Caspase-activating Complexes

描述（由申请人提供）：应激刺激，包括生长因子剥夺、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，这些刺激启动途径，最终导致线粒体外膜透化和细胞色素c从膜间隙释放到细胞质中。然后细胞色素c诱导（d）细胞凋亡蛋白酶激活因子-1（Apaf-1）的ATP依赖性寡聚化成多聚体“寡聚体”复合物，该复合物依次募集并激活起始剂胱天蛋白酶-9和效应剂胱天蛋白酶-3。尽管我们对这一极其重要的复杂系统的理解有所进步，但根本问题仍然没有答案。事实上，显著的争议围绕的组成和大小的线粒体，它仍然不清楚是否caspase-9经历激活的线粒体内的二聚化和/或Apaf-1诱导的构象变化。在目标#1中，我们将利用复杂的技术，包括分析性超离心、非天然/交叉互补氨基酸的位点特异性掺入和同步加速器蛋白足迹分析等，以确定Apaf-1溶酶体复合物中caspase-9的化学计量、二聚化和构象状态。类似地，许多先前的研究表明，许多激酶在不同位点对半胱天冬酶原-9的磷酸化抑制或激活该蛋白酶。然而，许多这些研究是有争议的，它仍然是完全未知的，在机制上，磷酸化如何改变蛋白酶原-9的激活/活性。在目标#2中，我们将确定半胱天冬酶原-9的磷酸化是否影响其对核糖体的亲和力，或其二聚化或经历活化所需的构象变化的能力。磷酸化对细胞凋亡的影响也将被评估，在某种程度上，通过将磷酸突变体重新引入半胱天冬酶-9缺陷细胞系。最后，我们最近已经证明Apaf-17胱天蛋白酶-9核糖体作为基于蛋白水解的“分子计时器”起作用，其中胱天蛋白酶-9的细胞内浓度设定计时器的总持续时间，胱天蛋白酶-9原自动加工激活计时器，以及加工的半胱天冬酶-9从复合物中解离的速率（并因此失去其激活半胱氨酸天冬氨酸蛋白酶原-3的能力）决定了计时器“滴答”的速度。在目标#3中，我们将使用新型caspase-9基因敲入小鼠评估这种分子计时器在体内的重要性，该小鼠可阻止caspase-9进行加工。我们将确定这种定时器的脱离是否使动物对发育或毒物诱导的细胞凋亡敏感。总之，这项资助申请的主要目标是利用一些高度新颖的方法，以前从未对Apaf-17 caspase-9的转录体复合物产生影响，以便在分子细节上表征介导这种关键的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引发剂半胱天冬酶如何在大蛋白复合物中被激活的一般理解，并将阐明它们在未来如何在治疗上被利用来治疗从癌症到神经退行性疾病的疾病。

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.