Mitophagy and Cardiac Myocyte Protein Aggregation

心肌细胞蛋白质质量控制是维持心脏收缩装置和心脏功能的关键 功能。影响蛋白质质量控制的伴侣蛋白突变导致蛋白毒性，肌节 功能障碍和心肌细胞死亡。人类中的一个这样的CryAB基因点突变导致 热休克蛋白β-5(R120G HSPB5)蛋白第120位精氨酸到甘氨酸的变化，以及 引发常染色体显性遗传性心肌病，导致心力衰竭，需要心脏移植和 过早死亡。预防蛋白毒性的策略主要集中在刺激自噬--溶酶体 去除蛋白质聚集体的途径；需要改进以最大限度地提高治疗效益。我们的研究 在目前的赠款资助期内，发现了TRAF2的一个重要作用，一种先天免疫 蛋白质，在介导心肌细胞的基础有丝分裂中。我们的初步数据显示有丝分裂的丧失 应用TRAF2消融心肌细胞可诱导心肌细胞内显著的蛋白质聚集，尽管 缺乏对一般宏观自噬的影响。在心肌细胞特异性人HSPB5的小鼠模型中 R120G突变蛋白的表达，我们发现了线粒体积累HSPB5的证据 蛋白质，以及多泛素化蛋白和p62(聚集体形成所必需的接头蛋白)。 伴随而来的是线粒体TRAF2表达的显著增加和减少 线粒体蛋白质含量，表明有丝分裂在处理蛋白质聚集病理中的作用。vt.给出 最近在酵母和哺乳动物细胞系中的观察表明，线粒体可能促进摄取和 胞浆蛋白聚集体的降解，我们假设有丝分裂促进胞浆的去除 HSPB5 R120G突变蛋白在心肌细胞内的蛋白聚集体预防心肌细胞死亡 和心肌病。在此续订应用程序中，我们生成了试剂并发展了协作，以 通过以下目标来检验这一假设。在目标1中，我们将研究是否需要有丝分裂来移除 小鼠体内的蛋白质聚集体。研究还将在人类诱导的多潜能干细胞来源中进行 CRISPR靶向消融TRAF2的心肌细胞(IPSC-CMS)，以及CRISPR敲入的心肌细胞 R120G突变。在目标2中，我们将研究TRAF2诱导的有丝分裂吞噬是否足以促进 小鼠和人IPSC-CMS中的蛋白质聚集体。目标3将侧重于机械学研究，以了解 线粒体摄取HSPB5 R120G突变蛋白的步骤。研究项目： 小鼠将重点放在破坏p62介导的蛋白质聚集上，以检测p62在线粒体中的作用 蛋白质聚集摄取。在酵母和小鼠模型系统中的实验将检查Hsp104，一种酵母 解聚酶蛋白被证明在哺乳动物细胞中具有功能，是线粒体所必需的，也是足够的。 蛋白质总摄取量。这些研究将确定有丝分裂在胞浆蛋白中的作用。 利用这一途径治疗蛋白中毒性心肌病的质量控制和疗效。

Cardiac myocyte protein quality control is critical for maintenance of the contractile apparatus and cardiac function. Mutations in chaperone proteins that affect protein quality control induce proteotoxicity, sarcomere dysfunction and cardiac myocyte cell death. One such point mutation in the CRYAB gene in humans, results in an arginine to glycine change at position 120 in the heat-shock protein beta-5 (R120G HSPB5) protein, and provokes autosomal dominant cardiomyopathy resulting in heart failure, need for cardiac transplantation and premature mortality. Strategies to prevent proteotoxicity have focused on stimulating the autophagy-lysosome pathway to remove protein aggregates; and need to be refined for maximizing therapeutic benefit. Our studies during the current grant-funding period have uncovered an essential role for TRAF2, an innate immunity protein, in mediating basal mitophagy in cardiac myocytes. Our preliminary data show that loss of mitophagy with TRAF2 ablation in cardiac myocytes induces marked protein aggregation in cardiac myocytes, despite a lack of effect on general macro-autophagy. In a mouse model of cardiac myocyte specific human HSPB5 R120G mutant protein expression, we have uncovered evidence for mitochondrial accumulation of HSPB5 protein, along with polyubiquitinated proteins and p62 (an adaptor protein essential for aggregate formation). This is accompanied with markedly increased TRAF2 expression in the mitochondria and reduced mitochondrial protein content, pointing to a role for mitophagy in handling protein aggregate pathology. Given recent observations in yeast and mammalian cell lines indicating that mitochondria may facilitate uptake and degradation of cytosolic protein aggregates, we hypothesize that mitophagy facilitates removal of cytosolic protein aggregates of HSPB5 R120G mutant protein in cardiac myocytes to prevent cardiac myocyte death and cardiomyopathy. In this renewal application, we have generated reagents and developed collaborations to test this hypothesis via the following aims. In aim 1, we will examine if mitophagy is required for removal of protein aggregates in mice. Studies will also be performed in human induced pluripotent stem cell-derived cardiac myocytes (iPSC-CMs) with CRISPR targeted ablation of TRAF2, and in those with CRISPR-knock-in of R120G mutation. In aim 2, we will examine if TRAF2-induced mitophagy is sufficient to facilitate removal of protein aggregates in mice and in human iPSC-CMs. Aim 3 will focus on mechanistic studies to understand the steps involved in mitochondrial uptake of HSPB5 R120G mutant protein uptake in mitochondria. Studies in mice will focus on impairing p62-mediated protein aggregation to examine the role of p62 in mitochondrial protein aggregate uptake. Experiments in yeast and mouse model systems will examine if hsp104, a yeast disaggregase protein shown to be functional in mammalian cells, is required and sufficient for mitochondrial protein aggregate uptake, respectively. These studies will determine the role of mitophagy in cytosolic protein quality control, and efficacy of therapeutically harnessing this pathway to treat proteotoxic cardiomyopathy.