Biochemical Characterization of the Iron-Regulatory Protein, RGMc/Hemojuvelin

铁调节蛋白 RGMc/血幼素的生化表征

• 批准号: 7779421
• 负责人: Mahta Nili
• 金额: $ 4.08万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7779421
• 关键词: Adolescent; Affinity; Amino Acid Substitution; Binding; Biochemical; Biological Assay; Disease; Event; Frameshift Mutation; Functional disorder; Goals; Growth Factor; Heart; Hemochromatosis; Hereditary Disease; Homeostasis; Inherited; Iron; Iron Overload; Iron-Regulatory Proteins; Knockout Mice; Lead; Life; Liver; Mass Spectrum Analysis; Measurement; Mediating; Monitor; Mutation; Nature; Organ; Pancreas; Patients; Pattern; Peptides; Phase; Play; Point Mutation; Production; Protein Isoforms; Public Health; Regulation; Role; Signal Transduction; Structure; Testing; base; bone morphogenetic protein 2; disulfide bond; hepcidin; iron metabolism; mutant; protein structure; reversed phase chromatography; solid solution

DESCRIPTION (provided by applicant): Hemochromatosis is a hereditary disorder in which the excessive accumulation of iron in organs such as the liver, heart, and pancreas leads to their damage and dysfunction. Mutations in repulsive guidance molecule c (RGMc) / hemojuvelin (HJV) cause juvenile hemochromatosis, an aggravated form of this disorder that presents earlier in life. Patients with juvenile hemochromatosis, and RGMc knockout mice, have diminished expression of the key iron-regulatory peptide, hepcidin, suggesting that RGMc plays a critical role in the regulation of iron homeostasis through hepcidin; however the mechanisms of RGMc actions are unknown. Recent studies have shown that RGMc binds to the growth factor, bone morphogenetic protein 2 (BMP2), and it has been postulated that this interaction regulates iron metabolism by controlling hepcidin production. The focus of this project is to define the structural determinants of RGMc that mediate its interaction with BMP2, with the long-range goal of understanding the role of RGMc in iron regulation and its dysregulation in disease. The major hypothesis to be tested is that RGMc mutations perturb protein structure such that binding to BMP2 is disrupted. To reach this goal, the follwing specific aims are proposed: I. To define the secondary and tertiary structure of RGMc. The secondary structure of soluble RGMc isoforms will be elucidated via CD measurements. Through a combination of enzymatic digests under reducing and non-reducing conditions, reverse-phase chromatography and mass spectrometry the disulfide bonding pattern of soluble RGMc isoforms will be established. Analogous studies performed on disease-associated amino acid substitution RGMc mutants will determine if point mutations perturb the structure of the protein. II. To characterize the nature of the interaction of RGMc with BMP2. Through competition binding assays the affinity of BMP2 for RGMc will be determined. Further binding studies combined with 2D analysis will determine if phosphorylated RGMc preferentially binds BMP2. By use of disease-associated frameshift mutations in solution- and solid-phase binding assays the binding domain of RGMc for BMP2 will be established. To determine if RGMc modulates BMP function, early events in BMP2 signaling will be monitored upon treatment with soluble RGMc isoforms and mutants. Relevance to public health: These studies will help to better understand the mechanisms of iron regulation, and identify what abnormalities lead to excessive iron accumulation in disease.

描述（申请人提供）：血色素沉着症是一种遗传性疾病，其中铁在肝脏、心脏和胰腺等器官中过度积累，导致其损伤和功能障碍。排斥性引导分子 c (RGMc)/血幼素 (HJV) 的突变会导致青少年血色素沉着症，这是这种疾病的一种加重形式，出现在生命早期。幼年血色病患者和 RGMc 敲除小鼠的关键铁调节肽铁调素的表达减少，表明 RGMc 通过铁调素在铁稳态调节中发挥着关键作用；然而，RGMc 的作用机制尚不清楚。最近的研究表明，RGMc 与生长因子、骨形态发生蛋白 2 (BMP2) 结合，并且推测这种相互作用通过控制铁调素的产生来调节铁代谢。该项目的重点是确定 RGMc 介导其与 BMP2 相互作用的结构决定因素，长期目标是了解 RGMc 在铁调节中的作用及其在疾病中的失调。要测试的主要假设是 RGMc 突变扰乱蛋白质结构，从而破坏与 BMP2 的结合。为实现这一目标，提出以下具体目标： 一、定义RGMc的二级和三级结构。可溶性 RGMc 亚型的二级结构将通过 CD 测量来阐明。通过结合还原和非还原条件下的酶消化、反相色谱和质谱分析，将建立可溶性 RGMc 亚型的二硫键模式。对与疾病相关的氨基酸取代 RGMc 突变体进行的类似研究将确定点突变是否会扰乱蛋白质的结构。二.表征 RGMc 与 BMP2 相互作用的性质。通过竞争结合测定，将确定 BMP2 对 RGMc 的亲和力。进一步的结合研究结合 2D 分析将确定磷酸化的 RGMc 是否优先结合 BMP2。通过在溶液和固相结合测定中使用与疾病相关的移码突变，将建立 RGMc 与 BMP2 的结合域。为了确定 RGMc 是否调节 BMP 功能，将在用可溶性 RGMc 亚型和突变体治疗后监测 BMP2 信号传导的早期事件。与公共卫生的相关性：这些研究将有助于更好地了解铁调节机制，并确定哪些异常导致疾病中铁过度积累。