Caspase-activating Complexes

描述(申请人提供)：应激性刺激，包括剥夺生长因子、辐射和化疗药物，通过细胞凋亡诱导细胞死亡。在大多数情况下，这些刺激启动最终导致线粒体外膜通透性的途径，并将细胞色素c从膜间隙释放到细胞质。然后，细胞色素c诱导(D)依赖于ATP的凋亡酶激活因子-1(APAF-1)寡聚形成一个多聚体“凋亡体”复合体，该复合体依次招募并激活启动子caspase-9和效应子caspase-3。尽管我们对这一极其重要的复杂问题的理解取得了进展，但根本问题仍然没有得到回答。事实上，围绕凋亡体的组成和大小存在重大争议，目前尚不清楚caspase-9是否在凋亡体内经历二聚化和/或APAF-1诱导的构象变化而被激活。在目标1中，我们将利用尖端技术，包括分析超速离心法、非天然/可交叉连接氨基酸的定点掺入和同步加速器蛋白质足迹分析等，来确定caspase-9在APAF-1凋亡体复合体中的化学计量、二聚化和构象状态。类似地，以前的一些研究表明，不同位置的原天冬氨酸氨基转移酶-9被一些激酶磷酸化，抑制或激活了这一酶。然而，这些研究中有一些是有争议的，从机制上讲，磷酸化如何改变原天冬氨酸蛋白酶-9的激活/活性仍然是完全未知的。在目标2中，我们将确定原天冬氨酸氨基转移酶-9的磷酸化是否影响其与凋亡体的亲和力，或影响其二聚化能力或经历激活所需的构象变化。通过将磷酸化突变体重新引入缺乏caspase-9的细胞系中，也将部分地评估磷酸化对细胞凋亡的影响。最后，我们最近证明了APAF-17caspase-9凋亡体的功能是一个基于蛋白水解酶的“分子定时器”，其中proaspase-9的细胞内浓度决定了定时器的总持续时间，前caspase-9的自动处理激活了定时器，而处理过的caspase-9从复合体中解离的速度(从而失去了激活proaspase-3的能力)决定了定时器“滴答”过去的速度。在目标3中，我们将使用一种新型的caspase-9敲入小鼠来评估这种分子计时器在体内的重要性，该小鼠可以阻止proaspase-9进行加工。我们将确定这种计时器的脱离是否会使动物对发育或毒物诱导的细胞凋亡敏感。综上所述，此次拨款申请的主要目标是利用一些前所未有的高度新颖的方法来研究APAF-17caspase-9凋亡体复合体，以便在分子细节上表征介导这一关键的caspase激活复合体的激活和调节的机制。此外，这些研究将提高我们对启动子caspase在大型蛋白质复合体中如何激活的总体理解，并将阐明未来如何利用它们进行治疗，以治疗从癌症到神经退化的各种疾病。

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.