Mechanism of cholera toxin retro-translocation

描述（由申请人提供）：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性的健康问题，阐明其基本的作用机制至关重要。为了使细胞中毒，CT从细胞表面转运到内质网（ER）的内腔。在这个区室中，CT的毒性CTA 1片段伪装成错误折叠的蛋白质，并劫持了通常将错误折叠的蛋白质从ER移动到胞质溶胶中以被蛋白酶体降解的细胞机器。然而，在到达细胞质时，CTA 1逃避蛋白酶体破坏并触发导致病理性水分泌（即腹泻）的信号级联，这在严重情况下可导致死亡。CTA 1是如何从内质网转移到胞质溶胶中的，这是一个决定性的中毒步骤，目前仍知之甚少。在本申请中，我们打算通过使用生物化学和细胞生物学方法的组合来解决这个问题。从历史上看，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，这些发现往往导致确定适合治疗干预的关键分子靶点。因此，我们预计，我们的研究结果可能揭示蛋白质跨生物膜转运的新机制，并确定可能作为可行的治疗靶点的新细胞因子。此外，由于其他毒素，如蓖麻毒素和滋贺毒素也经历ER-细胞质运输诱导细胞毒性，我们的研究结果应该提供深入了解其作用机制以及。 公共卫生相关性：霍乱毒素（CT）导致动物病理性水分泌（即腹泻），严重时可导致死亡。毒素依赖性感染过程中的决定性步骤是毒素穿过称为内质网（ER）的亚细胞区室的膜的运输。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阐明这些过程。

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.