Mechanisms of Activation of Ice-like Cysteine Proteases

在脊椎动物中，两个半胱天冬酶亚家族，半胱天冬酶-3 - 1样和半胱天冬酶-1样半胱天冬酶，发挥重要作用， 细胞死亡和炎症。与最近取得的重大进展不同， 多年来对细胞凋亡caspase-3- 1样激活和调节机制的理解 在这方面，关于促炎性半胱天冬酶-L样亚家族， 半胱天冬酶包括人的半胱天冬酶-1、-4和-5。半胱天冬酶-1是主要的细胞内蛋白酶， 将IL-1 β和IL-18的前体切割成活性细胞因子。最近的观察表明， 通过直接结合新发现的衔接蛋白Ipaf/CARD 12激活半胱天冬酶原-1。在 此外，其他初步结果表明，PYRIN-CARD蛋白ASC通过以下途径与半胱天冬酶-1相互作用： 卡-卡交互。这一观察结果暗示ASC作为激活半胱天冬酶-I的中心适配器 通过含PYRIN的蛋白DEFCAP/NAC/CARD-7和cryopyrirdPypafl，它们也与 ASC通过PYRIN-PYRIN结构域相互作用。因此，我们认为，在炎症过程中， 由Ipaf和/或ASC/DEFCAP/cryopyrin募集成类似于Apaf的大的“炎性体”复合物。 1-半胱天冬酶-9核糖体对促炎细胞因子的反应。为了验证这一假设， 本研究拟探讨Ipaf对caspase-1-like caspase的激活和调控机制， caspase-1相互作用的PYRIN蛋白。特别地，第一个具体目标描述了实验， 确定Ipaf在caspase-1激活机制中的作用。这些实验使用RNA 干扰（RNAi）技术沉默THP-1细胞中的Ipaf表达，显性负Ipaf突变体， 干扰半胱天冬酶-1的功能，并最终对半胱天冬酶-1和Ipaf-caspase-1复合物进行结构分析。 第二个具体目标是解决潜在的促炎功能和作用的PYRIN结构域 在半胱天冬酶-1、-4和-5活化机制中含有蛋白ASC、NAC和冷比林， 详细的结构-功能分析、RNAi实验和蛋白质-蛋白质相互作用的表征。 第三个具体目标将更详细地阐述体外细胞因子激活caspase-1的机制。 半胱天冬酶-1激活系统的重建和半胱天冬酶-1上游调节因子的鉴定 炎性小体结合这些研究，预计将产生重要的洞察机制， caspase-1被激活和调节，从而促进新治疗策略的长期设计 用于治疗炎症相关的或神经变性的人类疾病， 激活有牵连。

In vertebrates two subfamilies of caspases, the caspase-3-1ike and caspase-l-like caspases, play important roles in cell death and in inflammation, respectively. Unlike the significant progress which has been made in recent years towards understanding the mechanism of activation and regulation of the apoptotic caspase-3-1ike caspases, very little is known in this regard with respect to the pro-inflammatory caspase-l-like subfamily of caspases which include caspase-1, -4 and -5 in human. Caspase-1 is the major intracellular protease that cleaves the precursors of IL-1 beta and IL-18 into active cytokines. Recent observations suggest that procaspase-1 is activated through direct binding to the newly discovered adaptor protein Ipaf/CARD12. In addition other preliminary results suggest that the PYRIN-CARD protein ASC interacts with caspase-1 via CARD-CARD interactions. This observation implicates ASC as a central adaptor for activation of caspase-I by the PYRIN-containing proteins DEFCAP/NAC/CARD-7 and cryopyrirdPypafl which also interact with ASC via PYRIN-PYRIN domain interactions. Thus we propose that during inflammation procaspase-1 is recruited by Ipaf and/or ASC/DEFCAP/cryopyrin to a large "inflammasome" complex(s) similar to the Apaf- 1-caspase-9 apoptosome in response to proinflammatory cytokines. To test this hypothesis experiments are proposed to study the mechanism of activation and regulation of caspase-l-like caspases by Ipaf and the other caspase-1-interacting PYRIN containing proteins. In particular, the first specific aim describes experiments to define the role of Ipaf in the mechanism of activation of caspase-1. These experiments employ RNA interference (RNAi) techniques to silence Ipaf expression in THP-1 cells, dominant negative Ipaf-mutant to interfere with caspase-1 function and finally structural analysis of the caspase-1 and Ipaf-caspase-1 complex. The second specific aim addresses the potential pro-inflammatory function and role of the PYRIN-domain containing proteins ASC, NAC and cryopyrin in the mechanism of caspase-1, -4 and -5 activation, through detailed structure-function analyses, RNAi experiments and characterization of protein-protein interactions. The third specific aim will address in more detail the mechanism of activation of caspase-1 by in vitro reconstitution of a caspase-1 activation system and identification of upstream regulators of the caspase-1 inflammasome. Combined these studies are expected to yield important insight into the mechanisms by which caspase-1 is activated and regulated and thereby facilitate the long-term design of novel therapeutic strategies for treatment of inflammation-linked or neurodegenerative human disorders in which abnormal caspase-1 activation is implicated.