Cysteine Proteases in Apoptosis and Cancer

细胞凋亡是多细胞生物正常发育、细胞分化和体内平衡所必需的一个进化保守过程。细胞凋亡的失调与许多人类疾病有关，包括癌症、神经退行性疾病、自身免疫功能障碍和衰老。凋亡机制的一个关键步骤是通过凋亡刺激激活一类半胱氨酸蛋白酶，称为半胱天冬酶。半胱天冬酶的激活是由接合蛋白如FADD和Apaf-1调节的，它们以刺激依赖的方式与启动体半胱天冬酶的原结构域结合，并通过寡聚化促进其激活。caspase的活性也受到凋亡蛋白抑制剂（IAPs）和破坏IAP-caspase相互作用蛋白（iap抑制剂）数量之间的平衡的调节，如哺乳动物Smac/DIABLO和果蝇Reaper、Hid和Grim。本研究的总体目标是深入了解IAPs与iap抑制剂相互作用的分子基础，以及iap抑制剂激活caspase并诱导细胞凋亡的机制。第一个具体目标将通过详细的生化、生物学和基因转移实验，确定IAPs和Smac/DIABLO在细胞对死亡刺激的敏感性中的作用。这些实验包括研究Smac/DIABLO在凋亡刺激和促凋亡Bcl-2家族成员的作用下从线粒体释放，Smac与IAPs的相互作用，以及XIAP和Smac/DIABLO与Apaf-l-caspase-9凋亡体的相互作用。第二个具体目标将在功能和生物化学上表征本实验室鉴定的潜在IAP抑制剂。我们拟通过实验来表征这些蛋白与IAPs的相互作用，并确定它们在体内和体外的促凋亡和促caspase活性。其他的实验被提出来鉴定和表征其他新的哺乳动物IAP抑制剂。第三个具体目标将涉及设计和测试由iap抑制剂衍生的化学合成肽的凋亡活性。本研究的目的是基于IAP抑制剂的IAP结合基序设计短细胞可渗透肽，以验证小细胞可渗透IAP结合肽可以通过结合IAP分子中的重要口袋来阻断IAP功能的假设。这项工作有望为IAPs和iap抑制剂调控caspase活性和细胞凋亡提供重要信息。这将促进IAPs治疗抑制剂作为一种新的抗癌策略的发展。

Apoptosis is an evolutionarily conserved process essential for normal development, cellular differentiation and homeostasis of multicellular organisms. Dysregulation of apoptosis has been associated with many human diseases, including cancer, neurodegenerative disorders, autoimmune dysfunction and aging. A key step in the mechanism of apoptosis is activation of a class of cysteine proteases, called caspases, by apoptotic stimuli. Activation of caspases is regulated by adaptor proteins such as FADD and Apaf-1, which associate in a stimulus-dependent manner with the prodomains of the initiator caspases and promote their activation via oligomerization. The activity of caspases is also regulated by a balance between the amount of inhibitor of apoptosis proteins (IAPs) and proteins that disrupts IAP-caspase interaction (IAP-inhibitors), such as mammalian Smac/DIABLO and drosophila Reaper, Hid, and Grim. The overall objective of the proposed research is to gain insight into the molecular basis of the interactions between IAPs and IAP-inhibitors and the mechanism by which IAP-inhibitors activate caspases and induce apoptosis. The first specific aim will define the role of IAPs and Smac/DIABLO in the sensitivity of cells to death stimuli, through detailed biochemical, biological and gene transfer experiments. These experiments involve studying Smac/DIABLO release from the mitochondria in response to apoptotic stimuli and pro-apoptotic Bcl-2 family members, interactions of Smac with IAPs and detailed analysis of the interaction of XIAP and Smac/DIABLO with the Apaf-l-caspase-9 apoptosome. The second specific aim will functionally and biochemically characterize potential IAP inhibitors identified in this lab. Experiments are proposed to characterize the interaction of these proteins with IAPs and determine their apoptotic and caspase promoting activity in vivo and in vitro. Other experiments are proposed to identify and characterize additional novel mammalian IAP inhibitors. The third specific aim will involve designing and testing the apoptotic activity of chemically synthesized peptides derived from IAP-inhibitors. The goals of this aim are to design short cell permeable peptides based on the sequence of the IAP-binding motif of IAP-inhibitors to test the hypothesis that small cell permeable IAP-binding peptides could block IAP function by binding to an important pocket in the IAP molecule. This work is expected to yield important information on the regulation of caspase activity and apoptosis by IAPs and IAP-inhibitors. This will facilitate the development of therapeutic inhibitors of IAPs as a new anti-cancer strategy.