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

基本信息

批准号：
8850881
负责人：
Gang Ren
金额：
$ 30.95万
依托单位：
UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-05-01 至 2016-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8850881
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

项目摘要

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（申请人提供）：根据美国心脏协会的最新统计数据，每年有近 150,000 名低于退休福利年龄（<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。脂蛋白根据其密度分为高脂蛋白、低密度脂蛋白、中密度脂蛋白和极低密度脂蛋白（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。脂蛋白结构-功能关系提供了重要线索，有助于确定脂蛋白的作用 CVD 中的脂蛋白。低密度脂蛋白可以经历氧化修饰，介导低密度脂蛋白胆固醇在动脉壁中的积聚。 LDL 颗粒的大小、形状和成分各不相同，包括大 LDL (LDL1-2) 和小而密的 LDL (LDL3-7) 亚类；后者更容易氧化。每个 LDL 颗粒都含有一个载脂蛋白 B-100 (apoB-100) 分子，它是一种通过 LDL 受体肝脏清除血浆胆固醇的配体。 HDL 从周围组织（包括动脉壁）中隔离胆固醇，并将其转运至肝脏进行回收和处理，这一过程称为反向胆固醇转运 (RCT)。 HDL 亚种包含大小、形状和成分各异的颗粒。它们根据大小和脂质含量分为贫脂、新生和球形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 (ABCA1) 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 B 类清道夫受体 I 型 (SR-BI) 在肝脏中清除。传统技术（X 射线和 NMR）无法对脂蛋白颗粒进行结构测定，因为脂蛋白的大小、形状、成分和生物功能各不相同，并且本质上是动态的。电子显微镜（EM）作为一种新技术，可以直接观察单个颗粒。我们已经成功地观察了冷冻水合脂蛋白，而没有扭曲染色剂或固定剂，但对比度有限。尽管可以通过传统的冷冻电镜分类和平均方法来增强对比度，在传统的冷冻电镜分类和平均方法中，根据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张图像进行分组和平均，但这种策略对于异质颗粒群体是失败的。因此，我们发明了一种单独粒子电子断层扫描 (IPET) 技术，使我们能够使用来自一个目标单分子的基于中间分辨率 (~3nm) 的图像来获得 3D 冷冻电子断层扫描密度图 (PLoS One, 2012, 7:e30249, 1- 19）。尽管该方法听起来雄心勃勃且激进，但考虑到即使在 NIH 资助了 40 年之后，仍发现了非常有限的脂蛋白结构信息，我们的激进方法已经揭示了 100 多个 HDL 颗粒的 3D 密度图，这些颗粒的尺寸从 7 纳米到 20 纳米不等。最近两年。因此，值得期待的是，我们的 IPET 方法在 NIH 资助的支持下对脂蛋白结构进行了重大探索。审查委员会有必要以开放的态度考虑为以前从未使用过的全新方法提供资助的机会。尽管使用这种新方法可能会遇到意想不到的困难，但考虑到我过去几年在各种脂蛋白结构研究中丰富的冷冻电镜经验和成就（12篇同行评审文章，在高影响力领域领先5篇文章）期刊在没有重大资助条件下），我的经验应该足以进行故障排除以重现，甚至提高我们在 17nm HDL 中显示的分辨率，该分辨率已经足以适应 apoA-I/HDL 颗粒中的螺旋束域，提供了脂蛋白结构并回答脂蛋白机制中的主要生物学问题（提案中有更多详细信息）。作为备用方法，如果低对比度 CryoET 图像的分辨率出乎意料地太低而无法提供有用的脂蛋白结构，我们将使用我们报告的优化负染色 (OpNS) 对高对比度 NS 图像应用 IPET 重建协议。我们已经成功地从高对比度 OpNS 图像中获得了接近 1 nm 分辨率的图：例如，众所周知的动态分子、单个抗体（PLoS One，2012）的约 1.4 nm 分辨率图和约 1.1 nm 分辨率图更小的单分子，53kDa CETP 结构（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) 中出现的轮状伪影而开发的。作为一种额外的备用方法，关于 OpNS 干燥过程中的平坦度伪影，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。我们的冷冻 PS 图像可以提供小蛋白质的高对比度和令人惊叹的结构细节，例如 CETP（NCB，2012）和球形 HDL（JLR，2011）。可以合理地预期高分辨率的非平坦 3D 地图。提出了四个具体目标：1）通过医沛得和抗apoB抗体测试LDL的结构模型； 2) 用医沛得 (IPET) 测试八种新生 HDL 结构模型 3) 用医沛得 (IPET) 测试两种球形 HDL 结构模型； 4）通过IPET和apoA-I配体（LCAT和抗ApoA-I抗体）验证HDL的结构模型。