Characterization of Branched Chain Amino Acid Metabolism and Its Deficiency

支链氨基酸（BCAA）代谢障碍是最常见的有机代谢障碍。 通过新生儿筛查确定酸中毒。亮氨酸（LEU）、异亮氨酸（ILE）和缬氨酸（瓦尔）的催化剂 开始于一套常见的转运蛋白和酶定位于线粒体。然后 路径分叉。这些途径中的四种疾病共享巨大的未满足的治疗需求：枫糖浆 尿病（MSUD）和异戊酸（IVA）、丙酸（PA）和甲基丙二酸（MMA）。这是一 一个长期目标是表征支链氨基酸酸代谢及其 缺陷在原项目的每一个目标上都取得了重大进展，我们的成果 为研究新疗法提供了独特的机会。这项新的申请有三个具体目标。具体 具体目标1是继续表征BCAA代谢的线粒体结构。基于 根据我们的数据，我假设LEU和ILE/瓦尔的分解代谢酶由两个独立的代谢子组成 在线粒体基质中。具体目标1a是检查BCKADH，IVDH， 使用STED、cryo-CLEM和cryo-ET技术，将3 MCC作为推定的代谢子。具体目标1b是 使用蛋白质交联和共- 免疫沉淀研究。具体目标2是鉴定治疗IVDH和BCKADH的新型小分子 缺乏，作为底物抑制剂或化学伴侣蛋白，以改善IVDH的折叠，以恢复部分 通过亮氨酸途径。我假设BCAT的小分子抑制剂会减少 这些疾病的有毒中间体的积累，而LEU途径中的远端酶的抑制剂 将诱导近端底物的积累并稳定突变酶。具体目标2a是检查 一组BCAA催化剂抑制剂对IVDH和BCKADH的稳定性和功能的影响 IVA和MSUD患者。我预测这些分子将稳定突变的IVDH和BCKADH蛋白， 使它们成为进一步临床前药物开发的令人信服的候选者。具体目标2b是检查 BCAT抑制剂对通过BCAA催化剂的通量和细胞中毒性代谢物的积累的影响 来自IVA和MSUD患者。我预测，这种化合物将减少有毒代谢物的积累， 细胞，将其确定为IVA和MSUD的潜在疗法。具体目标3是检查线粒体ETC 以及PA和MMA患者细胞中TCA周期功能障碍。我假设，纠正这种 缺陷将允许开发用于这些疾病的治疗剂。具体目标3a是检查 使用新型线粒体抗氧化剂逆转MMA患者细胞中的继发性ETC功能障碍 和PA。具体目的3b是检查恢复来自患有以下疾病的患者的细胞中的TCA周期失衡的效果： PA和MMA对生物能量不平衡的影响。我建议研究一套新的合成回补剂， 患者细胞我假设这些化合物在逆转异常方面将优于现有的药物。

Disorders of the branched chain amino acid (BCAA) metabolism are the most common of the organic acidemias identified by newborn screening. The catabolism of leucine (LEU), isoleucine (ILE), and valine (VAL) begins through a set of common transporters and enzymes localized to the mitochondria. Thereafter, the pathways diverge. Four disorders in these pathways share enormous unmet needs for treatment: maple syrup urine disease (MSUD), and isovaleric (IVA), propionic (PA), and methylmalonic acidemias (MMA). This is a renewal application for a project with a long-term goal to characterize BCAA acid metabolism and its deficiencies. Significant progress has been made on each aim of the original project, and our results have provided unique opportunities to study novel therapies. This new application has three specific aims. Specific Specific Aim 1 is to continue characterization of the mitochondrial architecture of BCAA metabolism. Based on our data, I hypothesize that the catabolic enzymes of LEU and ILE/VAL comprise two separate metabolons within the mitochondrial matrix. Specific Aim 1a is to examine the physical interactions of BCKADH, IVDH, and 3MCC as a putative metabolon using STED and cryo-CLEM, and cryo-ET techniques. Specific Aim 1b is to examine the interactions of the enzymes involved in BCAA catabolism using protein cross linking and co- immunoprecipitation studies. Specific Aim 2 is to identify novel small molecules to treat IVDH and BCKADH deficiency, either as substrate inhibitors or chemical chaperonins to improve folding of IVDH to restore partial flux through the leucine pathway. I hypothesize that small molecule inhibitors of BCAT will reduce accumulation of toxic intermediates of these disorders, while inhibitors of distal enzymes in the LEU pathway will induce accumulation of proximal substrates and stabilize mutant enzymes. Specific Aim 2a is to examine effects of a panel of BCAA catabolism inhibitors on IVDH and BCKADH stability and function in cells from patients with IVA and MSUD. I predict that these molecules will stabilize mutant IVDH and BCKADH proteins, making them compelling candidates for further preclinical drug development. Specific Aim 2b is to examine the effect of a BCAT inhibitor on flux through BCAA catabolism, and accumulation of toxic metabolites in cells from patients with IVA and MSUD. I predict that this compound will reduce accumulation of toxic metabolites in cells, identifying it as a potential therapy for IVA and MSUD. Specific Aim 3 is to examine mitochondrial ETC and TCA cycle dysfunction in cells from patients with PA and MMA. I hypothesize that correction of such defects will allow development of therapeutic agents for these disorders. Specific Aim 3a is to examine the use of novel mitochondrial antioxidants to reverse secondary ETC dysfunction in cells from patients with MMA and PA. Specific Aim 3b is to examine the effect of restoring TCA cycle imbalance in cells from patients with PA and MMA on bioenergetic imbalance. I propose to study a set of novel synthetic anaplerotic agents in patient cells. I hypothesize that these compounds will be better than existing agent in reversing abnormalities.