Chemical Probes for Uncovering Differential Regulators o

描述(申请人提供)：尽管对细胞系的研究已经确定了哺乳动物细胞凋亡途径的主要成分，但不同原代细胞之间对凋亡调控的差异在很大程度上仍未被探索。例如，生化和分子研究已经确定caspase蛋白酶是细胞凋亡的中心执行者，线粒体全细胞色素c(血红素附着的细胞色素c)的释放是在细胞凋亡过程中激活caspase的关键触发因素。然而，尽管细胞色素c介导的caspase激活在哺乳动物细胞凋亡中具有重要作用，但我们对这一途径在原代细胞中如何调控的了解非常有限。我们最近的结果表明，在初级有丝分裂细胞和有丝分裂后细胞中，caspase激活的调控存在潜在的根本性差异。我们发现，尽管全细胞色素c本身是激活caspase并诱导有丝分裂细胞凋亡的必要条件和充分条件，但它是必要的但不足以诱导神经元、心肌细胞和肌管的凋亡。这种差异是显著的，表明了有丝分裂细胞和有丝分裂后细胞中新的、不同的凋亡调控机制。然而，不幸的是，我们对这种差异的分子基础的了解是有限的，因为目前在细胞色素c点激活细胞凋亡的唯一方法是通过单细胞显微注射全细胞色素c。因此，这些实验仅限于那些可以显微注射的细胞，它们与大规模的生化分析不兼容。为了克服这些限制并开发研究细胞凋亡调控的新探针，我们将采用高通量兼容性的无细胞caspase激活实验来筛选在细胞内线粒体细胞色素c释放点后触发细胞凋亡的小分子化合物。这些化学探针的发展在两个重要方面具有重要意义：a)它们是发现有丝分裂细胞和有丝分裂后细胞凋亡差异调控的分子机制的重要工具；b)这些探针被预测作为仅在选择性细胞中诱导凋亡的试剂具有治疗价值。

DESCRIPTION (provided by applicant): Although studies in cell lines have identified the main components of the mammalian apoptotic pathway, the differences in the regulation of apoptosis among various primary cells remain largely unexplored. For example, biochemical and molecular studies have identified the caspase proteases as central executioners of apoptosis and the release of mitochondrial holocytochrome c (heme-attached cytochrome c) as a critical trigger that activates caspases during apoptosis. However, despite the central importance of cytochrome c- mediated caspase activation in mammalian apoptosis, our understanding of how this pathway is regulated in primary cells is very limited. Our recent results point to a potentially fundamental difference in the regulation of caspase activation in primary mitotic versus postmitotic cells. We find that whereas holocytochrome c alone is necessary and sufficient to activate caspases and induce apoptosis in mitotic cells, it is necessary but not sufficient to induce apoptosis in neurons, cardiomyocytes, and myotubes. This difference is striking and is indicative of novel, differential mechanisms of apoptosis regulation in mitotic versus postmitotic cells. Unfortunately however, our understanding of the molecular basis for this difference is limited because the only current method for activating apoptosis at the point of cytochrome c is via single cell microinjection of holocytochrome c. Thus, these experiments are restricted to only those cells that can be microinjected and they are not compatible with large scale biochemical analysis. To overcome these limitations and to develop novel probes for studying apoptosis regulation, we will adapt a cell-free caspase activation assay for high throughput compatibility to screen for small-molecule compounds that trigger apoptosis after the point of mitochondrial cytochrome c release in cells. The development of these chemical probes is significant in two important aspects: a) They are essential tools for discovering the molecular mechanism of the differential regulation of apoptosis in mitotic and postmitotic cells, and b) These probes are predicted to have therapeutic value as reagents that induce apoptosis only in selective cells.