The biological activity of metal ion-gastrin complexes

描述（由申请人提供）：本项目的长期目标是了解金属离子与前激素前胃泌素衍生肽之间相互作用的生物学意义。来自该实验室的最新数据表明，前胃泌素衍生肽（PDP）选择性结合2个铁离子，并且重组人前胃泌素还在不同于铁离子结合位点的位点以高亲和力结合钙离子。该项目的具体目标是：（1）确定金属离子和PDP之间络合物的性质和结构，（2）确定金属离子在PDP生物活性中的作用，包括受体结合、细胞增殖和细胞迁移，以及（3）确定络合物在调节前胃泌素加工和胃肠道吸收金属离子中的作用。该项目的健康意义在于PDPs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，并可能参与铁稳态失调。研究设计反映了具体的目标。首先，金属离子和PDP之间的配合物的结构将通过荧光，EPR和NMR光谱的组合来确定。这些结构将用作设计具有单一氨基酸取代的重组和合成PDP的基础，所述单一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结合一组胃肠道细胞系、刺激其增殖和降低其粘附的能力。将通过测量细胞系、分离的隐窝和体内功能障碍的结肠中的DNA合成来评估增殖。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测定来评估粘附。第三，将在细胞系和铁状态改变的小鼠中测量三价铁离子浓度变化对前胃泌素加工的影响。还将确定PDPs在细胞摄取三价铁离子中的作用。铁离子和钙离子是生物活性形式的PDP的基本成分的证明将完全改变我们对金属离子在激素功能中的作用的理解。这些研究还可能揭示肽激素在铁离子体内平衡中的意想不到的作用。从长远来看，对PDP-金属离子复合物的高亲和力受体的定义可能允许开发用于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.