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

说明(申请人提供)：血色沉着症是一种遗传性疾病，铁在肝脏、心脏和胰腺等器官中过度聚集，导致它们的损害和功能障碍。排斥性引导分子c(RGMc)/血凝素(HJV)的突变会导致青少年血色沉着症，这是这种疾病的一种加重形式，在生命的早期出现。幼年血色沉着症患者和RGMc基因敲除小鼠的关键铁调节多肽--海普西丁的表达减少，这表明RGMc通过海普西丁在铁稳态的调节中发挥关键作用；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，推测这种相互作用通过控制海普西丁的产生来调节铁的代谢。该项目的重点是确定RGMc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。需要检验的主要假设是，RGMc突变扰乱了蛋白质结构，从而破坏了与BMP2的结合。为了实现这一目标，提出了以下具体目标：1.明确RGMc的二级结构和三级结构。可溶性RGMc亚型的二级结构将通过CD测量来阐明。通过还原和非还原条件下的酶消化、反相色谱和质谱分析相结合的方法，建立了可溶性RGMc亚型的二硫键模式。对疾病相关氨基酸替换RGMc突变体进行的类似研究将确定点突变是否扰乱了蛋白质的结构。II.研究RGMc与BMP2相互作用的性质。通过竞争结合实验确定BMP2与RGMc的亲和力。结合2D分析的进一步结合研究将确定磷酸化的RGMc是否优先与BMP2结合。通过在溶液和固相结合分析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，在用可溶性RGMc亚型和突变体处理后，将监测BMP2信号的早期事件。与公众健康相关：这些研究将有助于更好地了解铁的调节机制，并确定哪些异常会导致疾病中铁的过度积累。

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.