Biology subjects

Panja, S.

Publications and source records attributed to Panja, S..

5 recordsLinked to original sources

bioRxiv2025

Smooth muscle LRRC8A knockout reduces superoxide influx, inflammation, senescence and atherosclerosis

BackgroundLeucine Rich Repeat Containing 8A (LRRC8A) anion channels (VRACs) associate with NADPH oxidase 1 (Nox1) and support extracellular superoxide (O2{middle dot}-) production, inflammation, and contractility in vascular smooth muscle cells (VSMCs). We proposed previously that VRACs also support influx of O2{middle dot}- to localize cytoplasmic redox signals. MethodsWe assayed O2{middle dot}- influx, assessed changes in mRNA expression (RNAseq), and tested multiple phenotypes of cultured LRRC8A knockout (KO) VSMCs. Aortic atherosclerotic burden and inflammation, and mesenteric vascular reactivity were compared between wild type (WT) apolipoprotein E null (ApoE-/-) mice and VSMC-specific LRRC8A KO, ApoE-/- mice. ResultsKO cells were less permeable to extracellular O2{middle dot}-, produced less mitochondrial O2{middle dot}-, and experienced less oxidant stress (GSH/GSSG, lipid peroxidation, Nrf2 activity) than WT. RNAseq and reporter assays demonstrated reduced pro-inflammatory (NF-{kappa}B and Hif1) transcription. KO cells also under-expressed multiple senescence markers and had longer telomeres. Inflammation causes a metabolic shift from oxidative phosphorylation (OCR) to glycolysis (ECAR). Both pathways were less active in KO cells, as was expression of glycolytic enzymes. Mitochondrial membrane potential was lower in KO cells, but ADP/ATP and NADP+/NADPH were unaltered, suggesting lower energy demand. Consistent with this, proliferation and migration were both reduced in KO cells. Protein-protein interaction analysis of RNAseq data (PPI Hub) identified Epidermal Growth Factor Receptor (EGFR) signaling. Resting phosphorylation of EGFR (pY1068) and AKT (pS473 and pT308) were all reduced in KO cells, as was and EGF-induced pY1068 and pS308. Following 15 weeks of exposure to a high fat diet (42%) VSMC-specific LRRC8A-/- and 8A+/-, ApoE-/- mice had reduced atherosclerotic lesion area, aortic senescence ({beta}-Gal), inflammation (ICAM, VCAM) and proliferation marker (PCNA) expression compared to WT, ApoE-/- controls. Mesenteric artery vasomotor function was also preserved, and the abundance of MYPT1 and CPI17 was lower in KO vessels. Uptake of oxidized LDL (OxLDL) was significantly reduced in KO VSMCs and impaired by VRAC block in WT cells. ConclusionsLoss of LRRC8A reduced O2{middle dot}- influx, oxidative stress, inflammation and senescence, lowers energy demand, and in the setting of hypercholesterolemia impaired uptake of oxidized LDL and abrogated atherosclerosis. VSMC VRACs may be novel targets of vascular anti-inflammatory therapy. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIChronically inflamed vascular smooth muscle cells (VSMCs) are critical drivers of atherosclerotic plaque development. C_LIO_LIIn VSMCs, Nox1-derived superoxide drives redox signaling that promotes inflammation, phenotypic switching, and senescence. C_LIO_LILRRC8A family volume-regulated anion channels (VRACs) physically associate with Nox1 and support TNF-induced ROS production, receptor endocytosis, NF-{kappa}B activation, and proliferation. C_LIO_LIHypercholesterolemia causes VSMC inflammation via oxidized LDL (OxLDL) activation of scavenger receptors (e.g., LOX-1, CD36) that stimulate oxidant production and EGFR/PI3K-Akt-NF-{kappa}B signaling. C_LI What New Information Does This Article Contribute?O_LILRRC8A channels support influx of extracellular superoxide which impacts cytosolic redox status, metabolism and Nrf2, HIF-1, and NF-{kappa}B activity. C_LIO_LILoss of LRRC8A reprograms VSMCs into a low-oxidative-stress, low-energy-demand state, characterized by diminished proliferation and migration, attenuated senescence, and preservation of telomere length. C_LIO_LILoss of LRRC8A inhibits EGF-stimulated EGFR and Akt phosphorylation and uncouples oxidant-dependent growth factor signaling from downstream inflammatory and metabolic remodeling. C_LIO_LIVSMC-specific LRRC8A knockout or heterozygosity in hypercholesterolemic ApoE-/- mice reduces aortic inflammation and senescence, reduces atherosclerotic lesion burden, and preserves mesenteric artery vasomotor function. C_LIO_LILRRC8A/VRAC activity is necessary for uptake of oxidized but not native LDL in VSMCs. C_LIO_LILRRC8A is a novel regulator of scavenger receptor-dependent lipoprotein handling and a potential therapeutic target to uncouple oxidant signaling from VSMC lipid overload and inflammation in the vessel wall. C_LI

physiology↗

bioRxiv2025

Smooth muscle LRRC8A knockout preserves vascular function in AngII hypertension

Angiotensin II (AngII) causes hypertension and vascular inflammation both directly and indirectly via cytokines. In vascular smooth muscle cells (VSMCs) AngII and TNF activate NADPH oxidase 1 (Nox1) to produce superoxide. TNF receptors associate with Nox1 and Leucine Rich Repeat Containing 8A (LRRC8A) anion channels to modulate inflammation, as well as contractility in a RhoA-dependent manner. VSMC-specific LRRC8A knockout (KO) mesenteric arteries are protected from TNF-induced injury and vasodilated better. We hypothesized that LRRC8A KO would preserve vascular function and decrease blood pressure (BP) in AngII-infused mice. Wild type (WT) and LRRC8A KO mice received AngII infusions for 14 days. Systolic BP was not different, but KO mice had more BP "dipping" during inactive periods and dipping was preserved after AngII. Contraction of KO mesenteric vessels to AngII itself was not altered, however after AngII exposure the function of KO aortic and mesenteric vessels was less impaired as reflected by less augmented contraction to norepinephrine and serotonin and preserved relaxation to acetylcholine and sodium nitroprusside. Western blotting revealed increased soluble guanylate cyclase alpha and reduced CPI-17 in hypertensive KO aortae. Consistent with the presence of lower Rho kinase activity in KO VSMCs, phosphorylation of Ezrin/Radixin/Moesin (ERM) and Cofilin was reduced. Aortic wall/lumen was not different between hypertensive WT and KO mice, but AngII caused less proliferation (lower PCNA), and less induction of antioxidant enzymes and senescence markers in KO vessels. Thus, while AngII-induced contraction does not require LRRC8A, these channels support the associated inflammatory response which modifies BP dipping.

physiology↗

bioRxiv2023

Biophysical evolution of the receptor binding domains of SARS-CoVs

With hundreds of coronaviruses (CoVs) identified in bats that are capable of infecting humans, it is important to understand how CoVs that affected the human population have evolved. Seven known coronaviruses have infected humans, of which three CoVs caused severe disease with high mortality rates: SARS-CoV emerged in 2002, MERS-CoV in 2012, and SARS-CoV-2 in 2019. Both SARS-CoV and SARS-CoV-2 belong to the same family, follow the same receptor pathway, and use their receptor binding domain (RBD) of spike protein to bind to the ACE2 receptor on the human epithelial cell surface. The sequence of the two RBDs is divergent, especially in the receptor binding motif (RBM) that directly interacts with ACE2. We probed the biophysical differences between the two RBDs in terms of their structure, stability, aggregation, and function. Since RBD is being explored as an antigen in protein subunit vaccines against CoVs, determining these biophysical properties will also aid in developing stable protein subunit vaccines. Our results show that despite RBDs having a similar three-dimensional structure, they differ in their thermodynamic stability. RBD of SARS-CoV-2 is significantly less stable than that of SARS-CoV. Correspondingly, SARS-CoV-2 RBD shows a higher aggregation propensity. Regarding binding to ACE2, less stable SARS-CoV-2 RBD binds with a higher affinity than more stable SARS-CoV RBD. In addition, SARS-CoV-2 RBD is more homogenous in terms of its binding stoichiometry towards ACE2, compared to SARS-CoV RBD. These results indicate that SARS-CoV-2 RBD differs from SARS-CoV RBD in terms of its stability, aggregation, and function, possibly originating from the diverse RBMs. Higher aggregation propensity and decreased stability of SARS-CoV-2 RBD warrants further optimization of protein subunit vaccines that use RBD as an antigen either by inserting stabilizing mutations or formulation screening. Statement of SignificanceThis study holds significant relevance in the context of the COVID-19 pandemic and the broader understanding of coronaviruses. A comparison of the receptor binding domains (RBDs) of SARS-CoV and SARS-CoV-2 reveals significant differences in their structure, stability, aggregation, and function. Despite divergent sequences, the RBDs share a similar fold and ACE2 receptor binding capability, likely through convergent evolution. These findings are crucial for understanding coronavirus evolution, interactions with human receptors, and the spillover of coronaviruses from animals to humans. The study also has implications for vaccine design strategies for SARS-CoVs, where the RBD is used as an antigen in protein subunit vaccines. By anticipating future outbreaks and enhancing our understanding of zoonotic spillover, this research contributes to safeguarding human health.

biophysics↗

bioRxiv2021

Interplay between protein stability, binding to ACE2 and escape from neutralizing antibodies determines the natural selection of SARS-CoV-2 receptor binding domain variants

Emergence of new SARS-CoV-2 variants has raised concerns at the effectiveness of vaccines and antibody therapeutics developed against the unmutated wild-type virus. We examined the effect of 12 most commonly occurring mutations in the receptor binding domain on its expression, stability, activity, and antibody escape potential-some of the factors that may influence the natural selection of mutants. Recombinant proteins were expressed in human cells. Stability was measured using thermal denaturation melts. Activity and antibody escape potential were measured using isothermal titration calorimetry in terms of binding to ACE2 and to a neutralizing human antibody CC12.1, respectively. Our results show that variants differ in their expression levels with the two least stable variants showing lesser expression. Out of the 8 well-expressed mutants, only 2 (N501Y and K417T/E484K/N501Y) showed stronger affinity to ACE2, 4 (Y453F, S477N, T478I and S494P) have similar affinity, whereas the other 2 (K417N and E484K) have weaker affinity when compared to the wild-type. In terms of CC12.1 binding, when compared to the wild-type, 4 variants (K417N, Y453F, N501Y and K417T/E484K/N501Y) have weaker affinity, 2 (S477N and S494P) have similar affinity, and 2 (T478I and E484K) have stronger affinity. Taken together, these results indicate that multiple factors contribute towards the natural selection of variants, and all these factors need be considered to understand the evolution of the virus. In addition, since not all variants can escape a given neutralizing antibody, antibodies to treat new variants can be chosen based on the specific mutations in that variant.

biophysics↗

bioRxiv2019

Targeting EZH2 Increases Therapeutic Efficacy of Check-Point Blockade in Models of Prostate Cancer

Prostate cancers are considered immunologically cold tumors given the very few patients who respond to checkpoint inhibitor therapy (CPI). Recently, enrichment of interferon (IFN) response genes predicts a favorable response to CPI across various disease sites. The enhancer of zeste homolog-2 (EZH2) is over-expressed in prostate cancer and is known to negatively regulate IFN response genes. Here, we demonstrate that inhibition of EZH2 catalytic activity in prostate cancer models derepresses expression of double-strand RNA (dsRNA), associated with upregulation of genes involved in antigen presentation, Th-1 chemokine signaling, and interferon (IFN) response, including PD-L1. Similarly, application of a novel EZH2 derived gene signature to human prostate sample analysis indicated an inverse correlation between tumor EZH2 activity/expression with T-cell inflamed and IFN gene signatures and PD-L1 expression. EZH2 inhibition combined with PD-1 CPI significantly enhances antitumor response that is dependent on up-regulation of tumor PD-L1 expression. Further, combination therapy significantly increases intratumoral trafficking of activated CD8+ T-cells and M1 tumor associated macrophages (TAMs) with concurrent loss of M2 TAMs. Our study identifies EZH2 as a potent inhibitor of antitumor immunity and responsiveness to CPI. This data suggests EZH2 inhibition as a novel therapeutic direction to enhance prostate cancer response to PD-1 CPI.

cancer biology↗