Mitochondrial metabolism and the Lon-PDH axis

总结 来自题为“线粒体代谢和Lon-PDH轴”的母基金R 01 GM 136905 -01。 人Lon蛋白酶是线粒体蛋白质稳态的主要调节剂，其对于维持线粒体的蛋白质稳态是必需的。 调节线粒体能量代谢，缓解细胞应激。我们最近发现了一本小说 编码Lon的LONP 1基因的致病性变异，在两个患有严重神经系统疾病的兄弟姐妹中 损伤，大脑和小脑萎缩，其中761位的脯氨酸被亮氨酸取代 （Lon-P761L）。来自这些兄弟姐妹的原代皮肤成纤维细胞显示出显著降低的 丙酮酸脱氢酶（PDH）。我们的结果表明，PDH缺乏是由 Lon-P761 L不能降解磷酸化的PDH的E1 a亚基，其积累并 抑制PDH活性。PDH是连接糖酵解和三羧酸（TCA）的中心看门人 周期它也是葡萄糖和脂肪酸催化剂的关键调节节点。神经元产生ATP 几乎完全通过葡萄糖氧化，因此完全功能性的PDH活性是至关重要的。我们假设 野生型Lon调节PDH复合物的活性和结构， 线粒体代谢和能量学。在这个项目中，我们将采用（1）患者和父母来源的 成纤维细胞，和（2）从这些成纤维细胞重编程的诱导多能干细胞（iPSC）。的 iPSC将分化成神经元和星形胶质细胞。使用这些细胞，目标1将测试假设 Lon介导的降解调节PDH复合物的活性和结构;目标2将 鉴定Lon-PDH轴的上游和下游调节剂，其在表达 野生型Lon与Lon-P761 L;目的3将研究PDH的调节和失调， 野生型和突变型Lon在iPSC衍生的神经元和星形胶质细胞中的表达。我们的调查将建立新的 PDH的Lon-dependent调节的分子机制。获得的知识也将有助于 确定潜在的治疗性蛋白质靶点（如PDK、PDP、Lon），药理学和饮食 用于增加PDH活性和/或用于治疗与Lon相关的PDH缺乏的干预 功能障碍这些结果对理解PDH活动和 线粒体代谢可以在罕见和更常见的疾病中校准， 疾病、癌症和神经变性。

SUMMARY From the parent grant R01 GM136905-01 entitled, “Mitochondrial metabolism and the Lon-PDH axis”. The human Lon protease is a master regulator of mitochondrial proteostasis, which is essential for regulating mitochondrial energy metabolism and mitigating cell stress. We recently identified a novel pathogenic variant in the LONP1 gene encoding Lon, in two siblings with profound neurologic impairment, cerebral and cerebellar atrophy, in which proline at position 761 was replaced by leucine (Lon-P761L). Primary skin fibroblasts from these siblings, showed substantially reduced activity of pyruvate dehydrogenase (PDH). Our results demonstrated that PDH deficiency was caused by the failure of Lon-P761L to degrade the phosphorylated E1a subunit of PDH, which accumulates and inhibits PDH activity. PDH is the central gatekeeper linking glycolysis to the tricarboxylic acid (TCA) cycle. It is also a key regulatory node for glucose and fatty acid catabolism. Neurons generate ATP almost exclusively by glucose oxidation, thus fully functional PDH activity is crucial. We hypothesize that wild type Lon regulates the activity and architecture of the PDH complex and is crucial for calibrating mitochondrial metabolism and energetics. In this project, we will employ (1) patient- and parent- derived fibroblasts, and (2) induced pluripotent stem cells (iPSCs) reprogrammed from these fibroblasts. The iPSCs will be differentiated into neurons and astrocytes. Using these cells, Aim 1 will test the hypothesis that Lon-mediated degradation regulates the activity and architecture of the PDH complex; Aim 2 will identify the up- and down- stream modulators of the Lon-PDH axis, which are altered in cells expressing wild type Lon versus Lon-P761L; and Aim 3 will investigate the regulation and dysregulation of PDH by wild type and mutant Lon in iPSC-derived neurons and astrocytes. Our investigation will establish new molecular mechanisms for the Lon-dependent regulation of PDH. The knowledge gained will also help to identify potential therapeutic protein targets (e.g. PDK, PDP, Lon), pharmacologic and dietary interventions for increasing PDH activity and/or for treating PDH deficiency associated with Lon dysfunction. These outcomes have a broader impact for understanding how PDH activity and mitochondrial metabolism can be calibrated in both rare and more common disorders such as heart disease, cancer and neurodegeneration.