Biochemical Dissection of the Execution Step of Mammalian Necrotic Cell Death

 描述（由申请人提供）：坏死长期以来被认为是一种被动的、不受控制的细胞死亡形式。然而，最近的发现挑战了这一概念，提供证据表明坏死是一种严格调控的细胞死亡程序，涉及各种人类病理状况[1，2，3，4]。与细胞死亡对应物细胞凋亡类似，坏死可由一系列内在和外在应力信号诱导[5]。随后的形态学表现已得到很好的表征，包括细胞体积增加、细胞器肿胀、细胞膜破裂以及随后细胞内容物溢出到周围环境中[1]。由于缺乏关于坏死细胞死亡途径的分子机制的知识，坏死通常以这些特征为特征。然而，最近在模式生物秀丽隐杆线虫中进行的研究中出现了对坏死过程的见解，这些研究表明溶酶体是坏死应激途径的主要汇聚点[17，18]。在这些研究中，溶酶体膜透化（LMP）和半胱氨酸肽酶活性被广泛的细胞损伤所触发。有趣的是，LMP和坏死被阻断过表达一个单一的细胞内丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂），SRP-6，这表明坏死是由蛋白酶驱动的机制。我们的初步数据表明，类似的坏死途径在哺乳动物细胞中。在这个建议中，我们描述了一个研究策略，旨在确定坏死的LMP和刽子手蛋白酶的调节剂，使用两个独立的生化方法。首先，我们将利用一个新开发的LMP活性测定，以确定LMP监管机构从人类细胞提取物使用一系列的纯化步骤，然后通过质谱。第二个目的是利用蛋白酶抑制特性的人丝氨酸蛋白酶抑制剂，以丰富的坏死刽子手蛋白酶使用亲和标签下拉法。一种新的化合物，NB 24，将用于帮助稳定细胞坏死诱导后的丝氨酸蛋白酶抑制剂蛋白酶复合物。将通过质谱法分析丝氨酸蛋白酶抑制剂-蛋白质复合物的蛋白质组成。将使用基于细胞的存活测定法验证所有鉴定的候选蛋白质的功能，以确定其在坏死期间的功能和作用机制。最终，这些蛋白质的鉴定将导致新的治疗策略的开发，以防止LMP诱导的坏死，以及提供长期寻求的坏死的体内生物标志物，需要进一步揭示坏死途径类似于半胱天冬酶如何用于细胞凋亡研究。

 DESCRIPTION (provided by applicant): Necrosis has long been regarded as a passive, uncontrolled form of cell death. However, recent discoveries have challenged this notion providing evidence to show that necrosis is a tightly regulated cell death program with implication in various human pathological conditions [1, 2, 3, 4]. Similar to its cell death counterpart apoptosis, necrosis can be induced by an array of intrinsic and extrinsic stress signals [5]. The morphological manifestations that ensue have been well characterized which include increase in cell volume, swelling of the cellular organelles, rupturing of the cell membrane and subsequent spillage of their cellular contents into the surrounding environment [1]. Due to the lack of knowledge concerning the molecular mechanisms underlying necrotic cell death pathways, necrosis is often characterized by these features. However, insights into the necrotic process have recently emerged from studies conducted in the model organism Caenorhabditis elegans which have suggested that lysosomes are a major convergence point for necrotic stress pathways [17, 18]. In these studies, lysosome membrane permeabilization (LMP) and cysteine peptidase activity was triggered by a broad array of cellular insults. Interestingly, LMP and necrosis were blocked by overexpressing a single intracellular serine protease inhibitor (serpin), SRP-6, suggesting that necrosis is regulated by a proteases-driven mechanism. Our preliminary data indicates that similar necrotic pathways are employed in mammalian cells. In this proposal, we describe a research strategy aimed at identifying necrotic regulators of LMP and executioner proteases using two independent biochemical approaches. First, we will utilize a newly developed LMP activity assay to identify LMP regulators from human cell extracts using a series of purification steps followed by mass spectrometry. The second objective is to exploit the protease-inhibiting properties of human serpins in order to enrich for necrotic executioner proteases using an affinity tag pulldown method. A novel chemical compound, NB24, will be used to help stabilize serpin-protease complexes in the cell after necrosis induction. The protein composition of serpin-protein complexes will be analyzed via mass spectrometry. The function of all identified candidate proteins will be validated using cell-based survival assays to determine their function and mechanism of action during necrosis. Ultimately, the identification of these proteins will lead to the development of new therapeutic strategies to prevent LMP-induced necrosis, as well as provide the long-sought in vivo biomarkers of necrosis needed to further uncover necrotic pathways similar to how caspases have served for apoptosis research.