SLC4A11 Mitochondrial Uncoupling and ROS Production in Corneal Endothelium

SLC4A11 角膜内皮线粒体解偶联和 ROS 产生

• 批准号: 10615661
• 负责人: Joseph Aurelio Bonanno
• 金额: $ 42.48万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10615661
• 关键词: Acceleration; Aging; Amino Acids; Ammonia; Antioxidants; Apical; Apoptosis; Autophagocytosis; Back; Bicarbonates; Blindness; Buffers; Carcinoma; Catabolism; Cell Density; Cell Line; Cell membrane; Cells; Citric Acid Cycle; Colon Carcinoma; Consumption; Cornea; Corneal Endothelium; Corneal dystrophy; Corneal edema; Data; Defect; Down-Regulation; Electron Transport; Endothelial Cells; Endothelium; Feeds; Fuchs' Endothelial Dystrophy; Functional disorder; Genes; Glutamates; Glutamine; Goals; Human; Hydration status; In Vitro; Inflammation; Inherited; Knock-out; Knockout Mice; Laboratories; Lead; Malignant Epithelial Cell; Measurement; Measures; Medical; Membrane Potentials; Membrane Proteins; Membrane Transport Proteins; Metabolism; Mitochondria; Mitochondrial Membrane Protein; Modeling; Mus; Mutation; NADP; Na(+)-K(+)-Exchanging ATPase; Oryctolagus cuniculus; Pathology; Phenotype; Physiology; Production; Property; Proteins; Pump; Reactive Oxygen Species; Reduced Glutathione; Role; Source; Stimulus; Taurine; Testing; Tissues; Transfection; Transplantation; Trauma; UCP2 protein; Variant; Vision; Work; basolateral membrane; carbonate dehydratase; cell type; electron energy; in vivo; insight; loss of function; mitochondrial dysfunction; mitochondrial membrane; mouse model; novel; prevent; therapy development; transcriptome sequencing

“SLC4A11 Mitochondrial Uncoupling and ROS Production in Corneal Endothelium” ABSTRACT Defects in the gene SLC4A11 cause Congenital Hereditary Endothelial Dystrophy and some forms of Fuchs Dystrophy. The goal of this study is to understand the role of this membrane transporter in normal corneal endothelial metabolism and how SLC4A11 deficiency leads to Corneal Dystrophy. The corneal endothelial “pump” maintains corneal hydration and transparency. When the “pump” fails due to trauma, inflammation, ageing, or dystrophy, corneal edema ensues, transparency is lost, and vision is significantly degraded. The usual therapy is transplantation, which is not without significant compromises and complications. A hallmark of Corneal Endothelial Dystrophies is mitochondrial dysfunction. Our laboratory has shown that SLC4A11 is an NH3 dependent electrogenic H+ transporter. We have found that glutamine is actively metabolized by the endothelium producing NH3 and enhancing ATP formation. Slc4a11 knock out shows significant corneal edema, lactate accumulation, altered mitochondrial physiology, and ROS. These data have led to the overarching hypothesis that corneal endothelium actively metabolize glutamine and that the absence of SLC4A11 alters glutamine metabolism, leading to mitochondrial dysfunction, ROS, and eventual apoptosis. Preliminary data indicate that SLC4A11 is both a plasma membrane and a mitochondrial membrane protein, leading to the novel hypothesis that SLC4A11 is a mitochondrial uncoupler. Using multiple in vitro & in vivo complementary approaches these hypotheses will be tested in three aims. Aim 1 will determine how glutamine metabolism is facilitated in Corneal Endothelial mitochondria. The hypothesis is that Slc4a11 is an NH3 sensitive mitochondrial uncoupler that works in conjunction with Uncoupling Protein-2 and the potential mitochondrial buffer taurine to facilitate Glutamine catabolism. Aim 2 will examine the source of ROS and ROS as a stimulus to Apoptosis in Slc4a11 KO. Our hypothesis is that apoptosis is accelerated by ROS, which is generated by the interaction of NH3 with an energized electron transport chain and reduced by SLC4A11 uncoupling. Aim 3 will identify the cause of corneal edema in Slc4a11 KO Mice. We will test the hypothesis that loss of Slc4a11 secondarily induces downregulation of key proteins that facilitate lactate transport. Completion of this study will establish the role of SLC4A11 and glutamine in endothelial metabolism; provide new insight for mechanisms that facilitate glutamine metabolism yet alleviate NH3 induced ROS production that will be transferable to a wide array of glutamine metabolizing tissues; and provide insight for development of therapies for endothelial dystrophies.

“角膜内皮中的线粒体解偶联和ROS产生” SLC4A11” 抽象的 基因SLC4A11的缺陷导致先天性遗传性内皮营养不良和某些形式 富克斯营养不良。这项研究的目的是了解该膜转运蛋白在正常角膜中的作用 内皮代谢以及SLC4A11缺乏如何导致角膜营养不良。角膜内皮 “泵”保持角膜水合和透明度。当“泵”因创伤，炎症而失败时， 衰老或营养不良，随之而来的是角膜水肿，透明度会丧失，视力显着降解。通常 治疗是移植，这并非没有重大折衷和并发症。角膜的标志 内皮营养不良是线粒体功能障碍。我们的实验室表明SLC4A11是NH3 依赖性的电源H+转运蛋白。我们发现谷氨酰胺被内皮积极代谢 产生NH3并增强ATP形成。 slc4a11敲出显示明显的角膜水肿，乳酸 积累，线粒体生理改变和ROS。这些数据导致了总体假设 角膜内皮积极代谢谷氨酰胺，而SLC4A11的缺失会改变谷氨酰胺 代谢，导致线粒体功能障碍，ROS和最终凋亡。初步数据表明 SLC4A11既是质膜，也是线粒体膜蛋白，导致了新的假设 该SLC4A11是线粒体解耦合器。使用多个体外和体内完成方法 假设将以三个目标进行检验。 AIM 1将确定如何在角膜中制备谷氨酰胺代谢 内皮线粒体。假设是SLC4A11是一种有效的NH3敏感线粒体解耦合器 与解偶联蛋白2和电势线粒体缓冲牛磺酸结合使用以促进谷氨酰胺 分解代谢。 AIM 2将检查ROS和ROS的来源，作为SLC4A11 KO中凋亡的刺激。我们的 假设是凋亡是由ROS加速的，ROS是由NH3与A的相互作用产生的 电动电子传输链，并通过SLC4A11解耦合降低。 AIM 3将确定角膜的原因 SLC4A11 KO小鼠的水肿。我们将测试以下假设：SLC4A11次要损失会引起下调 促进乳酸转运的关键蛋白质。这项研究的完成将确定SLC4A11和 内皮代谢中的谷氨酰胺；为促进谷氨酰胺代谢的机制提供新的见解 然而，减轻NH3诱导的ROS产量将转移到广泛的谷氨酰胺代谢中 组织；并为内皮营养不良的疗法开发提供见解。

项目成果

Mitochondrial ROS in Slc4a11 KO Corneal Endothelial Cells Lead to ER Stress.

• DOI: 10.3389/fcell.2022.878395
• 发表时间: 2022
• 期刊: Frontiers in cell and developmental biology
• 影响因子: 5.5