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：
7385114
负责人：
WAYNE I LENCER
金额：
$ 64.83万
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/7385114
关键词：
Acute Adenylyl Cyclase 9 Affect Affinity Animal Model Bacterial Toxins Binding Biochemical Biological Assay Biology Cardiac Cell Separation Cell membrane Cell model Cell physiology Cell surface Cells Cellular biology Ceramides Cholera Cholera Toxin Cholesterol Cholesterol Homeostasis Complex Cultured Cells Cytosol Destinations Diarrhea Diffusion Disease Embryonic Development Endopeptidases Endoplasmic Reticulum Endoplasmic Reticulum Degradation Pathway Enterocytes Epithelial Epithelial Cells Escherichia coli Fourier Transform Ganglioside GM1 Gangliosides Gastrointestinal tract structure Genetic Models 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 Protein Sortings Proteins Range 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 degradation protein folding protein misfolding proteoliposomes receptor reconstitution research study retrograde transport trafficking transcytosis

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）入侵和中毒肠道细胞的分子基础。为了诱发疾病，CT（和其他 AB5 毒素）必须突破粘膜上皮屏障，而粘膜上皮屏障通常无法通过被动扩散渗透到大分子中。 CT 通过选择从质膜 (PM)、反式高尔基体到内质网 (ER) 的逆行脂质运输途径，进入肠上皮细胞，以稳定折叠的蛋白质复合物的形式实现这一点。从 PM 到 ER 的途径是一种特定的脂质，而不是“蛋白质”分选途径。它也是所有 AB5 毒素和多瘤病毒劫持导致疾病的通用途径。一旦进入内质网，CT 的一部分（即 A1 链）就会选择细胞机制，允许分泌途径中最终错误折叠的蛋白质穿过内质网膜，在胞质溶胶中降解，这一过程称为逆向易位。这些是上皮细胞功能的基本方面，与临床广泛相关，但人们了解甚少。在目标 1 中，我们将使用斑马鱼进行正向和反向遗传研究，以阐明 CT 毒性所涉及的分子成分。我们最近发现，斑马鱼模拟了被 CT 劫持导致哺乳动物细胞疾病的细胞生物学的各个方面，包括从 PM 逆行转运到 ER 以及逆向易位到胞质溶胶。在目标 2 中，我们将使用傅里叶变换质谱法来鉴定糖脂受体神经节苷脂 GM1 的结构异构体，从而解释上皮细胞如何将 GM1 特异性分类到逆行途径中。我们将在功能重建实验中使用合成的 GM1 同工型来确认这些结构的身份。我们将使用结合功能改变的野生型 (wt) 和突变型毒素来测量 CT-GM1 复合物在细胞膜中的扩散系数和聚集效率。这将测试 GM1 如何将毒素与脂筏偶联，脂筏似乎充当关键的运输平台。在目标 3 中，我们将使用 wt 和各种突变毒素对活细胞和固定细胞进行高级成像，并进行新型生化体外囊泡转运测定，以识别细胞内区室和对 CT 进行分类的分子机制-GM1复合物远离其他糖脂（及其货物）并进入逆行途径。细胞培养中的反向遗传学也将被使用。在目标 4 中，我们将在细胞培养和斑马鱼中使用反向遗传学，以及基于含有可裂解 HA 标签的突变毒素的新型体外蛋白酶保护测定。该测定将对逆向易位反应进行生化建模，以检查该过程所必需的分子成分。这些研究的意义在于它们与上皮粘膜生物学和广泛的临床重要疾病的相关性。此类疾病在全球范围内分布，包括急性感染性腹泻以及因与肠道菌群异常相互作用而导致的疾病，例如IBD。