The biological activity of metal ion-gastrin complexes

描述（由申请人提供）：该项目的长期目标是了解金属离子与原激素原泌素衍生肽之间相互作用的生物学意义。该实验室最近的数据表明，原胃泌素衍生肽（pdp）选择性地结合2个铁离子，重组人原胃泌素也在不同于铁离子结合位点的位置高亲和力地结合一个钙离子。该项目的具体目标是：(1)确定金属离子与pdp之间复合物的性质和结构；(2)确定金属离子在pdp的生物活性中的作用，包括受体结合、细胞增殖和细胞迁移；(3)确定复合物在调节前泌素加工和胃肠道金属离子摄取中的作用。该项目的健康意义在于pdp作为正常胃和结肠粘膜的生长因子，加速胃癌和结直肠癌的发展，并可能参与铁稳态紊乱。研究设计反映了具体的目标。首先，金属离子与pdp配合物的结构将通过荧光、EPR和NMR的组合来确定。这些结构将作为设计重组和合成pdp的基础，用单氨基酸取代防止铁离子或钙离子的结合。其次，将比较亲本和突变体pdp与胃肠道细胞系结合、刺激增殖和减少粘附的能力。增殖将通过测量细胞系、分离隐窝和体内失活结肠的DNA合成来评估。粘附性将通过粘附蛋白复合物的免疫化学分析，以及组织培养中的迁移和伤口愈合试验来评估。第三，铁离子浓度的变化对孕激素加工的影响将在细胞系和铁状态改变的小鼠中进行测量。pdp在细胞摄取铁离子中的作用也将被确定。铁离子和钙离子是pdp生物活性形式的重要组成部分的证明将彻底改变我们对金属离子在激素功能中的作用的理解。这些研究也可能揭示肽激素在铁离子稳态中的意想不到的作用。从长远来看，pdp -金属离子复合物高亲和受体的定义可能允许开发治疗铁代谢紊乱和胃肠道癌症的新疗法。

DESCRIPTION (provided by applicant): The long-term aim of this project is to understand the biological significance of the interactions between metal ions and peptides derived from the prohormone progastrin. Recent data from this laboratory has shown that progastrin-derived peptides (PDPs) selectively bind 2 ferric ions, and that recombinant human progastrin also binds a calcium ion with high affinity, at a site distinct from the ferric ion binding site. The specific aims of the project are: (1) to define the properties and structure of the complexes between metal ions and PDPs, (2) to define the role of metal ions in the biological activities of PDPs including receptor binding, cell proliferation and cell migration, and (3) to determine the role of the complexes in modulation of progastrin processing and in metal ion uptake by the gastrointestinal tract. The health significance of the project lies in the facts that PDPs act as growth factors for the normal gastric and colonic mucosa, accelerate the development of both gastric and colorectal cancer, and may be involved in disorders of iron homeostasis. The research design mirrors the specific aims. Firstly, the structures of the complexes between metal ions and PDPs will be determined by a combination of fluorescence, EPR and NMR spectroscopy. The structures will be used as the basis for the design of recombinant and synthetic PDPs with single amino acid substitutions which prevent the binding of either ferric or calcium ions. Secondly, the ability of the parent and mutant PDPs to bind to, stimulate proliferation in, and reduce adhesion of, a panel of gastrointestinal cell lines will be compared. Proliferation will be assessed by measurement of DNA synthesis in cell lines, isolated crypts, and the defunctioned colon in vivo. Adhesion will be assessed by immunochemical analysis of adhesion protein complexes, and migration and wound healing assays in tissue culture. Thirdly, the effect of changes in ferric ion concentration on progastrin processing will be measured in cell lines and in mice with altered iron status. The role of PDPs in the cellular uptake of ferric ions will also be determined. The demonstration that ferric and calcium ions are essential components of the biologically active forms of PDPs would alter completely our understanding of the role of metal ions in hormone function. The studies may also reveal an unexpected role for peptide hormones in ferric ion homeostasis. In the longer term definition of high affinity receptors for PDP-metal ion complexes may permit the development of novel therapies for treatment of disorders of iron metabolism and of gastrointestinal cancers.