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

描述（由申请人提供）：血色素沉着症是一种遗传性疾病，在这种疾病中，铁在肝脏、心脏和胰腺等器官中的过度积累导致它们的损害和功能障碍。排斥性引导分子c (RGMc) /血红少年素（HJV）突变可导致少年血色素沉着症，这是该疾病在生命早期出现的一种加重形式。少年血色素沉着症患者和RGMc敲除小鼠的关键铁调节肽hepcidin的表达减少，表明RGMc通过hepcidin在铁稳态调节中起关键作用；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 （bone morphogenetic protein 2, BMP2）结合，并假设这种相互作用通过控制hepcidin的产生来调节铁代谢。该项目的重点是确定RGMc的结构决定因素，介导其与BMP2的相互作用，其长期目标是了解RGMc在铁调节及其在疾病中的失调中的作用。要验证的主要假设是，RGMc突变扰乱了蛋白质结构，从而破坏了与BMP2的结合。为实现这一目标，提出了以下具体目标：1 .明确RGMc的二级和三级结构。可溶性RGMc异构体的二级结构将通过CD测量来阐明。通过还原和非还原条件下的酶切结合，反相色谱和质谱法将建立可溶性RGMc异构体的二硫键模式。对疾病相关氨基酸替代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。2。表征RGMc与BMP2相互作用的性质。通过竞争结合实验确定BMP2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的RGMc是否优先结合BMP2。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对BMP2的结合域。为了确定RGMc是否调节BMP功能，将在使用可溶性RGMc异构体和突变体治疗后监测BMP2信号的早期事件。与公共卫生的相关性：这些研究将有助于更好地理解铁调节的机制，并确定导致疾病中过量铁积累的异常。

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.