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

描述(申请人提供)：严格调控细胞凋亡的能力对神经元、心肌细胞和肌管等有丝分裂后细胞特别重要，因为这些细胞的再生潜力有限，并在生物体的整个生命周期中保持。未能限制细胞凋亡会导致细胞死亡的易感性增加，这在许多退行性疾病中都可以看到。因此，了解有丝分裂后细胞的存活机制具有重要的生理和病理意义。细胞色素c(Cytc)的线粒体释放是细胞凋亡过程中触发caspase激活的重要事件。在这里，我们已经确定了一种新的机制来调控细胞色素c介导的由神经元和其他有丝分裂后细胞参与的凋亡，其中胞浆细胞色素c本身是蛋白酶体降解的靶标。重要的是，虽然我们没有观察到原代有丝分裂细胞如成纤维细胞中细胞色素c的降解，但我们发现线粒体释放的细胞色素c在某些癌细胞中也被降解。由于逃避凋亡是有丝分裂后细胞和癌细胞的重要特征，靶向胞浆Cytc的降解可能是这些细胞共同使用的生存机制。在这项提案中，我们的目标是确定胞质Cytc被作为降解靶点的分子机制。在目标1中，我们将定义决定细胞是否针对胞浆Cytc进行降解的因素，包括检查细胞中低水平的APAF-1是否是Cytc降解的关键决定因素。在目标2中，我们将研究细胞色素c泛素化，并确定针对泛素化的细胞色素c的特定残基。我们的假设是泛素化的Cytc不能与APAF-1结合。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凋亡的脆弱性。我们在目标3中的重点是确定以Cyt c为降解目标的E3泛素连接酶。我们的初步结果表明，Cyt c与胞浆中的Hsp70和E3连接酶芯片(羧基热休克蛋白70相互作用蛋白)结合。因此，我们将测试在有丝分裂后细胞和癌细胞中，胞浆Cyt c的泛素化和降解是否由CHIP介导。 与公共卫生相关：我们的计划是研究哺乳动物细胞中细胞死亡途径是如何调节的。我们发现了一种新的机制，这种机制由神经元、心肌细胞和肌管等有丝分裂后细胞参与，高度限制细胞死亡，并可能确保它们的长期生存。了解有丝分裂后细胞的存活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、心脏病理和肌肉营养不良的重要病理基础。

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.