Membrane biophysics of enterotoxin mediated immunomodulation

描述（由申请人提供）：我们对肠毒素LT-II如何与细胞膜中的英脂苷结合调节免疫细胞膜的结构和功能（例如膜相关信号传导）的理解存在根本差距。有必要解决这一差距，以便了解和进一步使用这些分子作为佐剂来增强细胞免疫反应。这类佐剂是疫苗的基本成分。我们的长期目标是在分子水平上了解细胞膜超结构变化对免疫细胞膜信号传导的影响。本提案的目的是确定佐剂肠毒素LT-II如何改变膜超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和独立膜结构域或纳米簇之间相互作用的影响。根据初步数据，我们的假设是，与神经节苷脂结合的佐剂增加了胆固醇依赖性纳米簇的稳定性，或者受体蛋白与这些簇的关联。提出的研究的基本原理是，膜超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的佐剂增强。这一假设将通过量化肠毒素对免疫信号蛋白与脂质和蛋白膜结构域以及膜细胞骨架的大小、稳定性和关联的影响来验证；并通过确定哪些膜结合免疫信号过程受到肠毒素结合的影响。我们的方法是非常创新和新颖的：我们将建立先进的成像方法，使我们能够量化膜蛋白与胆固醇依赖性和非依赖性纳米团簇和完整细胞中细胞骨架的相互作用。我们的方法将首次实现肠毒素结合引起的膜结构调节的实时定量。此外，我们的研究特别适合于确定膜结构变化与膜功能调节的关系，例如细胞信号传导。通过建立响应外部扰动的结构和功能变化之间的直接联系，将实现对结构对功能的作用的理解。这项研究具有重要意义，因为它将增强我们对肠毒素类佐剂作用的基本分子认识，从而促进进一步研究优化所需的免疫增强功能。此外，我们的研究结果将扩大目前对膜结构（如脂质结构域和膜细胞骨架）调控的理解，并将阐明它们在自然免疫功能中的作用。

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.