Cytochrome c Degradation: A Mechanism to Restrict Apoptosis in Postmitotic Cells

描述（由申请人提供）：严格调节细胞凋亡的能力对于有丝分裂后细胞如神经元、心肌细胞和肌管特别重要，因为这些细胞具有有限的再生潜力，并且在生物体的寿命内得以维持。如在许多退行性疾病中所见，未能限制细胞凋亡可导致对细胞死亡的脆弱性增加。因此，了解有丝分裂后细胞的生存机制具有重要的生理和病理意义。 细胞色素c（cyt c）的线粒体释放是细胞凋亡过程中触发半胱天冬酶激活的关键事件。在这里，我们已经确定了一种新的机制，调节细胞色素c介导的细胞凋亡参与神经元和其他有丝分裂后的细胞，其中胞质细胞色素c本身是针对蛋白酶体降解。 重要的是，虽然我们没有观察到细胞色素c在原代有丝分裂细胞如成纤维细胞中的降解，但我们发现细胞释放的细胞色素c在某些癌细胞中也会降解。由于逃避凋亡是有丝分裂后细胞和癌细胞的一个重要特征，因此靶向胞浆细胞色素c降解可能是这些细胞生存的共同机制。 在这个提议中，我们的目标是确定细胞质中细胞色素c降解的分子机制。在目标1中，我们将定义决定细胞是否将靶向胞质cyt c降解的因素，包括检查细胞中低水平的Apaf-1是否是cyt c降解的关键决定因素。在目标2中，我们将检查细胞色素c泛素化，并确定特定的残基的细胞色素c的泛素化的目标。我们的假设是泛素化的cyt c不能结合Apaf-1。我们将测试这一点，并检查阻止细胞色素c降解的条件是否会增加有丝分裂后细胞对细胞凋亡的脆弱性。我们的目标3的重点是确定E3泛素连接酶的目标细胞色素c的降解。我们的初步结果表明，细胞色素c结合热休克蛋白70和E3连接酶CHIP（羧基热休克蛋白70相互作用蛋白）在胞质溶胶中。 因此，我们将测试在有丝分裂后和癌细胞中，胞质cyt c的泛素化和降解是否由CHIP介导。 公共卫生相关性：我们的计划是研究细胞死亡途径在哺乳动物细胞中是如何调节的。我们已经发现了一种新的机制，这种机制被有丝分裂后的细胞如神经元、心肌细胞和肌管所参与，它高度限制了细胞的死亡，并可能确保它们的长期存活。了解有丝分裂后细胞的生存机制具有巨大的临床意义，因为这些细胞的死亡增加是许多神经退行性疾病、心脏病和肌营养不良症的病理学核心。

DESCRIPTION (provided by applicant): The ability to strictly regulate apoptosis is particularly important for postmitotic cells such as neurons, cardiomyocytes and myotubes because these cells have limited regenerative potential and are maintained for the lifetime of the organism. Failure to restrict apoptosis can result in increased vulnerability to cell death, as seen in many degenerative diseases. Therefore, an understanding of survival mechanisms in postmitotic cells is of physiological and pathological importance. The mitochondrial release of cytochrome c (cyt c) is a crucial event that triggers caspase activation during apoptosis. Here, we have identified a novel mechanism for regulating cyt c-mediated apoptosis engaged by neurons and other postmitotic cells, where cytosolic cyt c itself is targeted for proteasomal degradation. Importantly, while we do not observe cyt c degradation in primary mitotic cells such as fibroblasts, we find mitochondrial-released cyt c to be also degraded in certain cancer cells. As evasion of apoptosis is an important feature of both postmitotic and cancer cells, targeting cytosolic cyt c for degradation could be a shared mechanism used by these cells for survival. In this proposal, our goals are to identify the molecular mechanisms by which cytosolic cyt c is targeted for degradation. In Aim 1, we will define the factors which determine whether or not a cell will target cytosolic cyt c for degradation, including examining whether low levels of Apaf-1 in cells is a key determinant for cyt c degradation. In Aim 2, we will examine cyt c ubiquitination and identify the specific residues of cyt c that are targeted for ubiquitination. Our hypothesis is that ubiquitinated cyt c is not able to bind Apaf-1. We will test this, and examine whether conditions that block cyt c degradation increase the vulnerability of postmitotic cells to apoptosis. Our focus in Aim 3 is to identify the E3 ubiquitin ligase that targets cyt c for degradation. Our preliminary results suggest that cyt c binds to Hsp70 and the E3 Ligase CHIP (Carboxyl Heat shock protein 70-Interacting Protein) in the cytosol. Therefore, we will test whether the ubiquitination and degradation of cytosolic cyt c is mediated by CHIP in postmitotic and cancer cells. PUBLIC HEALTH RELEVANCE: Our plans here are to investigate how the cell death pathway is regulated in mammalian cells. We have discovered a novel mechanism engaged by postmitotic cells such as neurons, cardiomyocytes and myotubes that highly restricts cell death and likely ensures their long term survival. Understanding the survival mechanisms used by postmitotic cells has enormous clinical significance because increased death of these cells is central to the pathology of many neurodegenerative diseases, cardiac pathologies and muscular dystrophies.