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: 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
• 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

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15万低于退休年龄(65岁)的美国人死于心血管疾病(CAD)。心血管疾病的主要危险因素是血浆脂蛋白水平。脂蛋白根据其密度被分类为高密度脂蛋白 低、中、极低密度脂蛋白(分别为高密度脂蛋白、低密度脂蛋白、低密度脂蛋白和极低密度脂蛋白)以及乳糜粒；高密度脂蛋白和低密度脂蛋白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 心血管疾病中的脂蛋白。低密度脂蛋白可以进行氧化修饰，调节动脉壁中低密度脂蛋白-胆固醇的增加。低密度脂蛋白颗粒的大小、形状和组成各不相同，包括大的低密度脂蛋白(LDL1-2)和小而致密的低密度脂蛋白(LDL3-7)亚类；后者更容易被氧化。每个低密度脂蛋白颗粒包含一个载脂蛋白B-100(apoB-100)分子，它是肝脏通过低密度脂蛋白受体清除血浆胆固醇的配体。高密度脂蛋白从包括动脉壁在内的外周组织中隔离胆固醇，并将其输送到肝脏进行回收和处置，这一过程被称为反向胆固醇运输(RCT)。高密度脂蛋白亚种包括大小、形状和组成各不相同的颗粒。它们按大小和脂量分布为贫脂、新生和球形的高密度脂蛋白。血浆高密度脂蛋白颗粒含有多种载脂蛋白，但含量最丰富的是载脂蛋白A-I。载脂蛋白A-I通过细胞内的三磷酸腺苷结合盒转运体A1(ABCA1)介导胆固醇外流，并产生新生的盘状高密度脂蛋白颗粒，然后被卵磷脂-胆固醇酰基转移酶(LCAT)转化为球形高密度脂蛋白。球形高密度脂蛋白是血浆中高密度脂蛋白的主要形式，在肝脏中被B类清道夫受体I型(SR-BI)清除，后者介导选择性胆固醇酯摄取。由于脂蛋白在大小、形状、成分和生物功能上的不同，并且在性质上是动态的，传统技术(X-射线和核磁共振)阻碍了对脂蛋白颗粒的结构测定。电子显微镜(EM)作为一种新技术，可以直接显示单个颗粒。我们已经成功地看到了冷冻水合的脂蛋白，而没有扭曲的染色或固定剂，但对比是有限的。虽然传统的CryoEM分类和平均方法可以增强对比度，在这些方法中，来自不同颗粒的数千张图像根据每两张图像之间的相似性(互相关系数)进行分组和平均，但这种策略对不同种类的颗粒群体无效。因此，我们发明了单粒子电子断层扫描(IPET)技术，使我们能够使用基于中等分辨率(~3 nm)的图像从一个目标单分子(PLoS One，2012，7：E30249，1-19)获得3D低温ET密度图。尽管这种方法听起来雄心勃勃且咄咄逼人，但考虑到即使在NIH资助了40年后也发现了非常有限的脂蛋白结构信息，我们的激进方法在过去两年已经揭示了100多张来自高密度脂蛋白颗粒的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，在NIH资金支持下，我们的IPET方法在脂蛋白结构方面进行了重大探索。审查委员会有必要以开放的心态考虑为一种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有意想不到的困难，但考虑到我在过去几年中在各种脂蛋白结构研究方面的丰富低温EM经验和成就(12篇同行评议文章，导致5篇高影响力文章 在没有重大资金条件的情况下，我的经验应该足以用于故障排除，复制甚至提高我们在17 nm高密度脂蛋白中所示的已实现的分辨率，该分辨率已经足以适合apoA-I/高密度脂蛋白颗粒中的螺旋束结构域，提供脂蛋白结构的总体框架，并回答脂蛋白机制中的主要生物学问题(更多详细信息在提案中)。作为一种备用方法，如果来自低对比度低温图像的分辨率出人意料地太低，无法提供有用的脂蛋白结构，我们将使用我们报道的优化负染(OPNS)协议在高对比度NS图像上应用IPET重建。我们已经成功地从高对比度的OPNS图像中获得了近1 nm的分辨率图：例如，一个已知的动态分子的~1.4 nm分辨率图，单个抗体(PLoS One，2012)和一个较小的单分子，53 kDa CETP结构的~1.1 nm分辨率图(PLoS One，2012，7：e30249，1-19)。这种分辨率足以提供螺旋束骨架和配体间距。我们报道的OPNS是为了消除传统NS EM(NS-EM)中出现的Rouleau伪影而开发的。作为另一种备份方法，考虑到OPNS干燥过程中产生的平坦性伪影，我们将对已报道的高对比度冷冻阳性染色(Cryo-PS)图像进行IPET重建。我们的低温光电子能谱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)和球形高密度脂蛋白(JLR，2011)。期待一张高分辨率的非平面度3D地图是合理的。提出了四个具体目标：1)用IPET和抗apoB抗体检验低密度脂蛋白的结构模型；2)用IPET检验新生的8种高密度脂蛋白的结构模型；3)用IPET检验两种球形高密度脂蛋白的结构模型；4)用IPET和apoA-I配体(LCAT和抗ApoA-I抗体)验证高密度脂蛋白的结构模型。