Apo-B Domains and Lipoprotein Structure and Assembly

Apo-B 结构域和脂蛋白结构与组装

• 批准号: 7140006
• 负责人: DONALD M SMALL
• 金额: $ 43.09万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7140006
• 关键词: X ray crystallography; abetalipoproteinemias; apolipoprotein B; blood lipoprotein biosynthesis; cardiovascular disorder; gene mutation; human tissue; molecular assembly /self assembly; molecular chaperones; nuclear magnetic resonance spectroscopy; phospholipids; protein binding; protein folding; protein structure function; proteolysis; structural biology; triglycerides; very low density lipoprotein

The assembly of triadyglycerol-rich lipoproteins (TAG-LP) involves the initial formation of a small HDL sized primordial particle with a neutral lipid core and a later process which adds TAG and phospholipid (PL) to create nascent VLDL. The N-terminal 20.5% of apoB(B0.5) has homology to lipovitellin (LV) that consists of an N-terminal beta barrel, a region of 17 amphipathic alpha helices (AaH) and 2 beta sheet domains, the A and C sheets. Proper folding of the N-terminal is necessary for secretion since certain point mutations and failure to form disulfide bonds target it to degradation. Limited proteolysis of 617 and B20.5 indicates that apoB homology to LV is approximate but not exact. Structural determination of isolated proteolytic and expressed domains using CD, NMR and X-ray diffraction will define B20.5 domains at the molecular level. The assembly with lipids as driven by the primary sequence of apoB is studied in MTP-deficient C127 cells transfected with C-terminally truncated forms. The role of molecular chaperones in folding, lipidation and in quality control is studied both in C127 and hepatoma-derived cells. The region from B20.5 to B29 recruits surface molecules, mainly PL to the nascent particle. As the peptide lengthens from B20.5 to B29 the number of surface molecules increases from approximately 5 to about 70 per particle. The sequence between B32 and B41 recruits almost 200 TAG (about 1 TAG/2.3 aa) which form nascent particles with a cryo-EM discernable core which is not seen in B32.5. Biophysical analysis shows that B37 and B41 peptides must interact directly with the core. This region contains 24 amphipathic beta strands (ApS) 12-15 aa long. Consensus 12 aa ApS and 27 aa p sheet peptides bind to oil/water (O/W) interfaces, are elastic and are not displaced at high pressure. ApoB also contains 2 major regions a2 and a3 of AaH. Consensus AaH peptides bind to O/W and air/water interfaces, but are ejected at a critical pressure into the aqueous phase. Native apoB also binds to the TAG/W interface and cannot be fully displaced by high pressure, but parts of the apoB come off when compressed and snap back on rapidly when the surfaces are again expanded. Thus, we suggest the flexible parts of apoB are AaH and the non-exchangeable part is AbetaS. This project dissects the complex pathway by which "bad cholesterol" (apoB associated cholesterol, VLDL, LDL) is made and secreted by the liver. The long term goal is to find procedures or drugs to reduce bad cholesterol, heart attack and stroke.

富含甘油三酸酯的脂蛋白（TAG-LP）的组装涉及小HDL大小的初始形成， 具有中性脂质核心的原始颗粒和随后的过程，该过程将TAG和磷脂（PL）添加到 产生新生的VLDL。apoB（B0.5）的N-末端20.5%与卵黄脂磷蛋白（LV）具有同源性， N-末端β桶，17个两亲性α螺旋（AaH）和2个β折叠结构域的区域，A和C 薄膜. N-末端的正确折叠对于分泌是必需的，因为某些点突变和失败 形成二硫键，使其降解。617和B20.5的有限蛋白水解表明apoB 与LV同源性是近似的，但不是精确的。分离的蛋白水解和表达的蛋白质的结构测定 使用CD、NMR和X-射线衍射确定B20.5结构域将在分子水平上确定B20.5结构域。的 在MTP缺陷的C127细胞中研究了由apoB的一级序列驱动的与脂质的组装 用C末端截短形式转染。分子伴侣在折叠、脂化和细胞凋亡中的作用 在C127和肝癌衍生细胞中研究了质量控制。从B20.5到B29区域招聘 表面分子，主要是PL的新生粒子。随着肽从B20.5延长至B29， 表面分子的数量从每个颗粒约5个增加到约70个。B32和B41之间的序列募集几乎200个TAG（约1个TAG/2.3 aa），其形成具有cryo-EM可辨别的核心的新生颗粒 这在B32.5中是看不到的。生物物理分析表明，B37和B41肽必须直接与 核心的该区域含有24个12-15个氨基酸长的两亲性β链（ApS）。共有12 aa ApS和 27个氨基酸的β折叠肽结合到油/水（O/W）界面，是弹性的，并且在高压下不移位。 ApoB还含有AaH的两个主要区域a2和a3。共有AaH肽与O/W和空气/水结合 界面，但在临界压力下喷射到水相中。天然的apoB也结合到 TAG/W界面，不能被高压完全置换，但当压力升高时， 当表面再次膨胀时，它们被压缩并迅速恢复。因此，我们建议灵活的 apoB的部分是AaH，不可交换部分是AbetaS。本项目通过以下方式剖析了复杂的路径： 其中“坏胆固醇”（载脂蛋白B相关胆固醇，VLDL，LDL）是由肝脏产生和分泌的。的 长期目标是找到程序或药物，以减少坏胆固醇，心脏病发作和中风。