Membrane biophysics of enterotoxin mediated immunomodulation

描述（由申请人提供）：我们对肠毒素 LT-II 与细胞膜中的血管苷脂结合如何调节免疫细胞膜的结构和功能（例如膜相关信号传导）的理解存在根本性差距。有必要解决这一空白，以便了解并进一步使用这些分子作为佐剂来增强细胞免疫反应。此类佐剂试剂是疫苗的重要组成部分。我们的长期目标是在分子水平上了解细胞膜超微结构的变化对免疫细胞膜信号传导的影响。该提案的目的是确定佐剂肠毒素 LT-II 如何改变膜超微结构以影响膜信号传导。特别是，我们的目标是确定肠毒素结合对膜蛋白与胆固醇依赖性和非依赖性膜结构域或纳米簇之间相互作用的影响。基于初步数据，我们的假设是佐剂与神经节苷脂的结合增加了胆固醇依赖性纳米簇的稳定性，或受体蛋白与这些簇的关联。该研究的基本原理是膜超微结构变化对细胞信号传导影响的生物物理模型将解释免疫信号传导的佐剂增强作用。该假设将通过量化肠毒素对免疫信号蛋白的大小、稳定性以及与脂质和蛋白质膜结构域以及膜细胞骨架的关联的影响来检验；并确定哪些膜结合免疫信号过程受到肠毒素结合的影响。 我们的方法极具创新性和新颖性：我们将建立先进的成像方法，使我们能够连续量化完整细胞中膜蛋白与胆固醇依赖性和非依赖性纳米簇和细胞骨架的相互作用。我们的方法将首次对肠毒素结合诱导的膜结构调节进行实时定量。此外，我们的研究特别适合确定膜结构变化与膜功能调节的关系，例如膜功能的调节。细胞信号传导。通过在结构和功能变化之间建立这种响应外部扰动的直接关联，将实现对结构对功能的作用的理解。这项研究意义重大，因为它将增强我们对肠毒素类佐剂作用的基本分子理解，从而有助于进一步研究优化所需的免疫增强功能。此外，我们的结果将扩大目前对脂质结构域和膜细胞骨架等膜结构调节的理解，并将阐明它们在自然免疫功能中的作用。 公共卫生相关性：通过实验揭示肠毒素类药物调节膜超微结构的分子过程以及这些变化影响免疫信号传导的机制，将指导未来免疫佐剂的设计，这将与公共卫生问题直接相关。该项目更广泛的影响将是加深对调节膜结构的药物如何影响细胞信号传导的理解。因此，拟议的研究与 NIH 的使命相关，即发展基础知识以帮助保护和改善人类健康。

DESCRIPTION (provided by applicant): There is a fundamental gap in our understanding of how Enterotoxin LT-II binding to angliosides in the cell membrane modulates the structure and function (e.g. membrane-associated signaling) of the immune-cell membrane. It is necessary to address this gap in order to understand and further the use of these molecules as adjuvants to potentiate the cellular immune response. Such adjuvant reagents are essential components of vaccines. Our long-term goal is to understand the impact of changes in cell membrane ultra-structure on immune-cell membrane signaling at the molecular level. The objective of this proposal is to determine how the adjuvant Enterotoxin LT-II alters the membrane ultra-structure to influence membrane signaling. In particular, we aim to determine the effects of Enterotoxin binding on interactions between membrane proteins and cholesterol-dependent and -independent membrane domains, or nanoclusters. Based on preliminary data, our hypothesis is that adjuvant binding to gangliosides increases the stability of cholesterol-dependent nanoclusters, or the association of the receptor proteins with these clusters. The rationale for the proposed research is that a biophysical model of the influence of changes in membrane ultra-structure on cellular signaling will explain adjuvant potentiation of immune signaling. This hypothesis will be tested by quantifying the effects of Enterotoxin on the size, stability, and association of immune signaling proteins with lipid- and protein-membrane domains, and the membrane cytoskeleton; and by indentifying which membrane bound immune signaling processes are influenced by Enterotoxin binding. Our approach is extremely innovative and novel: We will establish advanced imaging methods which allow us to quantify membrane protein interaction with cholesterol-dependent and -independent nanoclusters and cytoskeleton continuously in intact cells. Our methods will enable a first real-time quantification of membrane structure modulation induced by enterotoxin binding. In addition, our study is uniquely suited to identify the relationship of changes in membrane structure to modulation of membrane function, e.g. cell signaling. By establishing this direct association between structural and functional changes in response to external perturbation will achieve an understanding of the role of the structure for function. The proposed research is significant because it will enhance our basic molecular understanding of the effect of enterotoxin like adjuvants, thus facilitating further investigation into optimizing the desired immune potentiating function. In addition, our results will expand the current understanding of the regulation of membrane structures such as lipid domains and the membrane cytoskeleton, and will elucidate their roles in natural immune function. PUBLIC HEALTH RELEVANCE: Experiments to reveal the molecular processes by which enterotoxin-like agents modulate membrane ultra-structure and the mechanisms by which these changes influence immune signaling will guide the design of future immune adjuvants, which will have direct relevance to issues of public health. The broader impact of this project will be an improved understanding of how agents that modulate membrane structure influence cell signaling. Thus, the proposed research is relevant to NIH's mission that pertains to developing fundamental knowledge to help to protect and improve human health.