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: 6272624
• 负责人: DONALD M SMALL
• 金额: $ 33.49万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6272624
• 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 VLDL 是一个复杂的过程，需要脂质和 N- 末端 31-41% 的 apo-B (B)。 目前的假设表明，有 2 途径：如果有脂质，B 会分泌到富含 TG 的新生细胞上 颗粒 - 如果脂质缺乏，B 就会降解。 在分泌路径中 原始 HDL/LDL 大小的颗粒首先被释放到 ER 中，并且 然后它被放大形成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。 乳源性 C127 细胞不产生载脂蛋白，不分泌脂质，也不具有微粒体 TG 转移蛋白（MTP）。 当 C127 细胞转染 C 的 cDNA 时 截短的 B 形式，它们在脂质贫乏状态下有效分泌 B17 但仅在富含 TG 的原始颗粒上分泌 B41（约 125 埃）。 因此，C127 细胞翻译的 B41 也遵循两条途径： 分泌或降解。 与 TG 组装的信息， 与 ER 膜分离并分泌，因此存在于 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B21之间的两亲性β片层（ABS）非常丰富的区域 B41。 已鉴定出 35 条 12aa 链。 推测有 7 个 暂停序列也在这个区域与 5 个 ABS 重叠。 构造 表达 apo-B29 和 apo-B41 之间的截短形式 确定使 B 形成 a 所需的最小 N 端 富含TG的原始颗粒。 关键 ABS 域内的突变和暂停 B21-B41 中的序列将用于检查哪些 B 微观结构是 是允许与 TG 结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， 这个结构将通过X射线、核磁共振和冷冻电子来研究 显微镜。 共有序列将用于设计寡核苷酸 通过 beta 轮次连接 10 到 40 个共识链。 这些将是 连接到 B17 和 B29 以产生具有潜在能力的嵌合蛋白 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 3D 结构 腔内颗粒将通过冷冻电子显微镜进行探索。 我们的 假设暂停序列允许 ABS 插入 ER 易位子的膜区域。 如果 TG 相邻，ABS 将结合并且 开始形成原始粒子的核心。 如果脂质不是 发现，那么作为外来跨膜蛋白的蛋白质是 退化了。