FUNCTION OF THE HEMOCHROMATOSIS PROTEIN

描述(申请人提供)：遗传性血色沉着症(HH)是北欧后裔中最常见的遗传性疾病。超过83%的HH病例是由HH蛋白HFE中的Cys到Tyr的单一突变引起的。这种突变导致一种隐性疾病，导致铁在选定的组织中积累。铁超载会损害这些器官，导致肝硬变、糖尿病、心肌病和关节炎。HFe影响细胞和体内铁的动态平衡的机制仍然不清楚。人类缺乏功能性的HFE会在体内不同类型的细胞中产生相反的效果。在疾病的早期阶段，肝脏中的Kupffer细胞和肠细胞中的肠细胞铁耗竭，细胞内铁蛋白水平较低，而肝脏中的肝细胞铁超载，细胞内铁水平较高。这些是HFE功能的直接影响还是间接影响尚不清楚。为了解决这个问题，并了解HFe影响铁稳态的分子机制，已经发展了一些细胞系，当HFe转染后，细胞中的铁水平要么增加，要么降低。这项建议的目的集中在寻找HFE功能在不同细胞类型中相反影响的基础。HFe通过与不同细胞系中的不同蛋白质相互作用来改变铁的动态平衡的假设将得到验证。嵌合的HFE蛋白将被用来定义导致铁稳态变化的HFE结构域，并将使用定点突变来更精细地定位这些结构域。HFe蛋白复合体可能直接调节细胞内的铁水平，或者不依赖于细胞内的铁水平而发出信号。功能上与HFE偶联的新基因将在高通量筛选中被识别出来。HFe以铁依赖的方式改变基因表达的假设将通过表达和不表达HFe的细胞的微阵列分析来验证。最后，HFe仅通过控制细胞内铁水平来调节肝细胞中铁的动态平衡的假设将通过肝细胞系和胚胎肝细胞来验证。这项工作的长期目标是了解HFE的功能。

DESCRIPTION (provided by applicant): Hereditary hemochromatosis (HH) is the most common inherited disorder in people of Northern European descent. Over 83% of the cases of HH result from a single mutation of a Cys to Tyr in the HH protein, HFE. This mutation causes a recessive disease resulting in an accumulation of iron in selected tissues. Iron overload damages these organs leading to cirrhosis of the liver, diabetes, cardiomyopathy, and arthritis. The mechanism by which HFE influences iron homeostasis in cells and in the body remains elusive. Lack of functional HFE in humans produces the opposite effects in different cell types in the body. In the early stages of the disease, Kupffer cells in the liver and enterocytes in the intestine cells are iron depleted and have low intracellular ferritin levels, whereas hepatocytes in the liver are iron overloaded and have high intracellular iron levels. Whether these are direct or indirect effects of HFE function is not known. In order to address this question and to understand the molecular mechanisms by which HFE influences iron homeostasis, cell lines have been developed that when transfected with HFE show either increases or decreases in iron levels. The aims of this proposal are focused on finding the basis of the opposite effects of HFE function in different cell types. The hypothesis that HFE alters iron homeostasis by interacting different proteins in different cell lines will be tested. Chimeric HFE proteins will be made to define the domains of HFE responsible for the alterations in iron homeostasis and site-directed mutagenesis will be used for finer mapping of the domains. The HFE protein complex could be directly regulating intracellular iron levels or signaling independently of intracellular iron levels. New genes that functionally couple to HFE will be identified in a high throughput screen. The hypothesis that HFE alters gene expression in an iron-dependent manner will be tested by microarray analysis of cells expressing and not expressing HFE. Finally, the hypothesis that HFE acts to regulate iron homeostasis in hepatocytes solely through controlling intracellular iron levels will be tested using a hepatic cell line and orimary hepatocytes. The long term goal of this work is to understand the function of HFE.