MECHANISMS OF ACTIVATION OF ICE-LIKE CYSTEINE PROTEASES

描述（由申请人提供）：Caspase-1是先天免疫应答的重要组成部分，可抵抗致病性感染和细胞应激。caspase-1的激活可导致无活性的pro-IL-12和pro-IL-18分别加工产生活性细胞因子IL-12和IL-18，它们是炎症的有效介质，刺激发烧、免疫细胞的募集和激活、次生细胞因子的产生和细胞增殖。Caspase-1的激活也会导致一种炎症形式的细胞死亡，称为焦亡。申请人的实验室最近证明，这种形式的细胞死亡是由caspase-1接头蛋白ASC介导的，ASC组装了一种称为焦体的超分子结构，在焦体死亡期间激活caspase-1，以响应各种促炎和应激信号。他们还阐明了pyrin（一种在家族性地中海热中突变的蛋白）在人类自身炎症性PAPA综合征的caspase-1激活和炎症中的作用，并证明pyrin是细胞骨架组织蛋白PSTPIP1的同三聚体细胞质受体。PSTPIP1的连接通过揭露PYD结构域激活pyrin， PYD结构域随后与ASC相互作用，促进ASC寡聚化成活性焦体。在本应用中，我们提出了扩展这些正在进行的研究的具体目标，以进一步研究PSTPIP1与pyrin相互作用的调控和分子决定因素。特别是，我们的目标将集中在描述蛋白酪氨酸磷酸酶PTP-PEST和酪氨酸激酶c-Abl在pyrin介导的caspase-1激活途径中的确切作用，这两种蛋白酪氨酸磷酸酶和酪氨酸激酶c-Abl都被证明与PSTPIP1相互作用。研究人员还建议对ASC焦亡体上游的新型激活因子进行表征，并进一步研究TLR2、TLR4、cryopyrin和caspase-1介导的蛋白水解在巨噬细胞焦亡中的作用。最后，研究人员建议研究体外ASC焦体组装作为高通量筛选的潜在用途，以识别抑制和治疗炎症的小分子疗法。公共卫生相关性：炎症是先天免疫对感染和细胞应激反应的关键过程。本研究的重点是了解关键细胞蛋白在复杂的分子和细胞炎症机制中的作用。这项研究的结果将有助于设计和发现有效的治疗方法，以治疗与衰老、自身炎症性疾病和慢性炎症性疾病相关的炎症。

DESCRIPTION (provided by applicant): Caspase-1 is an important component of the innate immune response against pathogenic infection and cellular stresses. Activation of caspase-1 can lead to processing of the inactive pro-IL-12 and pro-IL-18 to produce the active cytokines IL-12 and IL-18, respectively, which are potent mediators of inflammation that stimulate fever, recruitment and activation of immune cells, production of secondary cytokines and cellular proliferation. Caspase-1 activation can also lead to an inflammatory form of cell death called pyroptosis. The applicant's laboratory demonstrated recently that this form of cell death is mediated by the caspase-1 adaptor protein ASC, which assembles a supramolecular structure termed the pyroptosome that activates caspase-1 during pyroptosis in response to diverse pro-inflammatory and stress signals. They have also elucidated the role of pyrin, a protein mutated in familial Mediterranean fever, in caspase-1 activation and inflammation in the human auto-inflammatory PAPA syndrome, and demonstrated that pyrin is a homotrimeric cytosolic receptor for the cytoskeleton- organizing protein PSTPIP1. Ligation by PSTPIP1 activates pyrin by unmasking of its PYD domain, which then interacts with ASC and facilitates ASC oligomerization into the active pyroptosome. In this application specific aims are proposed to extend these ongoing studies to further investigate the regulation and molecular determinants of the interaction between PSTPIP1 and pyrin. In particular, the aims will focus on characterizing the exact role of the protein-tyrosine phosphatase PTP-PEST and tyrosine kinase c-Abl, both of which have been shown to interact with PSTPIP1, in the pyrin-mediated caspase-1 activation pathway. It is also proposed to characterize novel upstream activators of the ASC pyroptosome and further investigate the role TLR2, TLR4, cryopyrin and caspase-1-mediated proteolysis in macrophage pyroptosis. Finally, studies are proposed to investigate the potential use of the in vitro ASC pyroptosome assembly as a high throughput screen to identify small molecule therapeutics to inhibit and treat inflammation. PUBLIC HEALTH RELEVANCE: Inflammation is a key process in the innate immune response to infections and cellular stress. This research focuses on understanding the role of key cellular proteins in the complex molecular and cellular inflammatory mechanisms. The results of this research will help in the design and discovery of effective therapeutics to treat inflammation associated with aging, auto-inflammatory disease and chronic inflammatory diseases.