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

项目摘要/摘要 虽然急性髓系白血病(AML)是成人最常见的白血病形式，但预后仍然很差。 一种以抑制丝氨酸蛋白酶DPP8和DPP9为中心的治疗AML的新策略 (DPP8/9)，它激活CARD8炎症体，并触发一种称为下垂的细胞死亡的溶解形式。 这一过程直接对AML细胞产生细胞毒作用，并间接激活宿主免疫反应，从而提供 抗癌活性的双重机制。我们的中心假设是，DPP8/9处理会使一些AS失活 未知底物(S)，当活性时，招募炎性小体并引发下垂。不幸的是， DPP8/9，像许多~550种人类蛋白水解酶一样，在其内源性方面仍然没有特征 底物。鉴定和鉴定DPP8/9的生理相关底物将是必要的 来理解和治疗利用这个嗜热性检查点。 尽管蛋白水解酶调节着无数重要的(病理)生理过程，但发现 底物具有极大的挑战性，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，我将提出一种实用的蛋白酶技术 底物识别。这个创新的平台使用N-末端的反应性化学部分来选择性地 标记复杂细胞裂解物中蛋白质的N-末端。将这种化学基团与生物素偶联将使 内源性蛋白酶底物的标记、浓缩和鉴定。在具体的目标2中，我将使用以下内容 技术，以及识别DPP8/9底物的互补策略，然后表征 这些底物如何激活CARD8炎症体。我预计这个项目将导致两个关键 结果：1)引入了一种实用的发现蛋白酶底物的方法，这将使 不带偏见地询问控制重要生物反应的许多复杂的蛋白质分解网络， 以及2)DPP8/9-CARD8焦磷酸检查点的特征，该检查点具有很高的启动潜力 调节这一焦链通路用于治疗急性髓系白血病。

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.