Intracellular Serpin Regulation of Intestinal Cell Necrosis

描述(申请人提供)：肠上皮细胞坏死是许多胃肠道疾病的共同病理特征，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尚不清楚，但危险因素包括等渗液、毒素、缺血再灌注损伤和活性氧。这些应激源扰乱了肽酶-抑制物的平衡，导致细胞内蛋白过度降解，膜完整性丧失，亚细胞结构溶解和坏死性细胞死亡。混杂的细胞内蛋白分解和细胞死亡之间的强烈联系表明，肽酶抑制剂是坏死性细胞死亡的调节剂。在最大的肽酶抑制剂家族中进行了广泛的搜索，蛇类(如抗凝血酶和11-抗胰蛋白酶)未能产生候选调控基因。然而，这一结果并不令人惊讶，因为大多数蛇类都是分泌的，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的细胞质中大量表达的一组蛇，包括哺乳动物的胃肠道上皮细胞。由于一些细胞内的丝氨酸(SerpinsIC)中和溶酶体半胱氨酸和丝氨酸肽酶，我们推测它们调节细胞内的蛋白分解并提高细胞存活率。这一假设得到了证实，表明线虫SerpinIC，SRP-6，通过阻断肠上皮细胞坏死而显示出促进生存的功能。在低渗休克、热应激、高氧、低氧或阳离子通道过度激活后，SRP-6缺失经历了一系列灾难性的事件，最终导致肠道细胞溶酶体破裂、细胞质蛋白分解和整个动物死亡。这种新定义的坏死性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来提供保护。综上所述，我们现在假设，多种伤害性刺激汇聚在一条进化上保守的、由肽酶驱动的核心应激反应途径上，在缺乏SerpinIC调控的情况下，该途径会导致坏死细胞死亡，而不是细胞生存。这项建议的目标是通过在线虫中使用强大的、无偏见的遗传方法来识别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的胞内肽酶靶标。最后，与哺乳动物系统的比较将确定这种进化上古老的SerpinIC抗肽酶防御系统是否调节高等脊椎动物的肠道上皮细胞适应性。这一途径的保守为最终利用线虫平台开发新型抗坏死疗法提供了理论基础。其具体目的是1)确定线虫肠道细胞坏死的遗传基础，并确定应激反应途径、丝氨酸IC和溶酶体多肽酶如何决定细胞活力，2)鉴定受SRP-6调控的坏死细胞死亡多肽酶，3)评估哺乳动物丝氨酸IC调节坏死细胞死亡的程度。与公共卫生相关：坏死是一种细胞死亡，会影响肠道和包括心脏和大脑在内的其他组织的衬里。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治疗这种非常常见的人类疾病的新方法。

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.