Lipoprotein Structure and Function by Individual Particle Electron Tomography

单粒子电子断层扫描的脂蛋白结构和功能

• 批准号: 8422818
• 负责人: Gang Ren
• 金额: $ 35.74万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-01 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8422818
• 关键词: ATP-Binding Cassette Transporters; Achievement; Age; Amaze; American; American Heart Association; Antibodies; Apolipoprotein A-I; Apolipoproteins; Apolipoproteins B; Applications Grants; Binding; Biological; Biological Process; CETP gene; Cardiovascular Diseases; Cholesterol; Cholesterol Esters; Cholesterol Homeostasis; Chylomicrons; Classification; Complex; Coronary Arteriosclerosis; Cryoelectron Microscopy; Development; Electron Microscopy; Environment; Fixatives; Freezing; Funding; Funding Opportunities; Goals; Heart Diseases; Hepatic; High Density Lipoprotein Cholesterol; High Density Lipoproteins; Housing; Image; Imagery; Individual; Journals; Knowledge; LDL Cholesterol Lipoproteins; Label; Ligands; Lipids; Lipoproteins; Liver; Low Density Lipoprotein Receptor; Low-Density Lipoproteins; Maps; Mediating; Methods; Mind; Modeling; Modification; Morphologic artifacts; N-terminal; Nature; Negative Staining; Peer Review; Pensions; Peripheral; Pharmaceutical Preparations; Phosphatidylcholine-Sterol O-Acyltransferase; Phospholipids; Plasma; Population; Positioning Attribute; Prevention; Procedures; Process; Production; Property; Proteins; Protocols documentation; Recycling; Reporting; Resolution; Review Committee; Risk; Risk Factors; Roentgen Rays; Role; SR-BI receptor; Scaffolding Protein; Shapes; Solar Flare; Staining method; Stains; Structural Models; Structure; Structure-Activity Relationship; Surface; Techniques; Testing; Tissues; Trefoil; United States National Institutes of Health; Very low density lipoprotein; apolipoprotein B-100; base; cardiovascular risk factor; density; electron tomography; enzyme substrate; experience; improved; killings; lipoprotein cholesterol; low density lipoprotein inhibitor; nanoscale; novel; novel strategies; oxidation; particle; protein structure; public health relevance; reconstitution; reconstruction; reverse cholesterol transport; screening; single molecule; sound; statistics; three dimensional structure; uptake; water solubility

DESCRIPTION (provided by applicant): Nearly 150,000 Americans younger than the retirement benefit age (<65 years) were killed each year by cardiovascular disease (CAD) according to the latest statistics from the American Heart Association. Major risk factors for CVD are the plasma lipoprotein levels. Lipoproteins are classified according to their densities as high , low-, intermediate- and very low-density lipoproteins (HDL, LDL, IDL and VLDL respectively), as well as chylomicrons; HDL and LDL are major players in plasma cholesterol metabolism. Lipoprotein structure-function relationships provide important clues that help identify the role of lipoproteins in CVD. LDL can undergo oxidative modifications that mediate the accretion of LDL-cholesterol in the arterial wall. LDL particles vary in size, shape, and composition, and comprise large LDL (LDL1-2) and small, dense LDL (LDL3-7) subclasses; the latter are more prone to oxidation. Each LDL particle contains one molecule of apolipoprotein B-100 (apoB-100), a ligand for hepatic clearance of plasma cholesterol via LDL receptors. HDL sequesters cholesterol from peripheral tissues, including the arterial wall, and transports it to the liver fo recycling and disposal, a process called reverse cholesterol transport (RCT). HDL subspecies comprise particles that vary in size, shape, and composition. They distribute according to size and lipid amount into lipid- poor, nascent and spherical HDL. Plasma HDL particles contain multiple apolipoproteins, but the most abundant is apoA-I. ApoA-I mediates cholesterol efflux via the cellular ATP-binding cassette transporter A1 (ABCA1), and produces nascent discoidal HDL particles that are then converted to spherical HDL by lecithin- cholesterol acyltransferase (LCAT). Spherical HDL is the dominant form of HDL in plasma and is hepatically removed by scavenger receptor class B, type I (SR-BI), which mediates selective cholesteryl ester uptake. Structural determination of lipoprotein particles has been frustrated by conventional techniques (X-ray and NMR) because lipoproteins vary in size, shape, components, and biological functions and are dynamic in nature. Electron microscopy (EM), as a novel technique, allows direct visualization of individual particles. We have successfully viewed frozen-hydrated lipoproteins without distorting stains or fixatives, but the contrast is limited. Although the contrast can be enhanced by conventional cryoEM classification and averaging methods in which thousands of images from different particles are grouped and averaged based on similarity (cross- correlation coefficient) between each two images, this strategy fails for heterogeneous particle populations. Thus, we invented an individual particle electron tomography (IPET) technique that allows us to obtain a 3D cryoET density map using intermediate resolution (~3nm) based images from one targeted single-molecule (PLoS One, 2012, 7:e30249, 1-19). Although the approach sounds ambitious and aggressive, considering that very limited lipoprotein structure information has been discovered even after NIH funding for four decades, our aggressive approach has revealed more than a hundred 3D density maps from HDL particles that vary in size from 7nm to 20nm in the last two years. Thus, it is worthy to expect a significant exploration in lipoprotein structure by our IPET approach supported by NIH funding. It is necessary for the review committee to have an open mind in considering a funding opportunity for a totally new approach that has never been used before. Although there may be unexpected difficulties in using this new approach, considering my rich cryoEM experience and achievement in various lipoprotein structure studies in the last few years (12 peer-reviewed articles,leading5 articles in high impact journals under no major funding condition), my experience should be sufficient for troubleshooting to reproduce and even improve our achieved resolution shown in 17nm HDL that is already sufficient to fit the helical bundle domain in apoA-I/HDL particles, provide an overall frame of lipoprotein structure and answer the major biological questions in lipoprotein mechanism (more details in proposal). As a backup approach, if the resolution from low-contrast cryoET images is unexpectedly too low to provide a useful structure of lipoprotein, we will apply the IPET reconstruction on the high-contrast NS images by using our reported optimized negative-staining (OpNS) protocol. We have successfully achieved near ~1nm resolution maps from high-contrast OpNS images: for example, a ~1.4 nm resolution map of a well-known dynamic molecule, a single antibody (PLoS One, 2012) and a ~1.1 nm resolution map of a smaller single molecule, 53kDa CETP structure (PLoS One, 2012, 7:e30249, 1-19). This resolution is sufficient to provide the helical bundle framework and ligand spacing. Our reported OpNS is developed to eliminate the rouleau artifact represented in the conventional NS EM (NS-EM). As an additional backup approach, concerning the flatness artifact from the drying procedure of OpNS, we will apply the IPET reconstruction on our reported high-contrast cryo-positive-staining (cryo-PS) images. Our cryo- PS images can provide high-contrast and amazing structural details of small proteins, such as CETP (NCB, 2012) and spherical HDL (JLR, 2011). It is reasonable to expect a high-resolution no-flatness 3D map. Four specific aims are proposed: 1) To test the structural model of LDL by IPET and anti-apoB antibodies; 2) To test eight structural models of nascent HDL by IPET 3) To test two structure models of spherical HDL by IPET; 4) To validate the structural model of HDL by IPET and apoA-I ligands (LCAT and anti-ApoA-I antibodies).

描述（由申请人提供）：根据美国心脏协会的最新统计数据，每年有近15万低于退休年龄（<65岁）的美国人死于心血管疾病（CAD）。CVD的主要危险因素是血浆脂蛋白水平。脂蛋白根据它们的密度被分类为高密度脂蛋白。 低密度、中密度和极低密度脂蛋白（分别为HDL、LDL、IDL和VLDL）以及乳糜微粒; HDL和LDL是血浆胆固醇代谢的主要参与者。脂蛋白的结构-功能关系提供了重要的线索，有助于确定的作用， 脂蛋白在CVD中的作用LDL可以进行氧化修饰，介导LDL-胆固醇在动脉壁中的积聚。LDL颗粒的大小、形状和组成各不相同，包括大LDL（LDL 1 -2）和小而致密的LDL（LDL 3 -7）亚类;后者更容易氧化。每个LDL颗粒含有一分子载脂蛋白B-100（apo B-100），一种通过LDL受体肝脏清除血浆胆固醇的配体。HDL从外周组织（包括动脉壁）中隔离胆固醇，并将其转运到肝脏进行回收和处置，这一过程称为胆固醇逆向转运（RCT）。HDL亚种包含大小、形状和组成不同的颗粒。它们按大小和脂质含量分为贫脂型、新生型和球形HDL.血浆HDL颗粒含有多种载脂蛋白，但最丰富的是apoA-I。ApoA-I通过细胞ATP结合盒转运蛋白A1（ABCA 1）介导胆固醇流出，并产生新生盘状HDL颗粒，然后通过卵磷脂-胆固醇酰基转移酶（LCAT）将其转化为球形HDL。球形HDL是血浆中HDL的主要形式，并通过介导选择性胆固醇酯摄取的I型B类清道夫受体（SR-BI）在肝脏中清除。脂蛋白颗粒的结构测定已被常规技术（X射线和NMR）所挫败，因为脂蛋白在大小、形状、组分和生物学功能上变化，并且在性质上是动态的。电子显微镜（EM），作为一种新的技术，可以直接可视化的单个颗粒。我们已经成功地观察了冷冻水合脂蛋白，没有扭曲的染色剂或固定剂，但对比度是有限的。尽管可以通过常规的cryoEM分类和平均方法来增强对比度，其中基于每两个图像之间的相似性（互相关系数）对来自不同颗粒的数千个图像进行分组和平均，但是该策略对于异质颗粒群体失败。因此，我们发明了单个粒子电子断层扫描（IPET）技术，该技术允许我们使用来自一个靶向单分子的基于中间分辨率（~ 3 nm）的图像获得3D cryoET密度图（PLoS One，2012，7：e30249，1-19）。尽管这种方法听起来雄心勃勃，但考虑到即使在NIH资助四十年后，也发现了非常有限的脂蛋白结构信息，我们积极的方法在过去两年中已经揭示了100多个HDL颗粒的3D密度图，这些颗粒的大小从7 nm到20 nm不等。因此，值得期待的是，我们的IPET方法在美国国立卫生研究院的资金支持下，在脂蛋白结构的显着探索。审查委员会必须以开放的态度考虑为以前从未使用过的全新方法提供资金的机会。尽管使用这种新方法可能会遇到意想不到的困难，但考虑到我丰富的cryoEM经验和过去几年在各种脂蛋白结构研究中取得的成就（12篇同行评审文章，领先5篇具有高影响力的文章 在没有重大资助条件下的期刊），我的经验应该足以故障排除，以再现甚至改进我们在17 nm HDL中所实现的分辨率，该分辨率已经足以适合apoA-I/HDL颗粒中的螺旋束结构域，提供脂蛋白结构的整体框架并回答脂蛋白机制中的主要生物学问题（更多细节在提案中）。作为一种备用方法，如果低对比度冷冻ET图像的分辨率出乎意料地太低，无法提供有用的脂蛋白结构，我们将通过使用我们报告的优化负染色（OpNS）方案在高对比度NS图像上应用IPET重建。我们已经成功地从高对比度OpNS图像中获得了近~ 1 nm分辨率的图：例如，一个众所周知的动态分子，一个单一抗体的~ 1.4nm分辨率的图（PLoS One，2012）和一个较小的单一分子，53 kDa CETP结构的~ 1.1nm分辨率的图（PLoS One，2012，7：e30249，1-19）。该分辨率足以提供螺旋束框架和配体间距。我们报道的OpNS是为了消除传统NS EM（NS-EM）中的Rouleau伪影而开发的。作为一种额外的备份方法，考虑到OpNS干燥过程中的平整度伪影，我们将在我们报告的高对比度冷冻阳性染色（cryo-PS）图像上应用IPET重建。我们的cryo-PS图像可以提供小蛋白质的高对比度和惊人的结构细节，例如CETP（NCB，2012）和球形HDL（JLR，2011）。期望高分辨率非平坦3D图是合理的。本文提出了四个具体目标：1）用IPET和抗apoB抗体验证LDL的结构模型; 2）用IPET验证新生HDL的八种结构模型; 3）用IPET验证球形HDL的两种结构模型; 4）用IPET和apoA-I配体（LCAT和抗ApoA-I抗体）验证HDL的结构模型。