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Functional consequences of autophagy mutations in Crohn's disease.

克罗恩病自噬突变的功能后果。

基本信息

批准号：
8306227
负责人：
DAVID L. BOONE
金额：
$ 33.87万
依托单位：
UNIVERSITY OF CHICAGO
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：
2009-08-01 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8306227
关键词：
Accounting Address Affect Amino Acids Antigen Presentation Autophagocytosis Autophagosome Bacteria Bacterial Infections C-terminal Cell Death Cell physiology Cells Code Crohn&apos s disease Cytoplasm Cytosol Defense Mechanisms Epithelial Cells Gene Mutation Gene Targeting Genes Genus Mycobacterium Health Homeostasis Human Immune Immune response Immunologic Receptors Infection Inflammation Inflammatory Inflammatory Response Intestines Ligands Linkage Disequilibrium Listeria Lysosomes MAP Kinase Gene Metabolism Microbe Mutation NF-kappa B Natural Immunity Nutrient Organelles Pathogenesis Pathology Play Predisposition Process Production Proliferating Proteins Regulation Role Salmonella Shigella Signal Transduction Somatic Cell Starvation Stress Testing Time Tumor Necrosis Factor Receptor Vacuole cytokine deprivation falls genome wide association study novel pathogen prevent protein complex protein degradation response

项目摘要

DESCRIPTION (provided by applicant): Genome wide scans (GWS) have implicated two autophagy genes, ATG16L1 and IRGM, in Crohn's disease (CD), thus implicating autophagy in the pathology of CD. The SNP in ATG16L1 falls within the coding sequence of ATG16L1 and is predicted to cause an amino acid change in the C-terminal half of human ATG16L1 protein (T300A). The SNP in IRGM is in perfect linkage disequilibrium with a 20 kB deletion that may affect the regulation of IRGM expression. Our objective is to determine the functional consequences of these mutations in human cells. Using somatic cell gene targeting, we will generate human intestinal epithelial cells (IEC) that lack ATG16L1, express the ATG16L1 T300A mutation or have the 20kB deletion in the upsteam region of IRGM. This approach will allow us to interrogate the function of these autophagy genes and the effects of the CD-associated mutations in autophagy in human cells. Autophagy is a cellular process that involves sequestration of cytosolic material in an autophagosome and destruction of its contents following fusion with the lysosome. Autophagy is thus employed by cells during times of nutrient deprivation to reclaim amino acids from cellular organelles. Autophagy is also involved in the turnover of large proteins or protein complexes, cell death and antigen presentation. Innate immune signals regulate autophagy and conversely, autophagy can regulate the response of cells to inflammatory signals. Lastly, autophagy is required for defense or against intracellular pathogens including species of Mycobacterium, Salmonella, Listeria and many other microbes. Alterations in any or all of these processes may account for a functional connection between the autophagy gene mutations identified in GWS and susceptibility to CD. Our hypothesis is that ATG16L1 and IRGM are required for autophagy in human cells and that CD-associated autophagy gene mutations alter autophagy and susceptibility of IEC to infection by intracellular bacteria. We will test this by generating ATG16L1+/-, ATG16L1-/-, and ATG16L1 T300A as well as IRGM-/- and IRGM 20kB human cells by somatic gene targeting and using these cells to address the following specific aims, namely: (1) Determine the effects of autophagy mutations on human cellular responses to stress and proinflammatory ligands; (2) Examine the effects of autophagy mutations on innate immune signaling in human cells and (3) determine the effect of autophagy mutations on bacterial infection and clearance in human cells. These studies will rapidly provide key information on whether the CD-associated mutations in human ATG16L1 and IRGM genes have functional consequences that affect metabolic processes, innate immune responses or host- pathogen interactions that contribute to CD. PUBLIC HEALTH RELEVANCE: Many new genes that increase susceptibility to Crohn's Disease have been discovered and the challenge now is to determine what these genes do and how mutations in these genes affect human cells. We are creating human intestinal cells that have these mutations and studying how the mutations affect responses to starvation and bacterial infection. These studies will help us to understand how mutations implicated in Crohn's disease affect cell functions to contribute to inflammation.

描述（由申请人提供）：全基因组扫描（GWS）表明克罗恩病（CD）中存在两种自噬基因，ATG 16 L1和IRGM，从而表明自噬在CD的病理学中存在。ATG 16 L1中的SNP福尔斯位于ATG 16 L1的编码序列内，并预测会导致人ATG 16 L1蛋白（T300 A）C-末端一半的氨基酸变化。IRGM中的SNP与可能影响IRGM表达调控的20 kB缺失处于完全连锁不平衡。我们的目标是确定这些突变在人类细胞中的功能后果。利用体细胞基因打靶技术，我们将获得缺失ATG 16 L1、表达ATG 16 L1 T300 A突变或IRGM上游20 kB缺失的人肠上皮细胞（IEC）。这种方法将使我们能够询问这些自噬基因的功能以及CD相关突变对人类细胞自噬的影响。自噬是一种细胞过程，涉及自噬体中胞质物质的隔离以及与溶酶体融合后其内容物的破坏。因此，细胞在营养缺乏期间利用自噬从细胞器中回收氨基酸。自噬还涉及大蛋白或蛋白复合物的周转、细胞死亡和抗原呈递。先天免疫信号调节自噬，相反，自噬可以调节细胞对炎症信号的反应。最后，自噬是防御或对抗细胞内病原体所必需的，包括分枝杆菌、沙门氏菌、李斯特菌和许多其他微生物。任何或所有这些过程的改变可能解释GWS中鉴定的自噬基因突变与CD易感性之间的功能联系。我们的假设是ATG 16 L1和IRGM是人类细胞自噬所必需的，CD相关的自噬基因突变改变了自噬和IEC对细胞内细菌感染的易感性。我们将通过体细胞基因靶向产生ATG 16 L1 +/-、ATG 16 L1-/-和ATG 16 L1 T300 A以及IRGM-/-和IRGM 20 kB人细胞来测试这一点，并使用这些细胞来解决以下具体目标，即：（1）确定自噬突变对人细胞对应激和促炎配体的反应的影响;（2）检查自噬突变对人类细胞中先天免疫信号传导的影响，以及（3）确定自噬突变对人类细胞中细菌感染和清除的影响。这些研究将迅速提供关于人ATG 16 L1和IRGM基因中CD相关突变是否具有影响代谢过程、先天免疫应答或导致CD的宿主-病原体相互作用的功能后果的关键信息。公共卫生相关性：已经发现了许多增加克罗恩病易感性的新基因，现在的挑战是确定这些基因的作用以及这些基因的突变如何影响人类细胞。我们正在创造具有这些突变的人类肠道细胞，并研究这些突变如何影响对饥饿和细菌感染的反应。这些研究将帮助我们了解克罗恩病中涉及的突变如何影响细胞功能以促进炎症。