Ferritin and Kininogen Interaction

描述(申请人提供)：铁蛋白是一种铁结合蛋白，存在于细胞内和细胞外。在炎症和恶性肿瘤中，铁蛋白水平显著升高。为了了解铁蛋白的结合伙伴，我们鉴定了H-激肽原(HK，高分子激肽原)及其裂解产物Hka为铁蛋白结合蛋白。HK是调节止血、炎症和血管重塑的关键蛋白。我们已经证明HK/Hka与铁蛋白结合，并且铁蛋白阻断了Hka对内皮细胞的凋亡效应。提示铁蛋白可能通过对HK/Hka的作用促进血管重塑和血管生成。这些发现将铁蛋白的作用扩展到铁储存之外，并在HK/Hka、铁蛋白和血管生成之间建立了新的联系。这项建议的目的是阐明铁蛋白与HK/Hka相互作用的机制，并了解其生物学后果。我们的具体目标是：(1)明确铁蛋白调节内皮细胞HK/Hka活性的机制(S)。具体地说，我们将测试铁蛋白对黏附信号和Hka诱导的氧化应激的影响。(2)探讨铁蛋白对HK/Hka抑制血管生成作用的调节作用。使用体外模型，我们将测试铁蛋白是否刺激经Hka处理的内皮细胞迁移或组织到血管中。我们还将研究铁蛋白是否能逆转Hka介导的体内血管生成抑制。(3)确定特定的氨基酸结构域在介导HK/Hka与铁蛋白相互作用中的作用。我们将绘制铁蛋白结合所需的HK结构域，评估铁蛋白对调节HK活性的重要特性，并利用溶液状态分析确定HK和HK-铁蛋白复合体中关键的分子内和分子间联系。总之，这些实验将首次确定铁蛋白在参与控制血管重塑和血管生成的基本生物学机制中的作用，并确定铁蛋白和HK/Hka中可用于调节这些过程的特定靶点。综述：血管生成是指从现有血管形成新的血管。这一过程必须仔细监管，因为过度的血管形成可能会导致糖尿病和癌症等疾病。另一方面，血管生成不足也可能产生负面影响，导致伤口愈合不良。我们发现，铁蛋白，一种自然存在的蛋白质，可能在调节血管形成方面发挥了以前没有被怀疑的作用。我们建议测试这种情况是如何发生的。最终，这将提高我们对健康和疾病中血管形成的理解，并可能导致控制这一过程的方法。

DESCRIPTION (provided by applicant): Ferritin is an iron binding protein that is present in both intracellular and extracellular compartments. Levels of ferritin are markedly elevated in inflammation and malignancy. Our work to understand the binding partners of ferritin led us to identify H-kininogen (HK, high molecular weight kininogen) and its cleavage product, HKa, as ferritin binding proteins. HK is a key protein that modulates hemostasis, inflammation, and vascular remodeling. We have shown that HK/HKa binds to ferritin and that ferritin blocks the apoptotic effects of HKa on endothelial cells. These results suggest that through its effects on HK/HKa, ferritin may promote vascular remodeling and angiogenesis. These findings expand the role of ferritin beyond that of iron storage, and forge a new link between HK/HKa, ferritin, and angiogenesis. The goal of this proposal is to elucidate the mechanism of the interaction between ferritin and HK/HKa, and to understand its biological consequences. Our Specific Aims are to (1) Identify the mechanism(s) by which ferritin regulates the activity of HK/HKa on endothelial cells. Specifically, we will test the effect of ferritin on adhesive signaling and HKa- induced oxidant stress. (2) Determine how ferritin regulates inhibitory effects of HK/HKa on angiogenesis. Using in vitro models, we will test whether ferritin stimulates migration or organization of HKa-treated endothelial cells into blood vessels. We will also examine whether ferritin reverses the HKa-mediated inhibition of angiogenesis in vivo. (3) Identify the role of specific amino acid domains in mediating the interaction between HK/HKa and ferritin. We will map HK domains required for ferritin binding, evaluate characteristics of ferritin important to regulation of HK activity, and define critical intra- and intermolecular contacts in HK and the HK-ferritin complex using solution state analysis. Collectively, these experiments will define for the first time the role of ferritin in basic biological mechanisms involved in the control of vascular remodeling and angiogenesis, and identify specific targets in both ferritin and HK/HKa that can be used to modulate these processes. Lay summary: Angiogenesis is the formation of new blood vessels from existing blood vessels. This process must be carefully regulated, because excessive blood vessel formation can contribute to diseases such as diabetes and cancer. On the other hand, insufficient angiogenesis can also have negative effects, contributing to poor wound healing. We have found that ferritin, a naturally occurring protein, may play a previously unsuspected role in regulating blood vessel formation. We propose to test how this occurs. Ultimately, this will improve our understanding of blood vessel formation in health and disease, and may lead to ways to control this process.