Mechanism of cholera toxin retro-translocation

描述(申请人提供)：细菌毒素是多种人类疾病的病原体。然而，在许多情况下，感染的分子基础仍然是个谜。霍乱弧菌产生的霍乱毒素(CT)是引起大量分泌性腹泻的毒力因子。由于这种疾病仍然是一个全球健康问题，阐明其基本作用机制至关重要。为了使细胞中毒，CT被从细胞表面运输到内质网(ER)的管腔。在这个隔间里，CT的有毒CTA1片段伪装成错误折叠的蛋白质，劫持了正常情况下将错误折叠的蛋白质从内质网转移到细胞质中由蛋白酶体降解的细胞机械。然而，一旦到达细胞质，CTA1就会逃脱蛋白酶体的破坏，并触发一系列信号级联反应，导致病理性水分泌(即腹泻)，在严重情况下可导致死亡。CTA1是如何从内质网转移到胞浆中的，这是一个决定性的醉人步骤，目前仍然知之甚少。在这项应用中，我们打算通过结合使用生化和细胞生物学的方法来解决这个问题。在历史上，对病原体-宿主细胞相互作用的研究阐述了基本的细胞过程。此外，这些发现经常导致确定可用于治疗干预的关键分子靶点。因此，我们预计，我们的发现可能会揭示蛋白质通过生物膜运输的新机制，并识别可能作为可行治疗靶点的新细胞因子。此外，由于其他毒素，如蓖麻毒素和志贺毒素也通过内质网到胞浆的转运来诱导细胞毒性，我们的结果也应该为深入了解它们的作用机制提供依据。 与公共卫生相关：霍乱毒素(CT)可引起动物的病理性水分泌(即腹泻)，严重时可导致死亡。毒素依赖感染过程中的一个决定性步骤是毒素通过被称为内质网(ER)的亚细胞室的膜运输。然而，CT穿透ER膜的分子细节尚不清楚。我们打算在本申请中澄清这些流程。

DESCRIPTION (provided by applicant): Bacterial toxins are the causative agents for a variety of human diseases. However, the molecular basis of infection, in many cases, remains enigmatic. Cholera toxin (CT) produced by Vibrio cholerae is the virulence factor responsible for massive secretory diarrhea. As this disease remains a global health issue, elucidating its basic mechanism of action is paramount. To intoxicate cells, CT is transported from the cell surface to the lumen of the endoplasmic reticulum (ER). In this compartment, the toxic CTA1 fragment of CT disguises as a misfolded protein and hijacks the cellular machinery that normally moves misfolded proteins from the ER into the cytosol for degradation by the proteasome. Upon reaching the cytosol, CTA1 however escapes proteasomal destruction and triggers a signaling cascade that leads to pathologic water secretion (i.e. diarrhea), which can lead to death in severe cases. How CTA1 is transferred from the ER into the cytosol, a decisive intoxication step, remains poorly understood. In this application, we intend to address this question by using a combination of biochemical and cell biological approaches. Historically, studies on pathogen-host cell interactions have expounded on basic cellular processes. Moreover, these findings often led to the identification of key molecular targets amenable for therapeutic intervention. Thus we anticipate that our findings are likely to reveal novel mechanisms of protein transport across biological membranes and to identify new cellular factors that may serve as viable therapeutic targets. In addition, as other toxins such as ricin and shiga toxin also undergo ER-to-cytosol transport to induce cytotoxicity, our results should provide insights into their mechanism of action as well. PUBLIC HEALTH RELEVANCE: Cholera toxin (CT) causes pathologic water secretion (i.e. diarrhea) in animals, which can lead to death in severe cases. A decisive step in the toxin-dependent infection process is transport of the toxin across the membrane of a sub-cellular compartment known as the endoplasmic reticulum (ER). However, the molecular details by which CT penetrate the ER membrane is not clear. We intend to clarify these processes in this application.