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

描述（由申请人提供）：本申请的总体目标是阐明霍乱毒素（CT） a1链从宿主细胞内质网（ER）腔内逆行转位到细胞质的分子基础。第二个目标是确定宿主细胞是否天生在内质网或在逆转录过程中感知毒素，从而诱导炎症或其他宿主防御反应。霍乱弧菌产生的CT突破肠上皮屏障，进入宿主上皮细胞引起疾病。CT属于毒素AB家族，其中A组分具有酶活性，B组分负责结合膜并介导毒素进入细胞；从质膜到高尔基体和内质网。到达内质网后，一部分a亚基被内质网伴侣PDI展开并与b亚基解离，靶向蛋白质传导通道，并运输到细胞质中。志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，以利用内质网的正常质量控制功能，识别和降解生物合成途径中最终错误折叠的蛋白质；这个过程被称为内质网相关降解（ERAD）或逆转录易位。在Aim 1中，我们将研究逆转录易位反应的三个步骤：将pdi - a1链复合物靶向到内质网管膜，通过蛋白质传导通道运输a1链，以及该反应的驱动力。我们的方法是制备一种突变毒素，该毒素被预测为在完整细胞中作为逆转录易位中间体被可逆捕获，以便识别与a1链相互作用的蛋白质；并且可以应用于使用内质网蛋白脂质体的无细胞模型。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Aim 2中，我们将使用缺乏所有酶活性、缺乏逆转录易位或无法结合GM1的突变毒素来测试CT是否诱导内质网应激或其他信号，以感知内质网中的细菌蛋白或在选择ERAD的过程中。由于CT是向宿主发出信号调节肠道生理和适应性粘膜免疫的重要细菌因子，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的相关性，包括急性感染性腹泻和炎症性肠病。CT是引起亚细亚霍乱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发生疫情。

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].