Intracellular Serpin Regulation of Intestinal Cell Necrosis

描述（由申请人提供）：肠上皮细胞坏死是许多胃肠道疾病的常见病理特征，包括炎症性肠病、感染性胃肠炎和坏死性小肠结肠炎。虽然肠细胞坏死的病因不明，但危险因素包括各向异性体液、毒素、缺血再灌注损伤和活性氧。这些应激源扰乱了肽酶-抑制剂的平衡，导致细胞内蛋白水解过度，膜完整性丧失，亚细胞结构溶解和坏死细胞死亡。混杂的细胞内蛋白水解与细胞死亡之间的密切联系表明肽酶抑制剂可作为坏死细胞死亡的调节因子。在最大的肽酶抑制剂家族中，蛇形蛋白（如抗凝血酶和11-抗胰蛋白酶）进行了广泛的搜索，未能产生候选调节基因。然而，这一结果并不令人惊讶，因为大多数蛇蛋白是分泌的，不太可能在细胞内发挥调节作用。最近，我们发现了在后生动物上皮细胞（包括哺乳动物胃肠道细胞）的细胞质中大量表达的蛇形蛋白亚群。由于这些细胞内蛇形蛋白（serpinsIC）中和溶酶体半胱氨酸和丝氨酸肽酶，我们假设它们调节细胞内蛋白水解并提高细胞存活率。这一假设被证实，表明秀丽隐杆线虫serpinIC， SRP-6，通过阻断肠上皮细胞坏死表现出促进生存的功能。低渗休克、热应激、高氧、缺氧或阳离子通道亢进后；Srp-6缺失者经历了一系列灾难性的事件，最终导致肠细胞溶酶体破坏、细胞质蛋白水解和整个动物死亡。这种新定义的坏死死亡表型依赖于钙蛋白酶和溶酶体半胱氨酸肽酶，这是SRP-6的两个体外靶点。SRP-6通过阻断溶酶体损伤的诱导和致死效应提供保护。综上所述，我们现在假设多种有害刺激聚集在进化上保守的、肽酶驱动的核心应激反应途径上，在缺乏蛇形蛋白酶调节的情况下，导致坏死细胞死亡而不是细胞存活。本提案的目标是通过在秀丽隐杆线虫中采用强大的无偏遗传方法来确定这种应激反应途径的核心分子成分。作为推论，我们将定义SRP-6的细胞内肽酶靶点。最后，与哺乳动物系统的比较将确定这种进化上古老的蛇形抗肽酶防御系统是否调节高等脊椎动物的肠上皮细胞适应性。这一途径的保存为最终利用秀丽隐杆线虫平台开发新的抗坏死疗法提供了理论依据。具体目的是：1)确定线虫肠细胞坏死的遗传基础，确定应激反应途径、serpinsIC和溶酶体肽酶如何决定细胞活力；2)鉴定SRP-6调控的坏死细胞死亡肽酶；3)评估哺乳动物serpinsIC调控坏死细胞死亡的程度。公共卫生相关性：坏死是一种细胞死亡，影响肠壁和其他组织，包括心脏和大脑。通过在一个更简单的模型系统中研究肠道坏死，我们将了解细胞死亡是如何发生的，这样我们就可以开始开发新的方法来治疗这种人类疾病的常见原因。

DESCRIPTION (provided by applicant): Intestinal epithelial cell necrosis is a common pathologic feature of many gastrointestinal disorders including inflammatory bowel disease, infectious gastroenteritis and necrotizing enterocolitis. Although the etiology of intestinal cell necrosis is uncertain, risk factors include anisotonic fluids, toxins, ischemia- reperfusion injury and reactive oxygen species. These stressors perturb peptidase-inhibitor balance, which leads to excessive intracellular proteolysis, loss of membrane integrity, dissolution of sub-cellular architecture and necrotic cell death. The strong association between promiscuous intracellular proteolysis and cell death suggests that peptidase inhibitors serve as necrotic cell death regulators. Extensive searches within the largest family of peptidase inhibitors, the serpins (e.g., antithrombin and 11-antitrypsin), failed to yield candidate regulatory genes. However, this result was not surprising as most serpins are secreted and unlikely to play a regulatory role intracellularly. Recently, we identified a subset of serpins that are abundantly expressed within the cytoplasm of metazoan epithelial cells, including those of the mammalian gastrointestinal tract. Since some of these intracellular serpins (serpinsIC) neutralize lysosomal cysteine and serine peptidases, we hypothesized that they regulate intracellular proteolysis and enhance cell survival. This hypothesis was confirmed by showing that the C. elegans serpinIC, SRP-6, exhibits a pro-survival function by blocking intestinal epithelial cell necrosis. After hypotonic shock, thermal stress, hyperoxia, hypoxia or cation channel hyperactivity; srp-6 nulls underwent a catastrophic series of events culminating in intestinal cell lysosomal disruption, cytoplasmic proteolysis and whole animal death. This newly defined necrotic death phenotype was dependent upon calpains and lysosomal cysteine peptidases, two in vitro targets of SRP-6. SRP-6 provided protection by blocking both the induction of, and the lethal effects from, lysosomal injury. Taken together, we now hypothesize that multiple noxious stimuli converge upon an evolutionarily conserved, peptidase-driven core stress-response pathway that, in the absence of serpinIC regulation, leads to necrotic cell death rather than cell survival. The goal of this proposal is to identify the core molecular components underlying this stress-response pathway by employing powerful unbiased genetic approaches in C. elegans. As a corollary, we will define the intracellular peptidase targets of SRP-6. Finally, comparisons to mammalian systems will determine whether this evolutionarily ancient, serpinIC anti-peptidase defense system regulates intestinal epithelial cell fitness in higher vertebrates. Conservation of this pathway provides the rationale for eventually developing novel anti- necrosis therapeutics using this C. elegans platform. The specific aims are to 1) define the genetic basis of C. elegans intestinal cell necrosis and determine how the stress-response pathway, serpinsIC and lysosomal peptidases determine cell viability, 2) identify the necrotic cell death peptidases regulated by SRP-6 and 3) assess the extent to which mammalian serpinsIC regulate necrotic cell death. PUBLIC HEALTH RELEVANCE: Necrosis is a type of cell death that affects the lining of the intestines and other tissues including the heart and brain. By studying intestinal necrosis in a simpler model system, we will learn how cell death occurs so we can begin to develop new means to treat this very common cause of human disease.