Mitochondrial metabolism and the Lon-PDH axis

摘要 出自母公司授权R01 GM136905-01，题为“线粒体新陈代谢和长PDH轴”。 人类Lon蛋白水解酶是线粒体蛋白平衡的主要调节者，线粒体蛋白稳定是 调节线粒体能量代谢，缓解细胞应激。我们最近确定了一部小说 两个神经系统疾病严重的同胞中编码LON的LONP1基因的致病变异 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活性 丙酮酸脱氢酶(PDH)。我们的结果表明，PDH缺乏是由 Lon-P761L不能降解PDH的磷酸化E1a亚基，它积累并 抑制PDH活性。PDH是连接糖酵解和三羧酸(TCA)的中央守门人。 周而复始。它也是葡萄糖和脂肪酸分解代谢的关键调节节点。神经元产生三磷酸腺苷 几乎完全通过葡萄糖氧化，因此全功能的PDH活性是至关重要的。我们假设 野生型Lon调节PDH复合体的活性和结构，对校准至关重要 线粒体新陈代谢和能量学。在这个项目中，我们将使用(1)患者和父母派生的 (2)诱导多能干细胞(IPSCs)从这些成纤维细胞重新编程。这个 IPSCs将分化为神经元和星形胶质细胞。使用这些细胞，目标1号将检验这一假设 Lon介导的降解调节PDH复合体的活性和结构；Aim 2将 识别Lon-PDH轴的上行和下行调节因子，它们在细胞中表达改变 野生型Lon与Lon-P761L；目标3将通过以下方式研究PDH的调节和失调 IPSC来源的神经元和星形胶质细胞中野生型和突变型Lon。我们的调查将建立新的 依赖Lon的PDH调节的分子机制。所获得的知识也将有所帮助 确定潜在的治疗蛋白质靶点(如PDK、PDP、LON)、药理和饮食 提高PDH活性和/或治疗与长时间相关的PDH缺乏症的干预措施 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进行校准，如心脏 疾病、癌症和神经退化。

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.