Intracellular Serpin Regulation of Intestinal Cell Necrosis

描述（由申请方提供）：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尚不确定，但危险因素包括非等渗液体、毒素、缺血-再灌注损伤和活性氧。这些应激源扰乱肽酶-抑制剂平衡，导致细胞内蛋白质过度水解、膜完整性丧失、亚细胞结构溶解和坏死性细胞死亡。混杂的细胞内蛋白水解和细胞死亡之间的强关联表明，肽酶抑制剂作为坏死性细胞死亡调节剂。在最大的肽酶抑制剂家族，丝氨酸蛋白酶抑制剂（例如，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，因为大多数丝氨酸蛋白酶抑制剂是分泌的，不太可能在细胞内发挥调节作用。最近，我们确定了一个子集的丝氨酸蛋白酶抑制剂，大量表达的后生动物上皮细胞的细胞质内，包括哺乳动物的胃肠道。由于这些细胞内丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂IC）中的一些中和溶酶体半胱氨酸和丝氨酸肽酶，我们假设，他们调节细胞内蛋白水解和提高细胞存活。这一假设得到了证实，表明C。elegans serpinIC，SRP-6，通过阻断肠上皮细胞坏死而表现出促存活功能。在低渗休克、热应激、高氧、缺氧或阳离子通道过度活跃后，srp-6的缺失经历了一系列灾难性的事件，最终导致肠细胞溶酶体破坏、胞质蛋白水解和整个动物死亡。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，这两种SRP-6的体外靶点。SRP-6通过阻断溶酶体损伤的诱导和致死作用提供保护。综上所述，我们现在假设，多种有害刺激汇聚在进化上保守的，肽酶驱动的核心应激反应途径，在没有丝氨酸蛋白酶抑制剂调节的情况下，导致坏死细胞死亡，而不是细胞存活。本研究的目的是通过采用强有力的无偏遗传学方法，在C.优雅作为推论，我们将定义SRP-6的细胞内肽酶靶标。最后，与哺乳动物系统的比较将确定这种进化上古老的丝氨酸蛋白酶抑制剂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。这一途径的保守性为最终利用这种C. elegans平台具体目的是：1）明确C. elegans肠细胞坏死，并确定应激反应途径、serpinsIC和溶酶体肽酶如何决定细胞活力，2）鉴定由SRP-6调节的坏死细胞死亡肽酶和3）评估哺乳动物serpinsIC调节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，影响肠道和其他组织（包括心脏和大脑）的衬里。通过在一个更简单的模型系统中研究肠坏死，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非常常见的人类疾病。

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.