Intestinal Disease - enterocyte toxin interaction

描述（由申请人提供）：本申请的目的是阐明霍乱毒素（CT）侵袭和中毒肠细胞的分子基础。为了诱发疾病，CT（和其他AB5毒素）必须突破粘膜上皮屏障，而这通常是大分子无法通过被动扩散的。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，到内质网（ER）的逆行脂质运输途径进入肠上皮细胞。从PM到ER的途径是一个特定的脂质分选途径，而不是一个“蛋白质”分选途径。它也是被所有AB5毒素和多瘤病毒劫持并导致疾病的一般途径。一旦进入内质网，一部分CT， 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，在细胞质中降解，这一过程被称为逆转录易位。这些是上皮细胞功能的基本方面，与临床广泛相关，但知之甚少。

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.