Intracellular Serpin Regulation of Intestinal Cell Necrosis

细胞内丝氨酸蛋白酶抑制剂对肠细胞坏死的调节

• 批准号: 8321042
• 负责人: GARY ARTHUR SILVERMAN
• 金额: $ 38.49万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8321042
• 关键词: Affect; Affinity Chromatography; Animals; Antithrombins; Architecture; Bacteria; Bacterial Toxins; Biological Models; Blood Circulation; Brain; Caenorhabditis elegans; Calpain; Cations; Cell Culture System; Cell Death; Cell Survival; Cells; Cessation of life; Chemicals; Cysteine; Cytoplasm; Epithelial Cells; Equilibrium; Etiology; Event; Exhibits; Failure; Family; Gastroenteritis; Gastrointestinal Diseases; Gastrointestinal tract structure; Genes; Genetic; Genetic Screening; Goals; Heart; Hyperactive behavior; Hyperoxia; Hypoxia; In Vitro; Inflammatory Bowel Diseases; Injury; Intestines; Invaded; Ischemic Bowel Disease; Learning; Liquid Chromatography; Liquid substance; Membrane; Molecular; Mucous body substance; Mutagenesis; Necrosis; Necrotizing Enterocolitis; Pathologic; Pathway interactions; Peptide Hydrolases; Peptide Signal Sequences; Phenotype; Play; Protease Inhibitor; Protein C Inhibitor; Proteolysis; RNA Interference; Reactive Oxygen Species; Recombinants; Regulation; Regulator Genes; Reperfusion Injury; Respiratory System; Respiratory tract structure; Risk Factors; Role; Septic Shock; Series; Serine; Serpins; Shock; Signal Recognition Particle; Stimulus; System; Therapeutic Uses; Tissues; Toxin; Transgenes; Uncertain Risk; Vertebrates; base; biological adaptation to stress; fitness; graft vs host disease; human disease; in vivo; novel; positional cloning; stressor; tandem mass spectrometry; thermal stress

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 ¿1-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.

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、传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，危险因素包括不等渗液体、毒素、缺血 再灌注损伤和活性氧。这些压力因素扰乱了肽酶抑制剂的平衡， 导致细胞内过度蛋白水解、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关联 表明肽酶抑制剂可作为坏死细胞死亡调节剂。在最大的范围内进行广泛的搜索 肽酶抑制剂家族，丝氨酸蛋白酶抑制剂（例如抗凝血酶和 ¿1-抗胰蛋白酶）未能产生候选物 调控基因。然而，这个结果并不令人惊讶，因为大多数丝氨酸蛋白酶抑制剂是分泌的，不太可能发挥作用 细胞内的调节作用。最近，我们鉴定了丝氨酸蛋白酶抑制剂的一个子集，它们在 后生动物上皮细胞的细胞质，包括哺乳动物胃肠道的细胞质。由于一些 我们假设这些细胞内丝氨酸蛋白酶抑制剂（serpinsIC）中和溶酶体半胱氨酸和丝氨酸肽酶 它们调节细胞内蛋白水解并增强细胞存活。这一假设得到了证实 显示秀丽隐杆线虫丝氨酸蛋白酶抑制剂 IC (SRP-6) 通过阻断肠上皮细胞表现出促生存功能 坏死。低渗休克、热应激、高氧、缺氧或阳离子通道过度活跃后； srp-6 无效 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。这种新定义的坏死性死亡表型取决于 钙蛋白酶和溶酶体半胱氨酸肽酶是 SRP-6 的两个体外靶标。 SRP-6 提供的保护 阻断溶酶体损伤的诱导和致命作用。综合起来，我们现在 假设多种有害刺激聚集在进化上保守的、肽酶驱动的核心上 应激反应途径，在缺乏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死亡 生存。该提案的目标是确定这种应激反应背后的核心分子成分 通过在秀丽隐杆线虫中采用强大的无偏见遗传方法来发现途径。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是否 这种进化古老的 serpinIC 抗肽酶防御系统可调节肠上皮细胞的适应性 高等脊椎动物。该途径的保护为最终开发新型抗- 使用该秀丽隐杆线虫平台进行坏死治疗。具体目标是 1) 定义 C 的遗传基础。 线虫肠细胞坏死并确定应激反应途径、丝氨酸蛋白酶抑制剂IC和溶酶体如何 肽酶决定细胞活力，2) 鉴定受 SRP-6 调节的坏死细胞死亡肽酶和 3) 评估哺乳动物丝氨酸蛋白酶抑制剂 IC 调节坏死细胞死亡的程度。