STRUCTURAL OF THE APOPTOSOME & ITS ROLE IN CELL DEATH

程序性细胞死亡（细胞凋亡）是个体细胞在胚胎发育和正常生长期间终止的过程，以使生物体受益。 一般来说，有3种途径响应凋亡信号并激活蛋白酶酶原（蛋白酶原），进而破坏确定的细胞靶点。 在线粒体途径中，促凋亡信号触发细胞色素c从线粒体膜间隙释放。 然后细胞色素c与细胞溶质中的凋亡蛋白酶激活因子-1（Apaf-1）相互作用以形成胞浆体。 随后，溶酶体募集半胱天冬酶原-9（pc-9）并介导自催化转化为功能性半胱天冬酶-9。 虽然胱天蛋白酶-9仍然结合到溶酶体上，但它可以激活胱天蛋白酶原-3（pc-3）以启动导致细胞死亡的蛋白水解级联反应。在这个提议中，将通过确定其三维（3D）结构来研究线粒体作为细胞死亡组织者的作用。 电子冷冻显微镜（EM）和单粒子图像处理将用于产生适合分子建模的3D图。 在初步的3D研究中，我们已经表明，Apaf-1分子由单个Apaf- 1分子组装而成，形成一个具有七个Y形辐条的中心双环（尺寸：约270 X 70埃单位;质量约1.1 Mda;对称性产生C7）。 基于这种结构，我们提出了一个细胞色素c激活Apaf-1的模型。 在该模型中，Apaf-1由于N-末端区域和作为分子内伴侣的两个WD 40结构域之间的相互作用而被自动抑制。 我们认为，细胞色素c可能对接到WD 40结构域内Apaf-1释放的N-末端区域。 这反过来又促进dATP/ATP与CED 4同源结构域的结合和功能性核糖体的组装。 我们还将确定3种不同复合物的结构，这些复合物包含pc-9，caspase-9/pc-3和caspase-9与小鼠X-连锁凋亡抑制剂（X-IAP），以提供对线粒体内功能相互作用的见解。 在平行的研究中，我们将尝试结晶用于X射线晶体学的晶体体，并利用约10埃单位分辨率的EM图通过分子置换开始相细化。 总之，这些研究将提供对线粒体及其与关键细胞死亡组分相互作用的详细了解。

Programmed cell death (apoptosis) is a process whereby individual cells are terminated during embryonic development and normal growth, to benefit the organism. In general, there are 3 pathways that respond to apoptotic signals and activate protease zymogens (procaspases), which in turn, destroy defined cellular targets. In the mitochondrial pathway, pro-apoptotic signals trigger the release of cytochrome c from the mitochondrial inter-membrane space. Cytochrome c then interacts with the Apoptosis protease activating factor-1 (Apaf-1) in the cytosol to form the apoptosome. Subsequently, the apoptosome recruits procaspase-9 (pc-9) and mediates auto-catalytic conversion to a functional caspase-9. Whilst caspase-9 remains bound to the apoptosome, it can activate procaspase-3 (pc-3) to initiate a proteolytic cascade that leads to cell death. In this proposal, the role of the apoptosome as a cell death organizer will be investigated by determining its 3-dimensional (3D) structure. Electron cryo-microscopy (EM) and single particle image processing will be used to produce 3D maps suitable for molecular modeling. In preliminary 3D studies, we have shown that the apoptosome is assembled from individual Apaf- 1 molecules, to form a central double ring with seven Y-shaped spokes (dimensions: approximately 270 X 70 Angstrom units; mass approximately 1.1 Mda; symmetry yields C7). Based on this structure, we have proposed a model for the activation of Apaf-1 by cytochrome c. In this model, Apaf-1 is auto-inhibited due to interactions between the N-terminal region and two WD40 domains which act as intramolecular chaperones. We suggest that cytochrome c may dock to the WD40 domains within Apaf-1 to release the N-terminal region. This in turn, promotes dATP/ATP binding to the CED4 homology domain and assembly of a functional apoptosome. We will also determine the structures of 3 distinct complexes that contain pc-9, caspase-9/pc-3, and caspase-9 with the mouse X-linked inhibitor of Apoptosis (X-IAP), to provide insights into functional interactions within the apoptosome. In parallel studies, we will attempt to crystallize the apoptosome for X-ray crystallography and utilize a approximately 10 Angstrom units resolution EM map to begin phase refinement by molecular replacement. Together, these studies will provide a detailed understanding of the apoptosome and its interactions with critical cell death components.