Intestinal Disease - enterocyte toxin interaction

肠道疾病 - 肠细胞毒素相互作用

基本信息

批准号：
7792223
负责人：
WAYNE I LENCER
金额：
$ 68.09万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-09-01 至 2011-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7792223
关键词：
Acute Adenylyl Cyclase 9 Affect Affinity Animal Model Bacterial Toxins Binding Biochemical Biological Assay Biology Cardiac Cell Culture Techniques Cell Separation Cell membrane Cell model Cell physiology Cell surface Cells Cellular biology Ceramides Cholera Cholera Toxin Cholesterol Cholesterol Homeostasis Complex Cytosol Destinations Diarrhea Diffusion Disease Embryonic Development Endoplasmic Reticulum Endoplasmic Reticulum Degradation Pathway Enterocytes Epithelial Epithelial Cells Escherichia coli Fourier Transform Ganglioside GM1 Gangliosides Gastrointestinal tract structure Genetic Glycolipids Goals Golgi Apparatus Image In Vitro Intestinal Diseases Intestines Intoxication Laboratories Life Lipids Mammalian Cell Mass Spectrum Analysis Measures Membrane Membrane Lipids Membrane Microdomains Membrane Protein Traffic Membrane Proteins Modeling Molecular Molecular Chaperones Movement Oligosaccharides Pathway interactions Peptide Hydrolases Pertussis Toxin Phenotype Play Polyomavirus Process Protein Isoforms Proteins Reaction Research Personnel Resistance Role Shigella Signal Transduction Simian virus 40 Sorting - Cell Movement Specificity Staging Structure Swelling System Testing Toxic effect Toxin Vertebrates Zebrafish base cell fixing clinically relevant glycolipid receptor immunological synapse in vitro Assay lipid transport macromolecule multicatalytic endopeptidase complex mutant novel positional cloning programs protein complex protein degradation protein misfolding proteoliposomes receptor reconstitution research study retrograde transport trafficking transcytosis

Acute Adenylyl Cyclase 9 Affect Affinity Animal Model Bacterial Toxins Binding Biochemical Biological Assay Biology Cardiac Cell Culture Techniques Cell Separation Cell membrane Cell model Cell physiology Cell surface Cells Cellular biology Ceramides Cholera Cholera Toxin Cholesterol Cholesterol Homeostasis Complex Cytosol Destinations Diarrhea Diffusion Disease Embryonic Development Endoplasmic Reticulum Endoplasmic Reticulum Degradation Pathway Enterocytes Epithelial Epithelial Cells Escherichia coli Fourier Transform Ganglioside GM1 Gangliosides Gastrointestinal tract structure Genetic Glycolipids Goals Golgi Apparatus Image In Vitro Intestinal Diseases Intestines Intoxication Laboratories Life Lipids Mammalian Cell Mass Spectrum Analysis Measures Membrane Membrane Lipids Membrane Microdomains Membrane Protein Traffic Membrane Proteins Modeling Molecular Molecular Chaperones Movement Oligosaccharides Pathway interactions Peptide Hydrolases Pertussis Toxin Phenotype Play Polyomavirus Process Protein Isoforms Proteins Reaction Research Personnel Resistance Role Shigella Signal Transduction Simian virus 40 Sorting - Cell Movement Specificity Staging Structure Swelling System Testing Toxic effect Toxin Vertebrates Zebrafish base cell fixing clinically relevant glycolipid receptor immunological synapse in vitro Assay lipid transport macromolecule multicatalytic endopeptidase complex mutant novel positional cloning programs protein complex protein degradation protein misfolding proteoliposomes receptor reconstitution research study retrograde transport trafficking transcytosis

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项目摘要

DESCRIPTION (provided by applicant): The goal of this application is to elucidate the molecular basis for invasion and intoxication of intestinal cells by cholera toxin (CT). To induce disease, CT (and the other AB5 toxins) must breech the mucosal epithelial barrier that is normally impermeant to macromolecules by passive diffusion. CT does this as a stably folded protein complex by entering the intestinal epithelial cell after co-opting a retrograde lipid trafficking pathway from the plasma membrane (PM), through the trans Golgi, to the endoplasmic reticulum (ER). The pathway from PM to ER is a specific lipid, not a "protein", sorting pathway. It is also a general pathway hijacked by all the AB5 toxins and the polyoma viruses to cause disease. Once in the ER, a portion of CT, the A1 chain, co-opts the cellular machinery that allows terminally misfolded proteins in the secretory pathway to cross the ER membrane for degradation in the cytosol, a process termed retro-translocation. These are fundamental aspects of epithelial cell function that are broadly clinically relevant and poorly understood. In Aim 1, we will use zebrafish in forward and reverse genetic studies to elucidate molecular components involved in CT toxicity. We have recently discovered that zebrafish model all aspects of the cell biology hijacked by CT to cause disease in mammalian cells, including retrograde transport from PM to ER and retro-translocation to the cytosol. In Aim 2, we will use fourier transform mass spectrometry to identify structural isoforms of the glycolipid receptor ganglioside GM1 that explain how the epithelial cell sorts GM1 specifically into the retrograde pathway. We will confirm the identity of these structures using synthetic GM1 isoforms in functional reconstitution experiments. We will use wild-type (wt) and mutant toxins with altered binding function to measure diffusional coefficients and clustering efficiencies of the CT-GM1 complex in the cell membrane. This will test how GM1 may couple the toxin to lipid rafts that appear to function as key trafficking platforms. In Aim 3, we will use wt and a variety of mutant toxins for advanced imaging of live and fixed cells, and for novel biochemical in vitro vesicular transport assays to identify the intracellular compartment(s) and molecular mechanism(s) that sort the CT-GM1 complex away from the other glycolipids (and their cargos) and into the retrograde pathway. Reverse genetics in cell culture will also be used. In Aim 4, we will use reverse genetics in cell culture and in zebrafish, and a novel in vitro protease protection assay based on a mutant toxin containing a cleavable HA-tag. The assay will biochemically model the retro-translocation reaction in order to examine the molecular components essential for this process. The significance of these studies pertains to their relevance to epithelial mucosal biology and a broad range of clinically important diseases. Such diseases are global in distribution and include acute infectious diarrheas as well as those that result from abnormal interactions with the intestinal microflora, such as IBD.

描述（由申请人提供）：本申请的目的是阐明霍乱毒素（CT）侵袭和陶醉肠细胞的分子基础。为了诱导疾病，CT（和其他AB5毒素）必须将粘膜上皮屏障通常通过被动扩散对大分子不透气。 CT通过进入逆行脂质运输途径从质膜（PM），通过反式高尔基人进入内质网（ER）后，将其作为稳定折叠的蛋白质复合物通过进入肠上皮细胞（PM）。从PM到ER的途径是特定的脂质，而不是“蛋白质”，分类途径。它也是所有AB5毒素和多瘤病毒劫持的一般途径，引起疾病。一旦进入ER，A1链的一部分CT选择了细胞机制，该细胞机制允许在分泌途径中终端错误折叠的蛋白质穿越ER膜以在细胞质中降解，这是一种称为retro-translosation的过程。这些是上皮细胞功能的基本方面，它们在临床上广泛相关且知名度不佳。在AIM 1中，我们将在前进和反向遗传研究中使用斑马鱼来阐明与CT毒性有关的分子成分。我们最近发现，斑马鱼模型由CT劫持的细胞生物学的各个方面引起哺乳动物细胞的疾病，包括从PM到ER的逆行转运以及恢复转移到胞质溶胶。在AIM 2中，我们将使用傅立叶变换质谱法来识别糖脂受体神经节GM1的结构同工型，以解释上皮细胞类别GM1如何专门进入逆行途径。我们将使用合成GM1同工型在功能重构实验中确认这些结构的身份。我们将使用具有改变结合功能的野生型（WT）和突变毒素来测量细胞膜中CT-GM1复合物的扩散系数和聚类效率。这将测试GM1如何将毒素与似乎充当关键运输平台起的脂质筏。在AIM 3中，我们将使用WT和各种突变毒素进行活细胞的先进成像，以及用于新型的生化体外体外囊泡传输测定法，以鉴定细胞内室（S）和分子机制，这些机制将CT-GM1与其他糖脂脱离的CT-GM1相分类，并将其分类为其他糖脂（以及其Cargos和其cargos）和cargrode cretrarde cretrograde。还将使用细胞培养中的反向遗传学。在AIM 4中，我们将在细胞培养和斑马鱼中使用反向遗传学，以及一种基于含有可裂解HA-TAG的突变毒素的新型体外蛋白酶保护测定法。该测定将在生化上对复古转换反应进行建模，以检查该过程必不可少的分子成分。这些研究的意义与它们与上皮粘膜生物学的相关性以及广泛的临床重要疾病。这些疾病在分布上是全球性的，包括急性感染性腹泻以及与肠道菌群异常相互作用所致的疾病，例如IBD。