Caspase-activating Complexes

描述（由申请人提供）：应激刺激，包括生长因子剥夺，照射和化疗药物，通过凋亡诱导细胞死亡。在大多数情况下，这些刺激启动的途径最终导致线粒体外膜通透和细胞色素c从膜间空间释放到细胞质中。然后细胞色素c诱导(d) atp依赖性凋亡蛋白酶激活因子-1 （Apaf-1）寡聚化成多聚体“凋亡体”复合体，该复合体依次招募并激活启动物caspase-9和效应物caspase-3。尽管我们对这一极其重要的复杂问题的理解有所进步，但基本问题仍未得到解答。事实上，围绕凋亡小体的组成和大小存在重大争议，并且尚不清楚caspase-9是否在凋亡小体中激活以响应二聚化和/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，包括分析超离心，非天然/交联氨基酸的位点特异性结合，同步加速器蛋白质足迹测定等，来确定Apaf-1凋亡复合物中caspase-9的化学计量学，二聚化和构象状态。同样，许多先前的研究表明，procaspase-9在不同位点被许多激酶磷酸化会抑制或激活该蛋白酶。然而，这些研究中有许多是有争议的，并且在机制上，磷酸化如何改变procaspase-9的激活/活性仍然完全未知。在目标#2中，我们将确定procaspase-9的磷酸化是否会影响其对凋亡细胞的亲和力，或其二聚体的能力或激活所需的构象变化。磷酸化对细胞凋亡的影响也将通过向caspase-9缺陷细胞系中重新引入磷化剂来部分评估。最后，我们最近证明了Apaf-17caspase-9凋亡细胞作为一个基于蛋白水解的“分子计时器”的功能，其中细胞内procaspase-9的浓度设置了计时器的总持续时间，前caspase-9的自动加工激活了计时器，加工的caspase-9从复合物中解离的速度（因此失去了激活procaspase-3的能力）决定了计时器“ticks”的速度。在目标#3中，我们将使用一种新的caspase-9敲入小鼠来评估这种分子计时器在体内的重要性，该敲入小鼠可以阻止procaspase-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性或毒物诱导的细胞凋亡敏感。总之，这项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凋亡复合物，以便从分子细节上描述介导这种关键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启动物半胱天冬酶如何在大蛋白复合物中被激活的一般理解，并将阐明如何在未来利用它们治疗从癌症到神经退行性疾病。

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.