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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）侵入和中毒肠细胞的分子基础。为了诱发疾病，CT（和其他AB 5毒素）必须通过被动扩散突破通常对大分子不渗透的粘膜上皮屏障。CT作为一种稳定折叠的蛋白质复合物，通过从质膜（PM）通过反式高尔基体（transGolgi）到内质网（ER）的逆行脂质运输途径进入肠上皮细胞来实现这一点。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AB 5毒素和多瘤病毒劫持导致疾病的一般途径。一旦进入ER，CT的一部分，即A1链，就会与细胞机制结合，使分泌途径中的末端错误折叠蛋白质穿过ER膜，在胞质溶胶中降解，这一过程称为逆向易位。这些都是上皮细胞功能的基本方面，广泛的临床相关性和知之甚少。在目标1中，我们将使用斑马鱼在正向和反向遗传学研究，以阐明参与CT毒性的分子成分。我们最近发现，斑马鱼模型的所有方面的细胞生物学劫持CT引起的疾病，在哺乳动物细胞，包括逆行运输从PM到ER和逆向易位到胞质溶胶。在目标2中，我们将使用傅立叶变换质谱法来鉴定糖脂受体神经节苷脂GM 1的结构异构体，以解释上皮细胞如何将GM 1特异性地分类到逆行途径中。我们将在功能重建实验中使用合成的GM 1亚型来确认这些结构的同一性。我们将使用野生型（wt）和突变体毒素改变结合功能，以测量扩散系数和细胞膜中的CT-GM 1复合物的聚集效率。这将测试GM 1如何将毒素偶联到脂筏上，脂筏似乎是关键的运输平台。在目标3中，我们将使用野生型和各种突变毒素对活细胞和固定细胞进行先进成像，并用于新型生物化学体外囊泡转运试验，以鉴定将CT-GM 1复合物从其他糖脂（及其货物）中分离出来并进入逆行途径的细胞内隔室和分子机制。还将使用细胞培养中的反向遗传学。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，以及基于含有可切割HA标签的突变毒素的新型体外蛋白酶保护测定。该测定将对逆易位反应进行生化建模，以检查该过程所必需的分子组分。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。这些疾病是全球性分布的，包括急性传染性腹泻以及与肠道微生物菌群异常相互作用引起的疾病，如IBD。