STRUCTURAL BIOLOGY--EARLY EVENTS IN LIPOPROTEIN ASSEMBLY

结构生物学——脂蛋白组装的早期事件

• 批准号: 6241705
• 负责人: DONALD M SMALL
• 金额: $ 32.32万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 1997-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6241705
• 关键词: X ray crystallography; apolipoproteins; blood lipoprotein biosynthesis; calorimetry; chimeric proteins; circular dichroism; electron microscopy; endoplasmic reticulum; gene mutation; genetic translation; lipid bilayer membrane; membrane lipids; membrane reconstitution /synthesis; molecular chaperones; nuclear magnetic resonance spectroscopy; oligonucleotides; phospholipids; protein folding; recombinant proteins; site directed mutagenesis; structural biology; transfection; triglycerides

The process of assembly and secretion of triglyceride-rich lipoproteins (TG-LP) such as VLDL is a complicated process requiring lipids and the N- terminal 31-41% of apo-B (B). Current hypotheses suggest that there are 2 pathways: if lipid is available, B is secreted on nascent TG-rich particles - if lipid is deficient, B is degraded. In the secretory path a primordial HDL/LDL sized particle is first released into the ER, and then it is enlarged to form VLDL. Several possible pause sequences in the N-terminal domain might facilitate lipid binding. Mammary derived C127 cells make no apolipoproteins, secrete no lipid nor have microsomal TG transfer protein (MTP). When C127 cells are transfected with cDNA for C truncated B forms, they effectively secrete B17 in the lipid poor state but secrete B41 exclusively on primordial TG-rich particles (about 125 angstroms). Thus, B41 translated by C127 cells also follows two pathways: secretion or degradation. The information for the assembly with TG, detachment from the ER membrane and secretion, therefore resides in the sequences between apo-B17 and apo-B41. A multialgorithm search for potential lipid binding sequences in B41 indicates that there is a large region very rich in amphipathic beta sheets (ABS) located between B21 and B41. Thirty-five 12aa strands have been identified. There are 7 putative pause sequences also in this region and 5 overlap ABS. Constructs expressing truncated forms between apo-B29 and apo-B41 will be made to determine the minimum N-terminal required to commit B to formation of a primordial TG-rich particle. Mutations within key ABS domains and pause sequences in B21-B41 will be made to examine which B microstructures are necessary to permit association with TG to form particles. Consensus ABS sequences will be synthesized and combined with membrane lipids and TG, and this structure will be studied by X-ray, NMR, and cryo-electron microscopy. Consensus sequences will be used to design oligonucleotides which link 10 to 40 consensus strands through beta turns. These will be ligated to B17 and B29 to produce chimeric proteins potentially capable of forming primordial TG-LP. Chaperones which bind to B during the assembly process will be probed. The 3D structure of the secreted particles and ER lumenal particles will be explored by cryo-electronmicroscopy. Our hypothesis is that pause sequences allow ABS to intercalate into the ER membrane region of the translocon. If TG is adjacent, ABS will bind and initiate formation of the core of a primordial particle. If lipid is not found, then the protein acting as a foreign transmembrane protein, is degraded.

富含甘油三酯的脂蛋白的组装和分泌过程 (TG-LP)如极低密度脂蛋白(VLDL)是一个复杂的过程，需要血脂和N- 载脂蛋白B(B)的31-41%的终端。目前的假说表明，存在 两条途径：如果有脂质，B会在新生的富含甘油三酯的体内分泌 颗粒--如果脂肪缺乏，维生素B就会被降解。在秘密之路上 一个原始的高密度脂蛋白/低密度脂蛋白大小的颗粒首先被释放到ER中，并且 然后它被放大形成极低密度脂蛋白。中的几个可能的暂停序列 N-末端结构域可能促进脂质结合。乳房衍生C127 细胞不产生载脂蛋白，不分泌脂质，也不分泌微粒体甘油三酯 转移蛋白(MTP)。C基因在C127细胞中的表达 截短的B型，它们在脂质贫乏的状态下有效地分泌B17 但仅在原始的富含甘油三酯的颗粒(约125)上分泌B41 埃)。因此，C127细胞翻译的B41也遵循两条途径： 分泌物分泌或降解。关于具有TG的组件的信息， 脱离内质网细胞膜和分泌物，因此驻留在 Apo-B17和apo-B41之间的序列。一种多算法搜索 B41中潜在的脂质结合序列表明，有一个很大的 位于B21和B21之间的富含两亲性β-折叠(ABS)的区域 B41。目前已鉴定出35条12AA链。有7个推定 暂停序列也在这个区域和5个重叠的ABS。构造 表达apo-B29和apo-B41之间的截短形式将使 确定使B形成A所需的最小N-末端 原始的富甘油三酯颗粒。关键ABS域内的突变和暂停 将制作B21-B41中的序列以检查哪些B微结构是 允许与Tg缔合形成颗粒所必需的。共识ABS 序列将被合成并与膜脂和TG结合， 这种结构将通过X射线、核磁共振和冷冻电子进行研究 显微镜。共识序列将被用来设计寡核苷酸 它将10到40条共识链通过Beta转弯连接起来。这些将是 连接到B17和B29，以产生潜在能够 形成原始的TG-Lp。在组装过程中与B结合的伴侣 将对过程进行调查。分泌颗粒的三维结构与内质网 腔内颗粒将通过低温电子显微镜进行观察。我们的 假设暂停序列允许ABS插入内质网 易位子的膜区。如果TG相邻，则ABS将结合并 启动原始粒子核心的形成。如果脂质不是 发现，然后作为外源跨膜蛋白的蛋白质，是 降级了。