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。无论是高密度脂蛋白本身直接预防心血管疾病，还是作为保护性抗炎或抗氧化蛋白质的附着平台，对高密度脂蛋白结构的了解都是重要的。本提案的目的是利用直接实验方法和计算机模拟相结合的方法来了解载脂蛋白A-I(apoA-I)动力学在高密度脂蛋白的两个重要生物学功能中的作用：i)卵磷脂酶的激活：胆固醇酰基转移酶(LCAT)，该酶负责在高密度脂蛋白组装过程中将新生的(盘状)高密度脂蛋白转化为循环(球状)高密度脂蛋白，这是反向胆固醇运输(RCT)和高密度脂蛋白重塑的重要步骤，在高密度脂蛋白组装和高密度脂蛋白组装中也很重要。由于apoA-I/高密度脂蛋白是一种软形式的凝聚态物质，容易受到热波动的变形，因此对高密度脂蛋白的更完整的理解将需要创新的方法。原则上，我们提出的实验方法和计算机模拟的协同使用可以显著有助于理解高密度脂蛋白的结构和动力学。基于我们最近对高密度脂蛋白的分子动力学(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高密度脂蛋白颗粒上的载脂蛋白A-I提供弹力式弹性(分子“Slinky”)和粘性(分子“魔术贴”)。2)高密度脂蛋白上apoA-I的末端结构域代表着一种重塑开关，它调节极性脂类的交换，并与其他载脂蛋白以及抗炎和抗氧化蛋白产生高亲和力的热点。3)apoA-I的反平行螺旋5结构域为未酯化胆固醇的疏水酰链和极性羟基从新生的高密度脂蛋白迁移到LCAT的活性部位创造了一个两亲性递呈隧道。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的高密度脂蛋白重塑中的作用。为了实现这一目标，我们将：i)利用我们的MD结果，通过对融合、交换、膜相互作用和蛋白质结合亲和力的分子模型进行定点突变来设计实验测试，重点放在N端的“粘性”假定的融合域和C端的“混杂”螺旋10的假定交换域。Ii)使用apoA-I/高密度脂蛋白系综(天然和突变)的所有原子和粗粒度模型，通过MD模拟进一步验证极性脂质重塑-开关假说。2)检测apoA-I的中心结构域在LCAT激活中的作用。酰化链和UC呈现隧道假说将通过基于所有原子和粗粒度MD模拟设计的定点突变体进行实验验证。由于对脂质相关apoA-I结构的详细预测，将湿实验室方法与我们建议并唯一定位的分子模拟相结合，可以为未来研究高密度脂蛋白结构-功能和动力学的分子机制提供分子路线图。 与公众健康相关：高密度脂蛋白是一种有益的胆固醇，是未来预防心脏病的药物的重要靶点。不幸的是，最近针对高密度脂蛋白的新药开发的所有尝试都没有成功。我们提出的对高密度脂蛋白的计算机和分子生物学研究的结合，这是我们实验室独有的组合，为针对高密度脂蛋白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.