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有丝分裂后细胞（如神经元、心肌细胞和肌管）尤其重要，因为这些细胞具有有限的再生潜力，并在生物体的整个生命周期内维持。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调节细胞c介导的凋亡的新机制，这种凋亡由神经元和其他有丝分裂后细胞参与，其中细胞质细胞c本身是蛋白酶体降解的目标。重要的是，虽然我们没有在原代有丝分裂细胞（如成纤维细胞）中观察到cyt - c降解，但我们发现线粒体释放的cyt - 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，靶向胞质cyt c降解可能是这些细胞生存的共同机制。在本提案中，我们的目标是确定胞质cyt c被降解的分子机制。在Aim 1中，我们将定义决定细胞是否会靶向胞质细胞c进行降解的因素，包括检查细胞中低水平的Apaf-1是否是细胞c降解的关键决定因素。在目标2中，我们将检查cyt c泛素化，并确定针对泛素化的cyt c的特定残基。我们的假设是泛素化的细胞c不能结合Apaf-1。我们将对此进行测试，并检查阻断cyt c降解的条件是否会增加有丝分裂后细胞凋亡的脆弱性。我们在Aim 3中的重点是鉴定靶向cyt c降解的E3泛素连接酶。我们的初步结果表明，cyt c在细胞质中与Hsp70和E3连接酶CHIP（羧基热休克蛋白70-相互作用蛋白）结合。因此，我们将在有丝分裂后细胞和癌细胞中测试CHIP是否介导胞质cyt c的泛素化和降解。

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.