Membrane biophysics of enterotoxin mediated immunomodulation

描述（由申请方提供）：我们对肠毒素LT-II与细胞膜中的神经节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）的理解存在根本性空白。有必要解决这一差距，以了解和进一步使用这些分子作为佐剂，以加强细胞免疫应答。这些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微结构的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膜蛋白和胆固醇依赖性和非依赖性膜结构域，或纳米簇之间的相互作用的影响。基于初步数据，我们的假设是，佐剂结合神经节苷脂增加胆固醇依赖性纳米簇的稳定性，或与这些簇的受体蛋白的协会。提出的研究的基本原理是，膜超微结构的变化对细胞信号传导的影响的生物物理模型将解释免疫信号传导的佐剂增强。将通过定量肠毒素对免疫信号蛋白的大小、稳定性和与脂质和蛋白质膜结构域以及膜细胞骨架的结合的影响，以及通过确定哪些膜结合免疫信号过程受肠毒素结合的影响，来检验这一假设。 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，使我们能够在完整的细胞中连续定量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。我们的方法将使第一个实时定量肠毒素结合诱导的膜结构调制。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（例如细胞信号传导）的关系。通过建立这种结构和功能变化之间的直接联系，以响应外部扰动，将实现对功能的结构的作用的理解。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理解，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的膜结构，如脂质结构域和膜细胞骨架的调节的理解，并将阐明它们在自然免疫功能中的作用。 公共卫生相关性：揭示肠毒素样制剂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的实验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。该项目的更广泛的影响将是对调节膜结构的试剂如何影响细胞信号传导的更好理解。因此，拟议的研究与NIH的使命有关，该使命涉及发展基础知识，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.