Dynamics of LCAT activation and lipoprotein remodeling

描述（由申请人提供）：高密度脂蛋白（HDL）是心血管疾病（CVD）药物治疗的有前途的靶点。无论HDL本身是否直接预防CVD或作为保护性蛋白或抗氧化蛋白附着的平台，HDL结构的知识都很重要。当前提议的目标是使用直接实验方法和计算机模拟的协同组合来理解载脂蛋白A-I（apoA-I）动力学在HDL的两个重要生物学功能中的作用：i）酶卵磷脂：胆固醇酰基转移酶（LCAT）的活化，该酶负责在HDL组装期间将新生（盘状）HDL转化为循环（球状）HDL，这是逆向胆固醇转运（RCT）中的重要步骤;以及ii）HDL重塑，在HDL组装和RCT中也很重要。由于apoA-I/HDL是一种软形式的凝聚态物质，很容易因热波动而变形，因此更完整地了解HDL将需要创新的方法。原则上，我们提出的使用实验方法和计算机模拟的协同组合可以显着有助于理解HDL结构和动力学。基于我们最近对HDL的分子动力学（MD）模拟，我们提出了三个工作假设：1）螺旋内和螺旋间盐桥的随机云分别为HDL颗粒上的apoA-I提供了弹簧样的弹性（分子“Slinky”）和粘性（分子“Velcro”）。2)HDL上apoA-I的末端结构域代表了一个重塑开关，其调节极性脂质的交换，并产生对其他载脂蛋白和抗氧化剂蛋白具有高亲和力的热点。3)apoA-I的成对反平行螺旋5结构域创建了一个两亲呈递通道，用于将未酯化胆固醇的疏水酰基链和极性羟基从新生HDL迁移到LCAT的活性位点。为了验证这些假设，我们提出了两个具体的目标：1）确定apoA-I的末端重叠结构域在新生HDL重塑中的作用。i）使用我们的MD结果，通过定点诱变融合、交换、膜相互作用和蛋白质结合亲和力的分子模型来设计实验测试，重点是N-末端“粘性”推定融合结构域和C-末端“混杂”螺旋10推定交换结构域。ii）使用apoA-I/HDL系综（天然的和突变的）的所有原子和粗粒模型以通过MD模拟进一步测试极性脂质重构转换假说。2)检测apoA-I中心区在LCAT激活中的作用。酰基链和UC介绍隧道假设将进行实验测试的基础上设计的所有原子和粗粒度的MD模拟定点突变体。由于脂质相关的apoA-I结构的详细预测，结合湿实验室的方法与分子模拟，我们建议，我们是唯一的定位，可以提供一个分子路线图，为未来的研究HDL的结构，功能和动力学的分子机制。 公共卫生相关性：高密度脂蛋白（HDL）是一种有益的胆固醇，是未来预防心脏病发作药物的重要靶点。不幸的是，所有最近的尝试在新的HDL靶向药物的开发都是不成功的。我们提出的HDL的计算机和分子生物学研究的结合，是我们实验室独有的结合，为针对HDL的未来药物开发提供了分子蓝图。

DESCRIPTION (provided by applicant): High density lipoproteins (HDL) are promising targets for pharmacological therapy of cardiovascular disease (CVD). Whether HDL itself directly prevents CVD or acts as a platform for attachment of protective antiinflammatory or antioxidant proteins, knowledge of HDL structure is important. The goal of the current proposal is to use a synergistic combination of direct experimental methods and computer simulations to understand the role of apolipoprotein A-I (apoA-I) dynamics in two important biological functions of HDL: i) activation of the enzyme lecithin:cholesterol acyl transferase (LCAT), the enzyme responsible for converting nascent (discoidal) HDL to circulating (spheroidal) HDL during HDL assembly, an important step in reverse cholesterol transport (RCT) and ii) HDL remodeling, also important in HDL assembly and RCT. Since apoA- I/HDL is a soft form of condensed matter easily deformable by thermal fluctuations, a more complete understanding of HDL will require innovative approaches. In principle, our proposed use of a synergistic combination of experimental methods and computer simulations can contribute significantly to understanding HDL structure and dynamics. Based upon our recent molecular dynamics (MD) simulations of HDL, we propose three working hypotheses: 1) A stochastic cloud of intrahelical and interhelical salt bridges, respectively, provide a spring-like elasticity (molecular "Slinky") and stickiness (molecular "Velcro") to apoA-I on HDL particles. 2) The terminal domains of apoA-I on HDL represent a remodeling-switch that regulates exchange of polar lipids and creates a hot spot with high affinity for other apolipoproteins and antiinflammatory and antioxidant proteins. 3) The pairwise antiparallel helix 5 domain of apoA-I creates an amphipathic presentation tunnel for migration of hydrophobic acyl chains and polar hydroxyl groups of unesterified cholesterol from nascent HDL to the active site of LCAT. To test these hypotheses we propose two specific aims: 1) To determine the role of the terminal overlap domain of apoA-I in nascent HDL remodeling. To achieve this aim, we will: i) Use our MD results to design experimental tests by site-directed mutagenesis of molecular models for fusion, exchange, membrane interactions and protein-binding affinity, focusing on the N- terminal "sticky" putative fusion domain and the C-terminal "promiscuous" helix 10 putative exchange domain. ii) Use all atom and coarse grained models of apoA-I/HDL ensembles (native and mutated) to further test the polar lipid remodeling-switch hypothesis by MD simulations. 2) To test the role of the central domain of apoA-I in LCAT activation. The acyl chain and UC presentation tunnel hypothesis will be tested experimentally by site-directed mutants designed on the basis of all atom and coarse grained MD simulations. Because of detailed predictions of lipid-associated apoA-I structure, the combination of wet lab approaches with molecular simulations that we propose and for which we are uniquely positioned can provide a molecular roadmap for future research into molecular mechanisms of HDL structure-function and dynamics. PUBLIC HEALTH RELEVANCE: HDL, the good cholesterol, is an important target for future drugs to prevent heart attacks. Unfortunately, all recent attempts at new HDL-targeted drug development have been unsuccessful. The combination of computer and molecular biology studies of HDL that we propose, a combination unique to our laboratory, provides a molecular blueprint for future drug development aimed at HDL.