Activation and in vivo Functions of Initiator Caspases

描述(申请人提供)：caspase在多种细胞过程中发挥重要作用，包括细胞凋亡、细胞生长和增殖以及细胞因子成熟。位于细胞凋亡信号级联顶端的半胱氨酸天冬氨酸酶称为启动子半胱氨酸蛋白酶。它们在诱导细胞凋亡信号的启动中起着关键作用。目前，启动子半胱氨酸天冬氨酸酶激活的确切分子机制尚未解决。此外，很难评估这些启动子半胱氨酸酶的体内功能，因为它们在小鼠身上的敲除会导致胚胎死亡。本研究拟通过实验研究启动子caspase激活的分子机制及其在体内的作用：1)检测依赖二聚体的caspase激活机制。晶体结构研究表明，半胱氨酸天冬氨酸酶在自激活之前形成同源二聚体。我们将使用可诱导的二聚体系统来研究二聚体中的两个caspase分子是如何相互作用导致caspase激活的。2)研究半胱氨酸氨基转移酶激活的级联反应。半胱氨酸天冬氨酸氨基转移酶可能形成一个信号级联来传递细胞凋亡信号。不同caspase之间的相互作用在细胞凋亡的信号级联中的作用还不是很清楚。我们将研究两种不同的半胱氨酸天冬氨酸酶是否在原天冬氨酸氨基转移酶水平上相互作用，以及在凋亡信号转导中是否相互作用。3)分析caspase-8和caspase-9在条件性基因敲除模型中的作用。Caspase-8和Caspae-9分别是死亡受体依赖性和线粒体依赖性细胞凋亡的启动子。然而，传统的caspase-8和caspase-9基因敲除会导致胚胎或围产期死亡，这使得评估这些caspase的体内功能变得困难。我们将在小鼠中产生caspase-8和caspase-9的条件性敲除，以研究它们在体内的功能：这些研究将有助于阐明caspase激活的机制和促凋亡caspase在体内调节T细胞功能的功能，从而促进针对这些caspase在自身免疫性疾病和癌症中的治疗方法的设计。

DESCRIPTION (provided by applicant): Caspases play important roles in multiple cellular processes, including apoptosis, cell growth and proliferation, and cytokine maturation. Caspases at the top of the apoptosis signaling cascade are called initiator caspases. They are critical in inducing the onset of apoptosis signaling. Currently, the precise molecular mechanisms for the activation of initiator caspases have not been resolved. Moreover, it has been difficult to assess the in vivo functions of these initiator caspases because their knockouts in mice cause embryonic lethality. In this proposal, experiments are proposed to study the molecular mechanisms of activation and in vivo functions of initiator caspases: 1) To test the mechanism of dimer-dependent caspase activation. Crystal structural studies suggest that caspases form homodimers prior to their autoactivation. We will use an inducible dimeric system to study how the two caspase molecules in the dimer interact to cause caspase activation. 2) To examine the caspase activation cascades. Caspases may form a signaling cascade to transmit apoptosis signals. The interactions between different caspases in the signaling cascade during apoptosis are not well understood. We will study whether two different caspases interact at the procaspase level and in apoptosis signaling cascade. 3) To dissect the functions of caspase-8 and caspase-9 in conditional knockout models. Caspase-8 and caspae-9 are the initiator caspases in death receptor- and mitochondrion-dependent apoptosis, respectively. However, conventional knockouts of caspase-8 and caspase-9 result in embryonic or perinatal lethality, making it difficult to assess the in vivo functions of these caspases. We will generate conditional knockouts of caspase-8 and caspase-9 in mice in order to study their functions in vivo: These studies will help to elucidate the mechanisms for caspase activation and the functions of pro-apoptotic caspases in regulating T cell functions in vivo, thereby facilitating the design of therapeutic approaches to target these caspases in autoimmune diseases and cancer.