Biology subjects

Pawar, M.

Publications and source records attributed to Pawar, M..

4 recordsLinked to original sources

bioRxiv2025

The Ca2+ Channel CYCLIC NUCLEOTIDE GATED CHANNEL13 (CNGC13) regulates systemic wound signalling and immunity against Spodoptera herbivory

Insect herbivory elicits rapid and complex signalling responses in plants, including membrane depolarization, cytosolic Ca2+ elevation and activation of jasmonate-mediated defense pathways in local and systemic leaves. These early events are orchestrated by calcium-permeable channels and regulatory components that generate stimulus-specific cytosolic Ca2+ signatures. However, the molecular mechanisms that generate long-distance Ca2+ propagation and systemic immune activation on herbivory remain poorly defined. Here, we identify CYCLIC NUCLEOTIDE GATED CHANNEL13 (CNGC13) as a novel plasma membrane-localized, phloem-expressed Ca2+ channel that is essential for systemic wound signalling and defense against lepidopteran pest, Spodoptera litura in Arabidopsis thaliana. CNGC13 is rapidly induced by wounding and herbivory, mediates cytosolic Ca2+ influx, and is required for efficient propagation of Ca2+ signals from wounded to distal leaves. Loss of CNGC13 compromises the thioglucosidase-dependent breakdown of aliphatic glucosinolates into aglucones/isothiocyanates within the vascular tissue, which are essential for wounding-induced systemic Ca2+ wave propagation. Consequently, cngc13 mutants display impaired systemic jasmonate accumulation in distal leaves, reduced glucosinolate levels and enhanced herbivore susceptibility. CNGC13 is also required for AtPep-induced Ca2+ elevation and Reactive Oxygen Species (ROS) signalling and interacts with the receptor kinase, PEPR2, implicating it in DAMP-mediated immune responses. Although CNGC13 and CNGC19 function cooperatively in herbivore defense, they do not physically interact, suggesting independent but converging roles. Our study identifies CNGC13 as a vasculature-localized Ca2+-permeable channel that functions downstream of PEPR2 receptor, to sustain systemic signalling, operating in a distinct yet converging pathway alongside known glutamate receptor-like channels during long-distance wound responses. SignificancePlants must detect and respond to insect herbivory rapidly by activating systemic immunity to trigger defense responses. Calcium (Ca2+) signalling plays a central role in this defense network, yet the identities and specific functions of Ca2+ channels involved in long-distance herbivory signalling remain poorly understood. Here, we identify the plasma membrane-localized channel CYCLIC NUCLEOTIDE GATED CHANNEL13 (CNGC13) as a key integrator of mechanical damage perception, ricca factor-mediated systemic signalling, and jasmonate-dependent defense against Spodoptera herbivory in Arabidopsis. CNGC13 facilitates systemic Ca2+ wave propagation, activates jasmonate and glucosinolate biosynthesis, and promotes the perception of damage-associated molecular pattern (DAMP) molecules to confer herbivore resistance. This work uncovers a previously uncharacterized calcium channel that plays a crucial dual role in systemic wound signalling.

plant biology↗

bioRxiv2025

Rewiring Catalytic Craters: A Path for Engineering β-Glucosidases to Improve Glucose Tolerance

Bioethanol, a sustainable alternative to fossil fuels, is produced from cellulose via enzymatic saccharification. {beta}-Glucosidase, a key enzyme in this process, hydrolyses disaccharides into glucose but is limited by feedback inhibition. This study investigated GH1 {beta}-glucosidase from a soil metagenome (UnBGl1) to understand and overcome this limitation. We solved the near-atomic resolution crystal structure of UnBGl1 at 1.15 [A] as an apo enzyme and report the first high-resolution crystal structures of the enzyme in its pre-hydrolytic state as a cellobiose complex and covalent intermediate-bound state, capturing key stages of its catalytic mechanism. Structural analysis revealed three crucial glucose-binding subsites in UnBGl1s crater. Glucose binding at the -1 subsite induced a 1.4 [A] shift in E370, increasing the distance between catalytic glutamates and reducing enzymatic activity. The C188V variant, generated by rational engineering, significantly improved glucose tolerance to 2.5 M. Additionally, the H261W mutation at the +2 subsite enhanced kinetic properties by improving cellobiose affinity (Km = 22.87 {+/-} 1.1 mM) and shifted the optimal pH to 5.5 from 6.0. Comparative structural analysis with other glucose-tolerant GH1 {beta}-glucosidases revealed conserved residues at the -1 subsite crucial for substrate stabilization and +1 subsite residues interacting with glucose, offering targets for further optimization. Engineered UnBGl1 variants retained high stability and activity on sugarcane bagasse, demonstrating their potential for industrial cellulase cocktails. These findings provide a robust framework for engineering {beta}-glucosidases with enhanced glucose tolerance and catalytic efficiency, paving the way for improved bioethanol production and contributing to sustainable energy solutions.

biochemistry↗

bioRxiv2019

Small molecule activation of metabolic enzyme pyruvate kinase muscle isozyme 2, PKM2, provides photoreceptor neuroprotection

Photoreceptor cell death is the ultimate cause of vision loss in many retinal disorders, and there is an unmet need for neuroprotective modalities to improve photoreceptor survival. Similar to cancer cells, photoreceptors maintain pyruvate kinase muscle isoform 2 (PKM2) expression, which is a critical regulator in aerobic glycolysis. Unlike PKM1, which has constitutively high catalytic activity, PKM2 is under complex regulation. Recently, we demonstrated that genetically reprogramming photoreceptor metabolism via PKM2-to-PKM1 substitution is a promising neuroprotective strategy. Here, we explored the neuroprotective effects of pharmacologically activating PKM2 via ML-265, a small molecule activator of PKM2, during acute outer retinal stress. We found that ML-265 increased PKM2 activity in 661W cells and in vivo in rat eyes without affecting the expression of genes involved in glucose metabolism. ML-265 treatment did, however, alter metabolic intermediates of glucose metabolism and those necessary for biosynthesis in cultured cells. Long-term exposure to ML-265 did not result in decreased photoreceptor function or survival under baseline conditions. Notably, though, ML-265-treatment did reduce entrance into the apoptotic cascade in in vitro and in vivo models of outer retinal stress. These data suggest that reprogramming metabolism via activation of PKM2 is a novel, and promising, therapeutic strategy for photoreceptor neuroprotection.

molecular biology↗

bioRxiv2019

Hexokinase 2 is dispensable for photoreceptor development but is required for survival during aging and outer retinal stress

Photoreceptor death is the ultimate cause of vision loss in many retinal degenerative conditions. Identifying novel therapeutic avenues for prolonging photoreceptor health and function has the potential to improve vision and quality of life for patients suffering from degenerative retinal disorders. Photoreceptors are metabolically unique among other neurons in that they process the majority of their glucose via aerobic glycolysis. One of the main regulators of aerobic glycolysis is hexokinase 2 (HK2). Beyond its enzymatic function of phosphorylating glucose to glucose-6-phosphate, HK2 has additional non-enzymatic roles, including the regulation of apoptotic signaling via AKT signaling. Determining the role of HK2 in photoreceptor homeostasis may identify novel signaling pathways that can be targeted with neuroprotective agents to boost photoreceptor survival during metabolic stress. Here we show that following experimental retinal detachment, p-AKT is upregulated and HK2 translocates to mitochondria. Inhibition of AKT phosphorylation in 661W photoreceptor-like cells results in translocation of mitochondrial HK2 to the cytoplasm, increased caspase activity, and decreased cell viability. Rod-photoreceptors lacking HK2 upregulate HK1 and appear to develop normally. Interestingly, we found that HK2-deficient photoreceptors are more susceptible to acute nutrient deprivation in the experimental retinal detachment model. Additionally, HK2 appears to be important for preserving photoreceptors during aging. We show that retinal glucose metabolism is largely unchanged after HK2 deletion, suggesting that the non-enzymatic role of HK2 is important for maintaining photoreceptor health. These results suggest that HK2 expression is critical for preserving photoreceptors during acute nutrient stress and aging. More specifically, p-AKT mediated translocation of HK2 to the mitochondrial surface may be critical for protecting photoreceptors from acute and chronic stress.

neuroscience↗