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: 7615836
• 负责人: Mahta Nili
• 金额: $ 4.06万
• 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/7615836
• 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（由申请人提供）：血色病是一种遗传性疾病，其中铁在肝脏、心脏和胰腺等器官中的过度积累导致其损伤和功能障碍。排斥性导向分子c（RGMc）/血幼素（HJV）的突变会导致青少年血色沉着病，这是一种在生命早期出现的这种疾病的加重形式。青少年血色素沉着症患者和RGMc基因敲除小鼠的关键铁调节肽hepcidin的表达减少，表明RGMc通过hepcidin在调节铁稳态中起关键作用;然而，RGMc作用的机制尚不清楚。最近的研究表明，RGMc结合到生长因子，骨形态发生蛋白2（BMP 2），它已被假定，这种相互作用通过控制铁调素生产调节铁代谢。该项目的重点是定义RGMc的结构决定因素，介导其与BMP 2的相互作用，长期目标是了解RGMc在铁调节及其在疾病中的失调中的作用。待检验的主要假设是RGMc突变扰乱蛋白质结构，从而破坏与BMP 2的结合。为实现这一目标，提出了以下具体目标：一、加强教育。定义RGMc的二级和三级结构。将通过CD测量阐明可溶性RGMc亚型的二级结构。通过在还原和非还原条件下结合酶促反应、反相色谱法和质谱法，确定可溶性RGMc亚型的二硫键模式。对疾病相关氨基酸取代RGMc突变体进行的类似研究将确定点突变是否扰乱蛋白质的结构。二.表征RGMc与BMP 2相互作用的性质。通过竞争结合测定，将确定BMP 2对RGMc的亲和力。结合2D分析的进一步结合研究将确定磷酸化RGMc是否优先结合BMP 2。通过在溶液和固相结合测定中使用疾病相关的移码突变，将建立RGMc对BMP 2的结合结构域。为了确定RGMc是否调节BMP功能，将在用可溶性RGMc同种型和突变体处理后监测BMP 2信号传导中的早期事件。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的机制，并确定哪些异常导致疾病中过量的铁积累。