BIOLOGY AND MOLECULAR BIOLOGY OF TRANSFORMING GROWTH FACTOR-BETA

转化生长因子-β的生物学和分子生物学

• 批准号: 3838326
• 负责人: A B ROBERTS
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 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/3838326
• 关键词: cell growth regulation; cell migration; chimeric proteins; growth factor receptors; growth inhibitors; high performance liquid chromatography; protein engineering; protein purification; protein sequence; protein structure function; receptor expression; recombinant DNA; transforming growth factors

The three isoforms of TGF-Beta expressed in mammals are 65-85% homologous to each other. Although they are functionally interchangeable in most in vitro assay systems, they have distinctive activities on certain cells such as endothelial cells where the activities of TGF-Beta 1 and TGF-Beta 3 on inhibition of growth and migration are approximately 100-fold more potent than that of TGF-Beta 2. To attempt to define specific regions of the TGF-Beta molecule responsible for these differences in activities, we have developed a system for expression of recombinant TGF-Betas engineered by recombinant DNA methodology to have portions of different isoforms spliced into a single chimeric molecule. Most interesting to date have been chimeras in which a region of the amino acid sequence of mature TGF- Beta 1 has been spliced into the TGF-Beta 2 molecule. Each recombinant TGF-Beta chimera has been purified to homogeneity using a series of high pressure liquid chromatography steps. We have shown that the middle third of the TGF-Beta molecule is sufficient to confer isoform-specific biological activity on endothelial cells. By making chimeras with only a few selected amino acid changes, we hope to identify specific amino acids responsible for this selective response. In a related aspect of this problem of isoform specificity, we are attempting to characterize the expression of TGF-Beta receptors and possible down-stream signalling intermediates to determine whether these will be common to all TGF-Beta isoforms or whether they will mediate isoform-specific effects.

在哺乳动物中表达的三种转化生长因子-β亚型具有65%-85%的同源性 为了彼此。尽管它们在功能上可以互换，但在 体外测试系统，它们对某些细胞具有独特的活性 如内皮细胞中的转化生长因子-β1和转化生长因子-β的活性 3对生长和迁移的抑制作用大约是100倍以上 比转化生长因子-β2更有效。试图定义 导致这些活动差异的转化生长因子-β分子，我们 我开发了一种表达重组转化生长因子-β的系统 通过重组DNA方法获得不同亚型的部分 拼接成单个嵌合分子。迄今为止最有趣的有 是嵌合体，其中成熟的转化生长因子的氨基酸序列的一个区域- β1已被拼接到转化生长因子-β2分子中。每一次重组 转化生长因子-β嵌合体通过一系列高纯度的 加压液相色谱步骤。我们已经证明了中间的三分之一 转化生长因子-β分子的活性足以赋予同种异构体特异性 对内皮细胞的生物活性。通过只用一种 少数精选氨基酸发生变化，我们希望鉴定出特定的氨基酸 对这种选择性反应负责。在与此相关的一个方面 异构体专一性的问题，我们正试图表征 转化生长因子-β受体的表达及其可能的下游信号转导 以确定这些中间体是否对所有转化生长因子-β都是通用的 异构体或它们是否会介导异构体特有的效应。