APPLICATION OF MASS SPECTROMETRY TO STRUCTURAL BIOLOGY

质谱在结构生物学中的应用

• 批准号: 2574385
• 负责人: K B TOMER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2574385
• 关键词: acylation; apolipoproteins; biomedical equipment; biomedical equipment development; blood lipoprotein biosynthesis; cholesterol; cysteine; electrospray ionization mass spectrometry; fatty acids; high density lipoproteins; insulin receptor; intermolecular interaction; intracellular transport; liver cells; mass spectrometry; polymers; posttranslational modifications; protein structure; site directed mutagenesis; structural biology; tissue /cell culture

Recent instrumental developments in mass spectrometry, such as matrix- assisted laser desorption and electrospray ionization, have enabled mass spectrometrists to investigate biological molecules with significantly higher Mr than previously possible. The combination of these techniques with chemical processing of large biopolymers such as proteins now enables mass spectrometry to play a significant role in the realm of structural biology. We are currently working on several projects: modifications on apolipoprotein A-1 (detailed below); developing the capability of probing non-covalent interactions between proteins and between proteins and DNA using mass spectrometry; and determining the cysteine residue in the insulin receptor that is susceptible to biotinylation. One project we are currently working on is the identification of the post-translational modifications, especially sites of fatty acid acylation, on apolipoprotein A-I secreted by Hep G2 human hepatoma cells. Apolipoprotein A-I (apo A-I) is the principal protein present in human high density lipoproteins. It not only serves as a structural protein for the integrity of the lipoprotein particle, it also is a cofactor for the enzyme lecithin cholesterol acyltransferase (LCAT) that esterifies cholesterol in the blood. Apo A-I has been proposed to play an important role not only in transporting cholesterol from nonhepatic tissues to the liver, but it may also be important in preventing the development of atherosclerosis. The 9 amphipathic alpha helices present in antiparallel array in this protein are critical for lipid binding, coactivator function of LCAT, and potentially for interaction with the cellular surface in order to facilitate cellular cholesterol transport. Because nascent, but not circulating, apo A-I is fatty acid acylated, Hoeg has proposed that the esterification of specific residues with fatty acids may be critical for protecting the hepatocyte and enterocyte, which secrete apo A-I into the circulatory system. Apo A-I is acylated only in primary hepatocyte cultures and in well-differentiated hepatoma cell lines indicating that acylation requires specific cellular factors. Transfected nonhepatic cell lines synthesize and secrete apo A-I without fatty acid acylation as defined by radio labeled fatty acid studies. These data indicate potential role(s) for the addition of these prosthetic groups. This could be altering the ability for apo A-I to induce transmembrane signaling and/or by altering the removal of cholesterol from intracellular membrane pools. Finally, nascent apo A-I may require fatty acyl groups for initial HDL particle assembly. By identifying the specific amino acids acylated, site-directed mutagenesis of apo A-I can be used to determine the physiological significance of acylation.

质谱仪的最新发展，如基质- 辅助激光解吸和电喷雾电离，使质量 光谱学家研究生物分子， 比以前可能的更高。 这些技术的结合 随着大型生物聚合物如蛋白质的化学加工， 使质谱分析在以下领域发挥重要作用： 结构生物学 我们目前正在进行几个项目： 对载脂蛋白A-1的修饰（详细信息见下文）;开发 探测蛋白质之间非共价相互作用的能力， 蛋白质和DNA之间的关系;并确定 胰岛素受体中的半胱氨酸残基， 生物素化 我们目前正在进行的一个项目是确定 翻译后修饰，特别是脂肪酸位点 酰化，对Hep G2人肝癌细胞分泌的载脂蛋白A-I的作用。 载脂蛋白A-I（apoA-I）是存在于人体内的主要蛋白质， 高密度脂蛋白 它不仅是一种结构蛋白 对于脂蛋白颗粒的完整性，它也是 卵磷脂胆固醇酰基转移酶（LCAT） 血液中的胆固醇 载脂蛋白A-I被认为是一种重要的 不仅在将胆固醇从非肝组织转运到 肝脏，但它也可能是重要的，在防止发展， 动脉粥样硬化 9个两亲性α螺旋以反平行方式存在， 这种蛋白质中的阵列对于脂质结合、辅激活因子 LCAT的功能，并可能与细胞相互作用 表面，以促进细胞胆固醇运输。 因为 新生的，但不是循环的，载脂蛋白A-I是脂肪酸酰化的，Hoeg有 提出了特定残基与脂肪酸的酯化 可能对保护肝细胞和肠上皮细胞至关重要， 将载脂蛋白A-I分泌到循环系统中。 Apo A-I仅酰化 在原代肝细胞培养物和高分化肝癌细胞中 线表明酰化需要特定的细胞因子。 转染的非肝细胞系合成并分泌载脂蛋白A-I， 脂肪酸酰化由放射性标记的脂肪酸研究定义。 这些数据表明了添加这些化合物的潜在作用。 假体组。 这可能会改变载脂蛋白A-I的能力， 诱导跨膜信号传导和/或通过改变 胆固醇从细胞内膜池。 最后，新生的载脂蛋白A-I 可能需要脂肪酰基用于初始HDL颗粒组装。 通过 鉴定特定的氨基酸酰化，定点诱变 载脂蛋白A-I的表达可用于确定 酰化反应。