STRUCTURE-FUNCTION OF TRANSFORMING GROWTH FACTOR BETA

多功能多肽，转化生长因子-β(TGF-β) Beta)，是从较大的 前体分子。先前的研究表明，亲本的二聚体 一段被称为β-潜伏期相关肽(β-LAP)的片段结合在一个 以非共价方式与较小的二聚体成熟生长因子结合。这 紧密而具体的交互完全归功于测试版，即 在加热和酸处理后变性；热和酸基本上 对成熟生长因子无影响。这样做的总体目标是 建议定义转化生长因子-β分子与 他们的测试圈，并研究潜在的激活机制。 这些研究的结果对于理解 转化生长因子-β生物活性的调节。为了检查每一个测试版- LAPS，我们将它们在转基因的CHO组织培养细胞中表达 独立于成熟的转化生长因子-β，允许生产大型 生物学的，功能性的测试圈的数量。此绑定源 蛋白质将允许进行测量解离常数的研究 成熟的转化生长因子-β及其多克隆和单抗的生产 抗体。抗体将允许研究评估结构 与其他测试圈的关联性。因为他们基本上不是 针对不同的β-圈反应的抗体，这些免疫球蛋白 将提供新的免疫试剂福特方法，如 免疫沉淀或组织分布研究。此外， 不同的，表达的测试圈将被提纯，使用已经 建立了纯化方案，并用来测试这些络合物是否 结合到细胞表面或细胞外基质上。最后，测试圈 来自转化生长因子-β2a和转化生长因子-β2b，转化生长因子-β3将被表达为 用Beta-Lap嵌合体来研究 延迟。这些研究应该会增进我们对潜伏期的理解 并为转化生长因子-β的调控提供了线索。

The multi-functional polypeptides, transforming growth factor betas (TGF- betas), are proteolytically derived from the carboxyl-terminus of a larger precursor molecule. Previous studies indicate that a dimer of the pro piece, known as beta-latency-associated peptide (beta-LAP), binds in a noncovalent manner with the smaller, dimeric, mature growth factor. This tight and specific interaction is entirely due to beta-LAP, being denatured upon heating and acid treatment; heat and acid have essentially no effects on the mature growth factor. The overall objective of this proposal is to define the molecular interaction of the TGF-betas with their beta-LAPs and to investigate potential activation mechanisms. Results from these studies are important for the understanding of the regulation of TGF-beta biological activity. To examine each of the beta- LAPs, we will express them in transfected CHO tissue culture cells independent of mature TGF-beta, allowing for the production of large amounts of a biologically, functional beta-LAP. This source of binding protein will allow for studies measuring the dissociation constants of mature TGF-beta and for the production of both polyclonal and monoclonal antibodies. Antibodies will permit studies to assess the structural relatedness with other beta-LAPs. Since their are essentially no antibodies reacting toward the diverse beta-LAPs, these immunoglobulins will provide new immunereagents ford approaches such as immuneprecipitation or tissue distribution studies. In addition, the different, expressed beta-LAPs will be purified, using an already established purification scheme, and used to test whether these complexes bind to cell surfaces or to extracellular matrix. Finally, the beta-LAPs from TGF-beta2a and TGF-beta2b, and TGF-beta3 will be expressed as chimeras with the beta-LAP to investigate structural regions necessary for latency. These studies should enhance our understanding of the latent complex and provide clues to the regulatory controls of the TGF-betas.