Characterizing the DPP8/9 Pyroptotic Checkpoint with Chemoproteomic Protease Substrate Profiling

项目总结/摘要 虽然急性髓性白血病（AML）是成人中最常见的白血病形式，但预后仍然很差。 一种新兴的AML治疗策略围绕抑制丝氨酸蛋白酶DPP 8和DPP 9 (DPP8/9），其激活CARD 8炎性体并触发称为细胞凋亡的裂解形式的细胞死亡。 这一过程对AML细胞具有直接细胞毒性，并间接激活宿主免疫应答， 抗癌活性的双重机制。我们的中心假设是，DPP 8/9处理使一些人失活， 未知的底物，当活性时，募集炎性小体并触发焦亡。不幸的是， 与约550种人类蛋白酶中的许多蛋白酶一样，DPP 8/9在其内源性方面仍然没有特征。 印刷受体.识别和表征DPP 8/9的生理相关底物将是必要的 来理解和治疗这个自燃检查点。 虽然蛋白酶调节无数重要的（病理）生理过程，发现蛋白酶 底物分析是非常具有挑战性的，并且最先进的蛋白酶底物分析平台 昂贵且不全面。在具体目标1中，我将提出蛋白酶的实用技术 底物识别这种创新平台使用N-末端反应性化学部分， 标记复杂细胞裂解物中蛋白质的N末端。将该化学基团与生物素偶联将使生物素能够与生物素结合。 内源性蛋白酶底物的标记、富集和鉴定。在第2章中，我将使用 技术，沿着互补策略以识别DPP 8/9的底物，然后表征 这些底物如何激活CARD 8炎性体。我预计，该项目将导致两个关键 结果：1）引入了一种实用的蛋白酶底物发现方法， 对控制重要生物反应的许多复杂蛋白水解网络的无偏询问， 和2）DPP 8/9 -CARD 8焦萎检查点的表征，其具有高潜力， 调节该热凋亡途径用于治疗AML。

Project Summary/Abstract While acute myeloid leukemia (AML) is the most common form of leukemia in adults, prognosis remains poor. An emerging strategy for the treatment of AML centers around inhibiting the serine proteases DPP8 and DPP9 (DPP8/9), which activate the CARD8 inflammasome and triggers a lytic form of cell death known as pyroptosis. This process is directly cytotoxic to AML cells and indirectly activates the host immune response, delivering a dual mechanism for anticancer activity. Our central hypothesis is that DPP8/9 processing inactivates some as of yet unknown substrate(s) that, when active, recruit the inflammasome and triggers pyroptosis. Unfortunately, DPP8/9, like many of the ~550 human proteases, remain uncharacterized with respect their endogenous substrates. Identifying and characterizing the physiologically relevant substrates of DPP8/9 will be necessary to both understand and therapeutically exploit this pyroptotic checkpoint. Although proteases regulate countless important (patho)physiological processes, discovering protease substrates is extraordinarily challenging and state-of-the-art protease substrate profiling platforms are expensive and incomprehensive. In Specific Aim 1, I will advance a practical technology for protease substrate identification. This innovative platform uses an N-terminal reactive chemical moiety to selectively label the N-termini of proteins in complex cell lysates. Coupling this chemical group to biotin will enable the labeling, enrichment, and identification of endogenous protease substrates. In Specific Aim 2, I will use this technology, along with complimentary strategies to identify the substrates of DPP8/9, and then characterize how these substrates activate the CARD8 inflammasome. I anticipate that this project will lead to two key outcomes: 1) the introduction of a practical method for protease substrate discovery that will enable the unbiased interrogation of the many complex proteolytic networks that control important biological responses, and 2) the characterization of the DPP8/9 – CARD8 pyroptotic checkpoint, which has high potential to enable modulation of this pyroptotic pathway for the treatment of AML.