Retrotranslocation of cholera toxin AI-chain from ER ot cytosol and cell response

描述(由申请人提供)：本申请的首要目标是阐明霍乱毒素(CT)A1链从宿主细胞内质网(ER)管腔内逆转移位到胞浆中的分子基础。第二个目标是确定宿主细胞是否天生感觉到内质网中的毒素或在逆转位过程中，从而诱导炎症或其他宿主防御反应。霍乱弧菌产生的CT穿过肠道上皮屏障，进入宿主上皮细胞致病。CT属于AB类毒素家族，其中A组分具有酶活性，B组分负责结合细胞膜并调节毒素进入细胞；从质膜进入高尔基体和内质网。在到达内质网后，A-亚基的一部分被内质网伴侣PDI解离并从B-亚基解离，靶向蛋白质传导通道，并运输到胞浆。志贺毒素和蓖麻毒素遵循相似的途径。这些毒素中的每一种在结构上都进化成利用内质网的正常质量控制功能，该功能识别和降解生物合成途径中末端错误折叠的蛋白质；这个过程被称为内质网相关降解(ERAD)或反向易位。在目标1中，我们将研究追溯易位反应的三个步骤：将PDI-A1链复合体靶向内质网腔膜，通过蛋白质传导通道运输A1链，以及这一反应的驱动力。我们的方法将是准备一种突变毒素，预计将被可逆地捕获在完整细胞中作为逆转录转位中间产物，以确定与A1链相互作用的蛋白质；并且可以应用于使用ER蛋白脂质体的无细胞模型中。使用shRNA技术的反向遗传学将被用来测试对某些已知的蛋白质的依赖性，这些蛋白质是逆转移位所必需的。在目标2中，我们将使用缺乏所有酶活性、逆转录易位缺陷或无法与GM1结合的突变毒素来测试CT是否诱导ER应激或其他信号，以感知ER中的细菌蛋白或在增选ERAD的过程中。由于CT是一种重要的细菌因子，在调节肠道生理和适应性粘膜免疫中向宿主发出信号，我们想知道CT在炎症反应中的信号转导是如何解释的。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有关，包括急性感染性腹泻和炎症性肠病。CT是导致亚洲霍乱大规模腹泻的致病因素。霍乱仍在亚洲、非洲和拉丁美洲的许多地区流行，疫情可能发生在受自然灾害、战争和饥荒影响的地区[1]。

DESCRIPTION (provided by applicant): The overarching goal of this application is to elucidate the molecular basis for retro-translocation of the cholera toxin (CT) A1-chain from within the lumen of the endoplasmic reticulum (ER) of host cells to the cytosol. A second goal is to determine if host cells innately sense the toxin in the ER or during the process of retro-translocation so as to induce an inflammatory or other host defense response. CT produced by Vibrio cholerae breeches the intestinal epithelial barrier and enters host epithelial cells to cause disease. CT belongs to the AB family of toxins, where the A component is enzymatically active and the B component is responsible for binding the membrane and mediating entry of the toxin into the cell; traveling from the plasma membrane to the Golgi and ER. After arrival in the ER, a portion of the A-subunit is unfolded and dissociated from the B-subunit by the ER chaperone PDI, targeted to a protein conducting channel, and transported to the cytosol. Shiga toxin and ricin follow a similar pathway. Each of these toxins has evolved structurally to exploit the normal quality control function of the ER that identifies and degrades terminally misfolded proteins in the biosynthetic pathway; the process is termed ER associated degradation (ERAD) or retro-translocation. In Aim 1, we will examine three steps in the retro-translocation reaction: targeting the PDI-A1-chain complex to the ER lumenal membrane, transporting the A1-chain through the protein-conducting channel, and the driving force for this reaction. Our approach will be to prepare a mutant toxin predicted to be reversibly trapped as a retro-translocation intermediate in intact cells so as to identify the proteins that interact with the A1-chain; and that can be applied in a cell-free model using ER proteoliposomes. Reverse genetics using shRNA technology will be used to test dependence on certain proteins known to be required for retro-translocation. In Aim 2, we will use mutant toxins lacking all enzymatic activity, deficient in retrotranslocation, or unable to bind GM1 to test if CT induces ER-stress or some other signal for sensing the bacterial protein in the ER or in the process of co-opting ERAD. Because CT is a prominent bacterial factor that signals the host in regulating intestinal physiology and adaptive mucosal immunity, we want to know how signal transduction by CT in the inflammatory response is explained. The significance of these studies pertains to their relevance to epithelial mucosal biology and a broad range of clinically important diseases, including acute infectious diarrheas and inflammatory bowel disease. CT is the virulence factor responsible for the massive diarrhea seen in Asiatic cholera. Cholera remains prevalent in many parts of Asia, Africa and Latin America and outbreaks can occur in areas affected by natural disasters, wars and famines [1].