Multiplexed Platform to Probe Interactions at the Model Cell Membrane Interface

用于探测模型细胞膜界面相互作用的多重平台

• 批准号: 8841783
• 负责人: Ryan C Bailey
• 金额: $ 29.88万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8841783
• 关键词: Affinity; Antibodies; Binding; Biochemical; Biological; Biological Assay; Biological Process; Blood Coagulation Factor; Blood coagulation; Cations; Cell membrane; Coagulation Process; Complex; DNA; Dependence; Development; Disease; Electrostatics; Ensure; Equilibrium; Ethanolamines; Factor VIIa; Health; Hemorrhage; Ions; Kinetics; Label; Lecithin; Life; Ligands; Light; Lipid Bilayers; Lipids; Mediating; Mediation; Mediator of activation protein; Membrane; Membrane Lipids; Membrane Proteins; Metals; Methods; Modality; Modeling; Monitor; Motivation; Nucleic Acids; Oxides; Phase; Phosphatidic Acid; Phosphatidylserines; Phospholipids; Polymers; Process; Protein C Inhibitor; Proteins; Prothrombin; Sampling; Signal Pathway; Signal Transduction; Silicon; Solutions; Surface; Technology; Therapeutic Agents; Therapeutic Intervention; Thrombosis; Time; Virus Diseases; Work; base; blood coagulation process; divalent metal; improved; metal complex; nanodisk; novel; novel therapeutics; photonics; receptor binding; response; screening; self assembly; sensor

DESCRIPTION (provided by applicant): Soluble proteins are key mediators of many biochemical signaling pathways via direct interaction with the lipid bilayer and via membrane-bound receptors. Components of the cell membrane are involved in many important biological processes including blood coagulation, viral infection, and signal transduction, and as such, they are common targets of therapeutic agents. Therefore, the development of analytical approaches to study interactions at the cell membrane is of critical importance. This proposal integrates two key technologies, silicon photonic microring resonator arrays and phospholipid bilayer Nanodiscs, which together allow multiplexed screening of soluble protein interactions with lipid and membrane-embedded targets. Microring resonator arrays are an intrinsically multiplexable, label-free analysis platform that has previously been applied to studying protein-protein, protein-nucleic acid, and nucleic acid-nucleic acid interactions. Nanodiscs are protein-stabilized lipid assemblies that represent a convenient construct to mimic the native phospholipid bilayer, investigate the effects of membrane composition, and solubilize membrane-embedded targets. Fused together, these technologies will offer an unsurpassed capacity to interrogate the biophysical modalities of membrane-ligand interactions in high-throughput, multiplexed, and information rich assay formats. The biological motivation for the proposed work is blood coagulation, which is a critical regulatory process that is a target for therapeutic intervention i thrombosis and bleeding disorders. In addition to protein- membrane protein interactions, the assembly of coagulation factors at the cell membrane surface is additionally regulated by protein-lipid interactions, and in particular interactions with anionic lipids that are critically-dependent upon divalent metal ions. However many details of protein-lipid interactions in blood clotting remain poorly understood, and an improved fundamental understanding might reveal new therapeutic strategies for life-threatening thrombotic diseases. Herein we propose to shed light onto complex lipid- and cation-dependent binding of blood coagulation factors (Factors VIIa and X; fVIIa and fX, respectively) and clotting inhibitors (activated protein C; aPC) through the use of multiplexed arrays of microring resonators functionalized with Nanodiscs that present variable, yet well-defined lipid composition. We also will implement a novel solution-phase gradient maker to dynamically control the concentration and identity of metal cations in solution while simultaneously monitoring the effects of these changes on protein-lipid interactions in real time. Together, these studies will provide unprecedented access to high throughput and multivariate interrogation of blood coagulation processes at model interfaces, and more generally will validate the integration of Nanodiscs and microring resonator arrays for highly multiplexed studies of interactions at the cell membrane.

描述（由申请人提供）：可溶性蛋白质通过与脂质双层的直接相互作用以及通过膜结合受体，是许多生化信号传导途径的关键介质。细胞膜的成分参与许多重要的生物过程，包括凝血、病毒感染和信号转导，因此它们是治疗药物的常见靶点。因此，开发研究细胞膜相互作用的分析方法至关重要。该提案集成了硅光子微环谐振器阵列和磷脂双层纳米圆盘这两项关键技术，它们共同允许对可溶性蛋白质与脂质和膜嵌入靶标的相互作用进行多重筛选。微环谐振器阵列是一种本质上可复用、无标记的分析平台，之前已应用于研究蛋白质-蛋白质、蛋白质-核酸和核酸-核酸相互作用。纳米圆盘是蛋白质稳定的脂质组件，代表了一种模拟天然磷脂双层、研究膜组成的影响以及溶解膜嵌入靶标的便捷结构。这些技术融合在一起，将提供无与伦比的能力，以高通量、多重且信息丰富的检测形式探究膜-配体相互作用的生物物理模式。 这项工作的生物学动机是凝血，这是一个关键的调节过程，是血栓形成和出血性疾病治疗干预的目标。除了蛋白质-膜蛋白质相互作用之外，凝血因子在细胞膜表面的组装还受到蛋白质-脂质相互作用的调节，特别是与严重依赖于二价金属离子的阴离子脂质的相互作用。然而，人们对血液凝固中蛋白质-脂质相互作用的许多细节仍知之甚少，而基础知识的改进可能会揭示危及生命的血栓性疾病的新治疗策略。 在此，我们建议通过使用微环谐振器的多路复用阵列来阐明凝血因子（分别为因子VIIa和X；fVIIa和fX）和凝血抑制剂（活化蛋白C；aPC）的复杂脂质和阳离子依赖性结合用纳米圆盘进行功能化，呈现可变但明确的脂质成分。我们还将实施一种新型溶液相梯度发生器，以动态控制溶液中金属阳离子的浓度和特性，同时实时监测这些变化对蛋白质-脂质相互作用的影响。总之，这些研究将为模型接口处的血液凝固过程的高通量和多变量询问提供前所未有的途径，并且更广泛地将验证纳米圆盘和微环谐振器阵列的集成，以用于细胞膜相互作用的高度多重研究。