STRUCTURAL OF THE APOPTOSOME & ITS ROLE IN CELL DEATH

凋亡体的结构

• 批准号: 6368554
• 负责人: CHRISTOPHER W AKEY
• 金额: $ 24.62万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6368554
• 关键词: X ray crystallography; adenosine triphosphate; apoptosis; biological signal transduction; cell component structure /function; cell growth regulation; computer data analysis; computer simulation; cryoelectron microscopy; crystallization; cysteine endopeptidases; cytochrome c; enzyme complex; enzyme induction /repression; inhibitor /antagonist; intermolecular interaction; model design /development; molecular assembly /self assembly; molecular dynamics; mutant; physical model; protein structure function; structural biology; zymogens

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.

程序性细胞死亡（凋亡）是一个过程，在该过程中，单个细胞在胚胎发育和正常生长过程中被终止以使生物体受益。 通常，有3种途径对凋亡信号响应并激活蛋白酶酶原（procaspases），而蛋白酶则酶（procaspases）反过来又破坏了定义的细胞靶标。 在线粒体途径中，促凋亡信号触发了从线粒体间膜间空间释放细胞色素c。 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，形成凋亡组。 随后，端子体募集了procaspase-9（PC-9）并介导自动催化转化为功能性caspase-9。 尽管Caspase-9保持与凋亡组的结合，但它可以激活procaspase-3（PC-3）启动导致细胞死亡的蛋白水解级联反应。在此提案中，将通过确定其3维（3D）结构来研究凋亡小体作为细胞死亡组织者的作用。 电子冷冻 - 微镜（EM）和单个颗粒图像处理将用于生成适合分子建模的3D地图。 在初步的3D研究中，我们表明，圆顶体是从单个APAF-1分子组装而成的，形成一个带有七个Y形辐条的中央双环（尺寸：大约270 x 70 Angstrom单元；质量约为1.1 MDA;对称性;对称性率c7）。 基于这种结构，我们提出了一个模型，用于通过细胞色素c激活APAF-1。 在此模型中，由于N末端区域和两个充当分子内伴侣的WD40结构域之间的相互作用，APAF-1自动抑制。 我们建议细胞色素C可能会将APAF-1中的WD40域停靠以释放N末端区域。 反过来，这促进了DATP/ATP与CED4同源域的结合和功能凋亡组的组装。 我们还将确定包含PC-9，caspase-9/pc-3和caspase-9与小鼠X连接凋亡的抑制剂（X-IAP）的3种不同的复合物的结构，以提供对凋亡组中功能相互作用的见解。 在平行研究中，我们将尝试将X射线晶体学的圆顶体结晶，并利用大约10个Angstrom单元分辨率EM图，以通过分子替代开始相结合。 总之，这些研究将提供对凋亡组及其与关键细胞死亡成分相互作用的详细理解。