Caspase-activating Complexes

Caspase 激活复合物

• 批准号: 8484933
• 负责人: Shawn B Bratton
• 金额: $ 29.61万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8484933
• 关键词: Caspase; activating; Complexes

DESCRIPTION (provided by applicant): Stressful stimuli, including growth factor deprivation, irradiation, and chemotherapeutic drugs, induce cell death via apoptosis. In most cases, these stimuli initiate pathways that culminate in mitochondrial outer membrane permeabilization and release of cytochrome c from the intermembrane space into the cytoplasm. Cytochrome c then induces (d)ATP-dependent oligomerization of apoptosis protease-activating factor-1 (Apaf-1) into a multimeric "apoptosome" complex that sequentially recruits and activates the initiator caspase-9 and the effector caspase-3. Despite advances in our understanding of this critically important complex, fundamental questions remain unanswered. Indeed, significant controversy surrounds the composition and size of the apoptosome, and it remains unclear whether caspase-9 undergoes activation within the apoptosome in response to dimerization and/or Apaf-1-induced conformational changes. In aim #1, we will utilize sophisticated techniques, including analytical ultracentrifugation, site-specific incorporation of an unnatural/cross-linkable amino acid, and synchrotron protein footprinting assays, among others, to determine the stoichiometric, dimerization, and conformational status of caspase-9 within the Apaf-1 apoptosome complex. Similarly, a number of previous studies suggest that phosphorylation of procaspase-9 at various sites by a number of kinases inhibits or activates this protease. However, a number of these studies are controversial, and it remains entirely unknown, mechanistically, how phosphorylation alters the activation/activity of procaspase-9. In aim #2, we will determine if phosphorylation of procaspase-9 impacts its affinity for the apoptosome, or its ability to dimerize or undergo conformational changes necessary for activation. The impact of phosphorylation on apoptosis will also be assessed, in part, through reintroduction of phosphomutants into caspase-9-deficient cell lines. Finally, we have recently demonstrated that the Apaf-17caspase-9 apoptosome functions as a proteolytic-based "molecular timer", wherein the intracellular concentration of procaspase-9 sets the overall duration of the timer, pro-caspase-9 autoprocessing activates the timer, and the rate at which processed caspase-9 dissociates from the complex (and thus loses its capacity to activate procaspase-3) dictates how fast the timer "ticks" over. In aim #3, we will assess the importance of this molecular timer in vivo using a novel caspase-9 knock-in mouse that prevents procaspase-9 from undergoing processing. We will determine if this disengagement of the timer sensitizes animals to developmental or toxicant-induced apoptosis. In summary, the major goal of this grant application is to utilize a number of highly novel approaches, never before brought to bear on the Apaf-17caspase-9 apoptosome complex, in order to characterize in molecular detail the mechanisms that mediate the activation and regulation of this critical caspase-activating complex. Moreover, these studies will improve our general understanding of how initiator caspases are activated within large protein complexes and will shed light on how they can be exploited therapeutically in the future to treat diseases ranging from cancer to neurodegeneration.

描述（由申请人提供）：压力刺激，包括生长因子剥夺，照射和化学治疗药物，通过细胞凋亡诱导细胞死亡。在大多数情况下，这些刺激启动了在线粒体外膜通透性中达到顶点的途径，并释放了从膜间空间进入细胞质的细胞色素C。然后，细胞色素C诱导凋亡蛋白酶激活因子-1（APAF-1）诱导（D）ATP依赖性的寡聚化，使其依次募集并激活启动器caspase-9和效应caspase-3。尽管我们对这一至关重要的复杂性的理解有所进步，但基本问题仍未得到答复。实际上，围绕呼吸体的组成和大小围绕着圆顶体的组成和大小，尚不清楚caspase-9是否在响应二聚化和/或APAF-1诱导的构象变化的情况下是否会在呼吸体内激活。在AIM＃1中，我们将利用复杂的技术，包括分析性超速离心，不自然/交叉链接氨基酸的特定地点合并以及同步蛋白蛋白质足迹测定等，以及其他确定caspase-9 Inspaf-1 apaf-apoptosome apoptosome secpase-9 Inspase-9 Inspase-9 Inspase-9 Inspase-9 Inspase-9同样，许多先前的研究表明，多个激酶在各个部位对procaspase-9的磷酸化抑制或激活该蛋白酶。但是，这些研究中的许多研究都是有争议的，从机械上讲，它仍然完全未知，磷酸化如何改变procaspase-9的激活/活性。在AIM＃2中，我们将确定procaspase-9的磷酸化是否会影响其对凋亡组的亲和力，或者其对激活所必需的构象变化的能力。磷酸化对凋亡的影响也将通过重新引入磷化剂中的部分评估，并将其重新引入CASPASE-9缺陷细胞系中。 Finally, we have recently demonstrated that the Apaf-17caspase-9 apoptosome functions as a proteolytic-based "molecular timer", wherein the intracellular concentration of procaspase-9 sets the overall duration of the timer, pro-caspase-9 autoprocessing activates the timer, and the rate at which processed caspase-9 dissociates from the complex (and thus loses its capacity to activate procaspase-3)决定计时器“滴答”的速度。在AIM＃3中，我们将使用一种新型的caspase-9敲入小鼠在体内评估该分子计时器的重要性，从而防止procaspase-9进行处理。我们将确定该计时器的这种脱离是否使动物对发育或有毒诱导的凋亡敏感。总而言之，该赠款应用的主要目的是利用许多高度新颖的方法，从未在APAF-17Caspase-9 apoptosome体中携带，以便用分子细节表征介导这种关键CASPase激活复合物的激活和调节的机制。此外，这些研究将提高我们对在大蛋白质复合物中如何激活引发剂胱天蛋白酶的一般理解，并将阐明如何在将来通过治疗方法剥削它们，以治疗从癌症到神经变性的疾病。