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: 6109584
• 负责人: DONALD M SMALL
• 金额: $ 34.7万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 1999-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6109584
• 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.

富含磷脂酰肌醇脂蛋白的装配和分泌过程 （TG-LP）如VLDL是一个复杂的过程，需要脂质和N-LDL。 apo-B（B）末端31-41%。 目前的假设表明， 2个途径：如果脂质可用，B在新生的富含TG的 颗粒-如果脂质缺乏，则B被降解。 在分泌途径 原始HDL/LDL大小的颗粒首先释放到ER中， 然后扩大形成VLDL。 中的几种可能的暂停序列 N端结构域可能促进脂质结合。 乳腺源性C127 细胞不产生载脂蛋白，不分泌脂质，也没有微粒体TG 转移蛋白（MTP）。 当C127细胞转染C 截短的B形式，它们在脂质贫乏状态下有效地分泌B17 而B41仅分泌在原始TG富集颗粒上（约125 埃）。 因此，C127细胞翻译的B41也遵循两条途径： 分泌或降解。 装配TG的信息， 从ER膜分离和分泌，因此驻留在 apo-B17和apo-B41之间的序列。 多算法搜索 B41中潜在的脂质结合序列表明存在大量 位于B21和B22之间的两亲性β折叠（ABS）非常丰富的区域 B41 已鉴定出35条12 aa链。 有7个假设 暂停序列也在这个区域和5重叠ABS。 构建体 表达apo-B29和apo-B41之间的截短形式， 确定使B形成所需的最小N-末端， 原始的富含TG的粒子 关键ABS结构域内的突变和暂停 将进行B21-B41中的序列，以检查哪些B微结构是 这是允许与TG缔合以形成颗粒所必需的。 共识ABS 序列将被合成并与膜脂质和TG结合， 这种结构将通过X射线，核磁共振和低温电子来研究 显微镜 共有序列将用于设计寡核苷酸 通过β转角连接10到40条共有链。 这些将是 与B17和B29连接以产生嵌合蛋白， 形成原始TG-LP。 在组装过程中与B结合的伴侣 过程将被探测。 分泌颗粒和ER的三维结构 将通过冷冻电子显微镜检查内腔颗粒。 我们 假设是暂停序列允许ABS插入ER 易位子的膜区域。 如果TG相邻，ABS将结合， 开始形成原始粒子的核心。 如果脂质不是 如果发现了，那么充当外源跨膜蛋白的蛋白质就是 退化了