Characterizing the Mechanism of DPP8/9 Inhibitor-Induced Pyroptosis

项目总结 几种人类模式识别受体检测细胞内危险相关信号，寡聚为 多蛋白复合体被称为炎性小体，并引发一种称为下垂的细胞死亡的溶解形式。 炎性小体参与增强对病原体的免疫反应，并维持机体 动态平衡，但它们的过度激活会导致癌症、自身免疫紊乱和代谢功能障碍。AS 因此，研究调控炎性小体激活的分子机制至关重要。 NLRP1和CARD8是形成炎症体的相关模式识别受体，但危险信号 他们认为这一点尚未完全确立。值得注意的是，与丝氨酸二肽基肽酶8结合的配体 和9(DPP8/9)，包括N-末端带有Xaa-Pro(其中Xaa是任何氨基酸)的内源肽，具有 据报道可以激活这些炎性小体。然而，为什么先天免疫系统监控XaA-Pro 多肽水平是未知的，构成了一个重大的知识鸿沟。最近，减量压力，还是深刻的 据报道，缺乏活性氧基(ROS)也可以激活NLPR1和CARD8炎症体。 这一应用的中心假设是还原应激和Xaa-Pro肽的积累是 密切相关的危险信号，共同构成了导致快速和充分的总体“危险状态” NLRP1和CARD8炎性小体激活。具体地说，它提出了许多无序区域 胞浆蛋白，包括CARD8自身抑制的N-末端区域，由分子内稳定 二硫键；还原应力取消了这些键，破坏了这些序列的稳定并触发了它们 蛋白酶体将蛋白质降解成多肽。脯氨酸是无序蛋白质中含量最丰富的氨基酸 区域，因此还原压力可能会产生许多Xaa-Pro多肽。通过这种方式，XaA-Pro肽 累积可以用来证实还原应力正在发生。这一中心假设一直是 根据申请人实验室提供并在本申请中描述的初步数据制定。 这个项目的目标是确定还原应激、无序蛋白质之间的关系 降解和XaA-Pro多肽积累。本项目由三个具体目标组成：1)确定 还原应激如何诱导蛋白酶体介导的CARD8降解，2)表征 还原应激与Xaa-Pro多肽蓄积的关系；3)确定两者的关系 细胞代谢和炎性小体激活之间的关系。这项工作的成功完成不仅将 阐明这些神秘的炎症体的原始功能，但也将揭示一个以前未知的 细胞内氧化还原状态与蛋白质稳定性之间的联系。此外，这项工作将提供 为今后通过治疗控制这些炎性小体治疗人类疾病的努力奠定了基础。

PROJECT SUMMARY Several human pattern-recognition receptors detect intracellular danger-associated signals, oligomerize into multiprotein complexes called inflammasomes, and trigger a lytic form of cell death called pyroptosis. Inflammasomes are involved in mounting immune responses to pathogens and in maintaining organismal homeostasis, but their hyperactivation can cause cancer, autoimmune disorders, and metabolic dysfunction. As such, it is critically important to characterize the molecular mechanisms that regulate inflammasome activation. NLRP1 and CARD8 are related pattern-recognition receptors that form inflammasomes, but the danger signals that they sense have not been fully established. Notably, ligands that bind to the serine dipeptidyl peptidases 8 and 9 (DPP8/9), including endogenous peptides with Xaa-Pro (where Xaa is any amino acid) N-termini, have been reported to activate these inflammasomes. However, why the innate immune system monitors Xaa-Pro peptide levels is unknown and constitutes a major knowledge gap. Recently, reductive stress, or a profound lack of reactive oxygen species (ROS), was also reported to activate the NLPR1 and CARD8 inflammasomes. The central hypothesis of this application is that reductive stress and Xaa-Pro peptide accumulation are intimately related danger signals that together comprise an overall “danger state” that causes rapid and full NLRP1 and CARD8 inflammasome activation. Specifically, it is proposed that the disordered regions of many cytosolic proteins, including the autoinhibitory N-terminal region of CARD8, are stabilized by intramolecular disulfide bonds; reductive stress abolishes these bonds, destabilizing these sequences and triggering their degradation into peptides by the proteasome. Proline is the most abundant amino acid in disordered protein regions, and therefore reductive stress likely generates many Xaa-Pro peptides. In this way, Xaa-Pro peptide accumulation can serve to confirm that reductive stress is occurring. This central hypothesis has been formulated based on preliminary data produced in the applicant’s laboratory and described in this application. The objective of this project is to determine the relationship between reductive stress, disordered protein degradation, and Xaa-Pro peptide accumulation. This project consists of three Specific Aims: 1) to determine how reductive stress induces the proteasome-mediated degradation of CARD8, 2) to characterize the relationship between reductive stress and Xaa-Pro peptide accumulation, and 3) to determine the relationship between cell metabolism and inflammasome activation. The successful completion of this work will not only clarify the primordial function of these enigmatic inflammasomes, but will also reveal a previously unknown a connection between intracellular redox state and protein stability. Moreover, this work will provide the foundation for future efforts to therapeutically control these inflammasomes for the treatment of human disease.