicated in oral carcinogenesis through the action of its virulence factor, pyocyanin--a redox-active phenazine that induces reactive oxygen species (ROS), promoting oxidative stress, immune dysregulation, and inflammation. Despite evidence linking pyocyanin to ROS-related pathways, its molecular interaction with key host proteins like NADPH oxidase and Toll-like receptor 4 (TLR4) remains unexplored. The present study aims to investigate the molecular interactions of pyocyanin with NADPH oxidase and TLR4 through molecular docking, aiming to elucidate its role in oxidative and inflammatory mechanisms associated with oral cancer pathogenesis. The structures of pyocyanin and the NADPH oxidase (NOX2) and TLR4 target proteins were retrieved from PubChem and RCSB PDB, respectively. CASTp was used to identify potential binding pockets. Molecular docking was performed using the HADDOCK server. The docked complexes were analyzed based on HADDOCK scores, binding energies, RMSD, and buried surface area. Interaction profiles were visualized using BIOVIA Discovery Studio. Among the docking clusters, Cluster 6 for the NOX2-pyocyanin complex showed a favorable HADDOCK score (84.4 {+/-} 2.9), low RMSD (0.2 {+/-} 0.0), and substantial buried surface area (456.1 {+/-} 13.2 [A]2). For the TLR4-pyocyanin complex, Cluster 1 exhibited the most favorable interaction profile with a HADDOCK score of 25.0 {+/-} 5.5 and a Z-score of -2.3. Binding site prediction and hydropathy analysis supported the structural feasibility of pyocyanin interaction with both targets. This study provides structural insights into the potential interaction of pyocyanin with NADPH oxidase and TLR4, supporting its role in modulating oxidative stress and inflammatory signaling pathways. These findings enhance our understanding of oral microbiome-host interactions and their contribution to oral cancer pathogenesis.

bioinformatics↗

bioRxiv2024

The Orphan G Protein-Coupled Receptor GPR52 is a Novel Regulator of Breast Cancer Multicellular Organization

Statement of SignificanceWe showed that loss of the orphan G protein-coupled receptor GPR52 in human breast cell lines leads to increased cell clustering, hybrid/partial EMT, and increased tumor burden in zebrafish. BackgroundG protein-coupled receptors (GPCRs) are the largest class of membrane-bound receptors that transmit critical signals from extracellular to intracellular spaces. Transcriptomic data of resected breast tumors show that low mRNA expression of orphan GPCR GPR52 correlates with reduced overall survival in patients with breast cancer, leading to the hypothesis that loss of GPR52 supports breast cancer progression. MethodsCRISPR-Cas9 was used to knockout GPR52 in the human triple-negative breast cancer (TNBC) cell lines MDA-MB-468 and MDA-MB-231, and in the non-cancerous breast epithelial cell line MCF10A. 2D and 3D in vitro studies, electron microscopy, Matrigel culture, and a zebrafish xenograft model were used to assess the morphology and behavior of GPR52 KO cells. RNA-sequencing and proteomic analyses were also conducted on these cell lines, and transcriptomic data from The Cancer Genome Atlas (TCGA) database were used to compare GPR52-null and wild-type (WT) signatures in breast cancer. ResultsLoss of GPR52 was found to be associated with increased cell-cell interaction in 2D cultures, altered 3D spheroid morphology, and increased propensity to organize and invade collectively in Matrigel. Furthermore, GPR52 loss was associated with features of EMT in MDA-MB-468 cells, and zebrafish injected with GPR52 KO cells developed a greater total cancer area than those injected with control cells. RNA sequencing and proteomic analyses of GPR52-null breast cancer cells revealed an increased cAMP signaling signature. Consistently, we found that treatment of wild-type (WT) cells with forskolin, which stimulates the production of cAMP, induces phenotypic changes associated with GPR52 loss, and inhibition of cAMP production rescued some GPR52 KO phenotypes. ConclusionGPR52 is an orphan GPCR and its role in cancer progression has not been previously characterized. We found that GPR52 loss in breast cancer cells can lead to increased cell clustering, collective invasion, and EMT in vitro. These are features of increased cancer aggression. Our results reveal that GPR52 loss is a potential mechanism by which breast cancer progression may occur and support the investigation of GPR52 agonism as a therapeutic option for breast cancer. O_FIG O_LINKSMALLFIG WIDTH=169 HEIGHT=200 SRC="FIGDIR/small/604482v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@8b0f55org.highwire.dtl.DTLVardef@19bd139org.highwire.dtl.DTLVardef@c52117org.highwire.dtl.DTLVardef@1ffa2bd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

cancer biology↗

bioRxiv2023

Fatty acid synthesis promotes inflammasome activation through NLRP3 palmitoylation.

Inflammasomes are multi-protein complexes assembled by NOD-like receptor (NLR) family of proteins, which play critical roles in infectious, inflammatory and metabolic diseases. The assembly of the NLRP3 inflammasome is triggered upon recognition of an apt stimulus by the sensor protein, resulting in binding to pro-caspase-1 via the adaptor protein ASC. Inflammasome activation results in the maturation of the precursor forms of cytokines IL-1{beta} and IL-18, along with caspase-1-dependent pyroptosis, a pro-inflammatory form of cell death. Emerging evidence suggests the involvement of lipid metabolism in inflammasome activation; however, the precise mechanisms by which lipids regulate the NLRP3 inflammasome remain ambiguous. A multi-enzyme protein, fatty acid synthase (FASN) is a central regulator of lipid metabolism partaking an essential role in fatty acid biosynthesis pathway by catalysing the production of palmitic acid. Palmitic acid acts as a precursor to long-chain fatty acids and additionally regulates cellular functions by palmitoylation, a process in which palmitate is reversibly added to cysteine residues of target proteins, modifying protein localization and function. Here, we undertook a pharmacological approach to investigate the roles of fatty acid biosynthetic pathway in NLRP3 inflammasome activation. Our results demonstrated that inhibition of FASN in primary mouse and human macrophages abrogates the activation of the NLRP3 inflammasome, resulting in blunted caspase-1 activation. Furthermore, this phenomenon relied on protein palmitoylation as in vitro and in vivo abrogation of palmitoylation similarly reduced NLRP3 activation, which could be restored by exogenously supplementing palmitate in cultured cells. Consequently, an acyl biotin exchange assay corroborated NLRP3 palmitoylation. Notably, activation of the dsDNA sensing AIM2 inflammasome remained unaltered when either FASN or palmitoylation was blocked. These results therefore highlight the pivotal role of FASN and palmitoylation, shedding new mechanistic insights into the activation of the NLRP3 inflammasome.

immunology↗

bioRxiv2020

Phosphatidylinositol Acyl Chains Configure TLR-Dependent Priming and Activation of the NLRP3 Inflammasome

Imbalance in lipid homeostasis is associated with discrepancies in immune signalling and is tightly linked to metabolic disorders. The diverse ways in which lipids impact immune signalling, however, remain ambiguous. The phospholipid phosphatidylinositol (PI), which is implicated in numerous immune disorders, is chiefly defined by its phosphorylation status. By contrast, the significance of the two fatty acid chains attached to the PI remains unknown. Here, by employing a mass-spectrometry-based assay, we demonstrate a role for PI acyl group chains in regulating both the priming and activation steps of the NLRP3 inflammasome in mouse macrophages. In response to NLRP3 stimuli, cells deficient in ABC transporter ABCB1, which effluxes lipid derivatives, revealed defective inflammasome activation. Mechanistically, Abcb1-deficiency shifted the total PI configuration exhibiting a reduced ratio of short-chain to long-chain PI-acyl lipids. Consequently, Abcb1-deficiency resulted in rapid degradation of TIRAP, the TLR adaptor protein which binds PI(4,5)-phosphate. Moreover, this accompanied increased NLRP3 phosphorylation at the Ser293 position and blunted inflammasome activation. Exogenously supplementing WT cells with linoleic acid, but not arachidonic acid, reconfigured PI acyl chains. Accordingly, linoleic acid supplementation increased TIRAP degradation, elevated NLRP3 phosphorylation, and abrogated inflammasome activation. Altogether, our study reveals a novel metabolic-inflammatory circuit which contributes to calibrating immune responses.

immunology↗