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

基本信息

批准号：
7729625
负责人：
GARY ARTHUR SILVERMAN
金额：
$ 41.66万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-01 至 2013-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7729625
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 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 public health relevance stressor tandem mass spectrometry thermal stress

项目摘要

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.

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。尽管肠细胞坏死的病因尚不确定，但危险因素包括各向异性液体，毒素，缺血再灌注损伤和活性氧。这些应激源扰动肽酶抑制剂平衡，导致细胞内蛋白水解，膜完整性的丧失，亚细胞结构的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，肽酶抑制剂是坏死细胞死亡调节剂。在最大的肽酶抑制剂家族中，SERPINS（例如抗凝结蛋白和11-抗抗蛋白酶）的广泛搜索未能产生候选调节基因。但是，这一结果并不奇怪，因为大多数Serpins是分泌的，并且不太可能在细胞内发挥调节作用。最近，我们确定了一部分丝丁蛋白，这些子集在后生细胞的细胞质中大量表达，包括哺乳动物胃肠道的细胞。由于这些细胞内丝丁（Serpinsic）中和溶酶体半胱氨酸和丝氨酸肽酶中和，我们假设它们调节细胞内蛋白水解并增强细胞的存活。通过证明秀丽隐杆线虫SRP-6通过阻止肠上皮细胞坏死，证实了这一假设。低渗性休克，热应力，高氧，缺氧或阳离子通道多动症过度； SRP-6无效发生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取决于CALPAIN和溶酶体半胱氨酸肽酶，这是SRP-6的两个体外靶标。 SRP-6通过阻止溶酶体损伤的诱导和致命作用来提供保护。综上所述，我们假设多种有害刺激会在进化保守的肽酶驱动的核心应力反应途径上汇聚，这些途径在没有蛇纹管调节的情况下会导致坏死细胞死亡而不是细胞存活。该提案的目的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力反应途径的核心分子成分。作为推论，我们将定义SRP-6的细胞内肽酶靶标。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较高脊椎动物的肠上皮细胞适应性。该途径的保护为最终使用此秀丽隐杆线虫平台开发新型抗坏死疗法提供了理由。具体目的是1）定义秀丽隐杆线虫肠道细胞坏死的遗传基础，并确定应激反应途径如何确定细胞活力，2）确定由SRP-6和3）评估对乳腺胞和培养基的程度来确定坏死性细胞死亡肽酶。公共卫生相关性：坏死是一种细胞死亡，会影响肠道和其他组织在内的内壁，包括心脏和大脑。通过研究更简单的模型系统中的肠道坏死，我们将学习细胞死亡的发生方式，以便我们开始开发新的手段来治疗这种非常普遍的人类疾病原因。