STRUCTURAL OF THE APOPTOSOME & ITS ROLE IN CELL DEATH

程序性细胞死亡(细胞凋亡)是指单个细胞在胚胎发育和正常生长过程中被终止，从而使有机体受益的过程。一般说来，有3条途径对细胞凋亡信号作出反应并激活蛋白酶酶原(原天冬氨酸氨基转移酶)，进而破坏特定的细胞靶点。在线粒体途径中，促凋亡信号触发细胞色素c从线粒体膜间间隙释放。然后，细胞色素c与胞浆中的凋亡蛋白激活因子-1(APAF-1)相互作用，形成凋亡体。随后，凋亡体招募原天冬氨酸蛋白酶-9(PC-9)，并介导自动催化转化为功能性的caspase-9。虽然caspase-9仍然与凋亡体结合，但它可以激活Proaspase-3(PC-3)，启动蛋白水解级联反应，导致细胞死亡。在这项提案中，将通过确定凋亡体的三维结构来研究凋亡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被用于制作适合于分子建模的3D地图。在初步的3D研究中，我们已经表明凋亡体由单个APAF-1分子组装而成，形成一个具有七个Y型辐条的中心双环(尺寸：约270×70埃单位；质量约1.1mda；对称产生C7)。基于这种结构，我们提出了细胞色素c激活APAF-1的模型。在该模型中，APAF-1是由于N-末端区域与作为分子内伴侣的两个WD40结构域相互作用而被自动抑制的。我们认为细胞色素c可能与APAF-1中的WD40结构域对接，从而释放N-末端区域。这反过来促进dATP/ATP与CED4同源结构域的结合和功能凋亡体的组装。我们还将确定3个不同的复合体的结构，它们包含PC-9、caspase-9/PC-3和caspase-9与小鼠X连锁的凋亡抑制物(X-IAP)，以提供对凋亡体内功能相互作用的见解。在平行研究中，我们将尝试使X-射线结晶学的凋亡体结晶，并利用大约10埃单位的分辨率EM图开始通过分子置换进行相细化。总之，这些研究将提供对凋亡体及其与关键细胞死亡成分的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.