STRUCTURE, FUNCTION, AND REGULATION OF COPPER AND IRON PROTEINS

我们一直在研究哺乳动物细胞的结构、功能和调节 与Cu和Fe相关的蛋白质，特别是铁蛋白（参与 铁储存）和铜蓝蛋白（参与铜转运）。 未来 几年后，MBRS的学生可能会学习其中一个方面， （a）两个机构的结构、职能和管理 在大鼠和人血浆中发现的血浆铜蓝蛋白（Cp）的主要同种型;（B） 我们假设牛奶Cp的结构和功能是一种 新生儿铜的重要来源;（c）潜在的 铁蛋白合成和分泌的刺激释放的淋巴因子 炎症，我们已经假设涉及一种新形式的铁蛋白 mRNA在膜结合的多聚核糖体上翻译。 在（a）中，氨基酸 组成、N-末端序列、表观分子量和 大鼠和人中两种主要Cp亚型的碳水化合物和铜含量 血清将被确定和比较，因为将是他们的相对丰度 在各种生理状态（炎症、癌症和雌激素 处理），以及它们在向培养的 静脉输注后细胞和组织。 在（B）中，我们将量化 并从结构上分析不同物种的牛奶中的Cp（但 特别是猪），并在大鼠和猪中测试牛奶Cp 是新生儿特别可用的铜来源，通过 特定的肠道受体。 关于（c），我们将测试 假设铁蛋白mRNA中没有铁调节元件（IRE）， 其5 'UTR（但具有信号序列）在细胞中的浓度增加。 肝脏对炎性淋巴因子的反应，并在ER上翻译， 结合多核糖体，导致“血清型”分泌增加 铁蛋白进入血液 这将在组织培养中进行研究， 肝癌细胞和IL-1，IL-6和TNF，使用免疫沉淀， 免疫测定（检测、定量和表征分泌的铁蛋白）， 来自游离和膜结合多核糖体的RNA的cDNA杂交（至 观察铁蛋白mRNA是否被驱动到ER结合的多聚核糖体， 在体内），以及在IRE-存在和不存在的情况下的体外翻译。 结合蛋白和Fe螯合剂（以检查IRE是否存在于 来自结合的多聚核糖体的mRNA），以及Fe是否介导了 对淋巴因子的反应。 我们希望这些研究能表明， 血浆铜蓝蛋白和铁蛋白具有额外的和意想不到的功能 与发育和疾病有关。

We have been studying the structure, function and regulation of mammalian proteins associated with Cu and Fe, particularly ferritin (involved in iron storage) and ceruloplasmin (involved in Cu transport). In the next few years, MBRS students are likely to be studying aspects of one of the following projects: (a) The structure, function and regulation of two major isoforms of ceruloplasmin (Cp) found in rat and human plasma; (b) the structure and function of milk Cp, which we have postulated is a significant source of Cu for the newborn; and (c) the potential stimulation of ferritin synthesis and secretion by lymphokines released in inflammation, which we have postulated involves a new form of ferritin mRNA translated on membrane-bound polyribosomes. In (a), the amino acid composition, N-terminal sequence, apparent molecular weight, and carbohydrate and copper content of two major Cp isoforms in rat and human serum will be determined and compared; as will be their relative abundance in various physiological states (inflammation, cancer and estrogen treatment), and their relative effectiveness in donating Cu to cultured cells and to tissues after intravenous infusion. In (b), we will quantify and structurally analyse Cp in the milk from various species (but especially the pig), and test in rats and pigs the hypothesis that milk Cp is an especially available source of Cu for the newborn, delivered via specific intestinal receptors. Concerning (c), we will test the hypothesis that a ferritin mRNA having no iron-regulatory element (IRE) in its 5'UTR (but having a signal sequence) increases in concentration in the liver in response to inflammatory lymphokines, and is translated on ER- bound polyribosomes, resulting in increased secretion of "serum-type" ferritin into the blood. This will be studied in tissue culture, with rat hepatoma cells and IL-I, IL-6 and TNF, using immunoprecipitation and immunoassays (to detect, quantify and characterize the ferritin secreted), cDNA hybridization of RNA from free and membrane bound polyribosomes (to observe whether ferritin mRNA is driven to the ER-bound polyribosomes, as in vivo), and in vitro translation in the presence and absence of IRE- binding protein and Fe chelators (to examine whether and IRE is present in the mRNA from the bound polyribosomes) ,and whether Fe is mediating the response to lymphokine. We expect these studies will show that ceruloplasmin and ferritin have additional and unexpected functions relating to development and disease.