MYRISTYLATION OF PROTEINS IN TRANSFORMED CELLS

转化细胞中蛋白质的十四烷基化

• 批准号: 3197117
• 负责人: MARILYN D RESH
• 金额: $ 17.88万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-03-01 至 1995-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3197117
• 关键词: autoradiography; binding proteins; biological signal transduction; cell growth regulation; chimeric proteins; computer assisted sequence analysis; enzyme linked immunosorbent assay; enzyme mechanism; enzyme structure; fatty acylation; fluorescence microscopy; gel electrophoresis; gene expression; high performance liquid chromatography; immunological substance; immunoprecipitation; ion exchange chromatography; laboratory mouse; laboratory rabbit; membrane fusion; molecular cloning; monoclonal antibody; myristates; neoplastic cell; nucleic acid sequence; oncogenes; protein purification; protein sequence; protein structure function; transferase; virus protein; vole

The long-term goal of the proposed research is to understand how modification of proteins by myristylation affects protein structure and function. This will be accomplished by studying N-myristyl transferase (NMT), the enzyme which catalyzes attachment of myristate to proteins, as well as its protein substrates. NMT will be purified from mammalian liver, and used as an antigen for production of polyclonal and monoclonal antisera. Anti-NMT antibodies will be employed, in conjunction with NMT activity assays, to probe the subcellular localization of NMT in normal and Rous sarcoma virus-transformed cells. In addition, the gene encoding NMT will be cloned and used to express NMT. Cellular substrates for NMT will be identified by immunoreaction with anti-myristylglycine antibodies, and effects of oncogenic transformation on the expression and distribution of myristylated proteins will be studied. Additional experiments will focus on the myristylated transforming protein of Rous sarcoma virus, pp60(v-src), which requires myristylation in order to bind to membranes and elicit transformation. The effect of altering fatty acid hydrophobicity, chain length, and protein sequence context of the myristate moiety on pp60(v-src) will be tested in an in vitro membrane binding assay. Since myristate is attached to numerous viral structural proteins and oncogenes, as well as to proteins involved in growth control and signal transduction, the proposed studies should aid in understanding the basic biochemical mechanisms which regulate cell growth.

拟议研究的长期目标是了解如何 通过肉豆蔻酰化修饰蛋白质会影响蛋白质结构和 功能。这将通过研究 N-肉豆蔻基转移酶来完成 (NMT)，催化肉豆蔻酸与蛋白质附着的酶，如 及其蛋白质底物。 NMT将从哺乳动物肝脏中纯化， 并用作生产多克隆和单克隆抗体的抗原 抗血清。抗 NMT 抗体将与 NMT 结合使用 活性测定，以探测正常和正常细胞中 NMT 的亚细胞定位 劳斯肉瘤病毒转化的细胞。此外，编码NMT的基因 将被克隆并用于表达 NMT。 NMT 的细胞基质将是 通过抗肉豆蔻酰甘氨酸抗体的免疫反应进行鉴定，并且 致癌转化对表达和分布的影响 将研究肉豆蔻酰化蛋白质。额外的实验将集中于 劳斯肉瘤病毒的肉豆蔻酰化转化蛋白，pp60(v-src)， 需要肉豆蔻酰化才能与膜结合并引发 转变。改变脂肪酸疏水性、链的影响 pp60(v-src) 上肉豆蔻酸酯部分的长度和蛋白质序列背景 将在体外膜结合测定中进行测试。由于肉豆蔻酸酯是 附着在许多病毒结构蛋白和癌基因上，以及 参与生长控制和信号转导的蛋白质，建议 研究应有助于理解基本的生化机制 调节细胞生长。