STRUCTURAL OF THE APOPTOSOME & ITS ROLE IN CELL DEATH

细胞程序性死亡（凋亡）是个体细胞在胚胎发育和正常生长过程中终止的过程，有利于生物体。一般来说，有3种途径响应凋亡信号并激活蛋白酶酶原（原aspase），进而破坏确定的细胞靶标。在线粒体途径中，促凋亡信号触发细胞色素c从线粒体膜间空间释放。然后细胞色素c与细胞质中的凋亡蛋白酶激活因子-1 （Apaf-1）相互作用形成凋亡小体。随后，凋亡细胞募集procaspase-9 （pc-9）并介导自催化转化为功能性caspase-9。虽然caspase-9仍然与凋亡细胞结合，但它可以激活原caspase-3 （pc-3）启动蛋白水解级联反应，导致细胞死亡。在这一建议中，凋亡小体作为细胞死亡组织者的作用将通过确定其三维（3D）结构来研究。电子冷冻显微镜（EM）和单粒子图像处理将用于制作适合分子建模的3D地图。在初步的3D研究中，我们发现凋亡细胞由单个Apaf- 1分子组装而成，形成一个有7个y形辐条的中心双环（尺寸：约270 X 70埃；质量约1.1 Mda；对称性为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。在这个模型中，由于n端区域和两个作为分子内伴侣的WD40结构域之间的相互作用，Apaf-1被自动抑制。我们认为细胞色素c可能停靠在Apaf-1内的WD40结构域，释放n端区域。这反过来又促进了dATP/ATP与CED4同源结构域的结合和功能性凋亡细胞的组装。我们还将确定含有pc-9、caspase-9/pc-3和caspase-9的3种不同复合物与小鼠X-linked inhibitor of Apoptosis （X-IAP）的结构，以深入了解凋亡中的功能相互作用。在平行研究中，我们将尝试对凋亡细胞进行x射线晶体学结晶，并利用大约10埃单位分辨率的EM图开始通过分子替换进行相细化。总之，这些研究将提供对凋亡及其与关键细胞死亡成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.