CAREER: Investigating the Molecular Factors that Underlie Myelin-Associated Glycoprotein Binding

生物分子之间的相互作用支配着所有的生命维持过程。这个项目将研究与构成神经系统细胞边界的类脂膜相关的生物分子之间的相互作用。胆固醇是脂膜的主要成分，它可以改变其他膜相关分子的结构和功能，如脂类、碳水化合物和蛋白质。具体地说，这个项目将研究胆固醇如何改变含有碳水化合物的脂类与调节神经系统中细胞与细胞相互作用的蛋白质的结合。处于这些研究中心的蛋白质，髓鞘相关糖蛋白(MAG)，是神经元生长的抑制因子，确定其相互作用受胆固醇和与其聚集的蛋白质调节的分子因素将阐明其作用机制。与研究活动相配合，PI将设计教学实验室模块，演示脂类和膜的性质和分析(脂质体表征、支持膜形成、分子扩散)、生物传感器和荧光显微镜。此外，还将开发一门关于生物传感器的研究生高级课程，以将该项目的影响范围扩大到课堂和实验室。本课程将在很大程度上依赖于主动学习教学方法的使用，它将使化学、生物化学、生物学和工程学学生接触到可用于检测和表征生物分子的各种技术。本项目的总体目标是通过定量分析测量来确定调节生物分子相互作用的因素，这些因素导致脂膜相互粘连。该项目的具体重点是定量分析一种膜上的MAG与其神经节苷脂和另一种膜上的蛋白质受体之间的相互作用。MAG是一种唾液酸结合的免疫球蛋白型凝集素(Siglec)，发现于神经系统的髓鞘上，其功能之一是维持髓鞘和轴突之间的粘附性相互作用。MAG与神经节苷脂(GD1a和GT1b)和神经元上Nogo受体1(NgR1)的结合抑制轴突生长，是神经系统修复的重要障碍。该项目的第一个目标将确定胆固醇如何调节MAG-神经节苷脂结合事件及其引起的膜-膜相互作用。第二个目标将确定膜相分离成类脂筏结构域如何影响MAG-神经节苷脂相互作用的空间和时间方面。最终目标将确定MAG受体复合体(NgR1/p75/LINGO-1)的化学计量比如何影响MAG的结合亲和力，从而影响膜-膜相互作用。抗LINGO-1抗体破坏MAG-NgR1相互作用的机制也将被确定。该项目将邀请本科生和研究生参与设计、执行和解释通过石英晶体微天平(QCM-D)生物传感、Förster共振能量转移(FRET)、总内反射荧光(TIRF)显微镜和平衡波动分析获得的数据。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Interactions between biomolecules govern all life sustaining processes. This project will investigate the interactions between biomolecules that are associated with the lipid membranes that form the boundaries of cells in the nervous system. Cholesterol is a major component of the lipid membranes, and it can alter the structure and function of other membrane-associated molecules, like lipids, carbohydrates, and proteins. Specifically, this project will investigate how cholesterol alters the binding of lipids that contain carbohydrates to proteins that regulate cell-cell interactions in the nervous system. The protein that is at the center of these studies, myelin-associated glycoprotein (MAG), is an inhibitor of neuron growth, and determining the molecular factors by which its interactions are modulated by cholesterol and the proteins it clusters with will illuminate the mechanisms through which it acts. Synergistically with research activities, the PI will design teaching laboratory modules that demonstrate the properties and analysis of lipids and membranes (liposome characterization, supported membrane formation, molecular diffusion), biosensors, and fluorescence microscopy. Additionally, an advanced graduate level course on biosensors will be developed to expand the reach of this project to the classroom as well as the laboratory. This course will rely heavily on the use of active learning teaching methods, and it will expose chemistry, biochemistry, biology, and engineering students to the wide variety of techniques that can be used to detect and characterize biomolecules.The overarching goal of this project is to determine, through quantitative analytical measurements, the factors that modulate biomolecular interactions responsible for the adhesion of lipid membranes to one another. The specific focus of the project is on the quantitative analysis of interactions between MAG on one membrane and its ganglioside and protein receptors on another. MAG is a sialic acid-binding immunoglobulin-type lectin (Siglec) found in the nervous system on myelin where one of its functions is to maintain adhesive interactions between myelin and axon membranes. Biding of MAG to gangliosides (GD1a and GT1b) and Nogo receptor 1 (NgR1) on neurons inhibits axonal growth and is a significant obstacle to nervous system repair. The first aim of this project will determine how cholesterol modulates MAG-ganglioside binding events and the membrane-membrane interactions they cause. The second aim will determine how membrane phase separation into lipid raft-like domains influences spatial and temporal aspects of MAG-ganglioside interactions. The final aim will determine how the stoichiometry of the MAG receptor complex (NgR1/p75/LINGO-1) influences the MAG’s binding affinity and thus membrane-membrane interactions. The mechanism by which anti-LINGO-1 antibodies disrupt MAG-NgR1 interactions will also be determined. This project will involve undergraduate and graduate students in the design, execution, and interpretation of data obtained with quartz crystal microbalance (QCM-D) biosensing, Förster resonance energy transfer (FRET), total internal refection fluorescence (TIRF) microscopy, and equilibrium fluctuation analysis.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.