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）腔内逆向易位至胞质溶胶的分子基础。第二个目标是确定宿主细胞是否在内质网中或在逆转位过程中天生地感知毒素，从而诱导炎症或其他宿主防御反应。霍乱弧菌产生的霍乱毒素突破肠上皮屏障，进入宿主上皮细胞引起疾病。CT属于毒素的AB家族，其中A组分具有酶活性，而B组分负责结合膜并介导毒素进入细胞;从质膜行进到高尔基体和ER。在到达ER后，A亚基的一部分被解折叠并通过ER伴侣PDI与B亚基解离，靶向蛋白质传导通道，并转运至胞质溶胶。滋贺毒素和蓖麻毒素遵循类似的途径。这些毒素中的每一种都在结构上进化以利用ER的正常质量控制功能，其识别并降解生物合成途径中的末端错误折叠蛋白;该过程被称为ER相关降解（ERAD）或逆向易位。在目标1中，我们将研究逆向易位反应中的三个步骤：将PDI-A1链复合物靶向ER内腔膜，通过蛋白质传导通道转运A1链，以及该反应的驱动力。我们的方法将是制备一种突变型毒素，该毒素被预测为在完整细胞中可逆地被捕获为反向易位中间体，以便鉴定与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用于测试对已知逆转录易位所需的某些蛋白质的依赖性。在目标2中，我们将使用缺乏所有酶活性、缺乏逆转录转运或不能结合GM 1的突变毒素来测试CT是否诱导ER应激或一些其他信号，用于感应ER中或在选择ERAD的过程中的细菌蛋白。由于CT是一个突出的细菌因子，在调节肠道生理和适应性粘膜免疫的信号主机，我们想知道如何解释炎症反应中的CT信号转导。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（包括急性感染性肠炎和炎症性肠病）的相关性。CT是引起亚细亚霍乱中大量腹泻的毒力因子。霍乱在亚洲、非洲和拉丁美洲的许多地区仍然流行，在受自然灾害、战争和饥荒影响的地区可能会爆发[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].