Ferritin and Kininogen Interaction

描述（由申请人提供）：铁蛋白是一种存在于细胞内和细胞外区室中的铁结合蛋白。铁蛋白水平在炎症和恶性肿瘤中显著升高。我们的工作，以了解铁蛋白的结合伙伴，使我们确定H-激肽原（HK，高分子量激肽原）和它的切割产物，HKa，作为铁蛋白结合蛋白。HK是调节止血、炎症和血管重塑的关键蛋白。我们已经表明，HK/HKA结合铁蛋白，铁蛋白阻断HKA对内皮细胞的凋亡作用。这些结果表明，铁蛋白可能通过其对HK/HKa的影响促进血管重塑和血管生成。这些发现扩展了铁蛋白的作用，超出了铁储存，并建立了一个新的联系HK/HKA，铁蛋白，血管生成。本研究的目的是阐明铁蛋白与HK/HKa之间相互作用的机制，并了解其生物学后果。我们的具体目的是（1）确定铁蛋白调节HK/HKa对内皮细胞活性的机制。具体来说，我们将测试铁蛋白对粘附信号传导和HKa诱导的氧化应激的影响。(2)确定铁蛋白如何调节HK/HKa对血管生成的抑制作用。使用体外模型，我们将测试铁蛋白是否刺激HKa处理的内皮细胞迁移或组织进入血管。我们还将研究铁蛋白是否逆转HKa介导的体内血管生成抑制。(3)确定特定氨基酸结构域在介导HK/HKa和铁蛋白之间相互作用中的作用。我们将映射铁蛋白结合所需的HK结构域，评估铁蛋白对调节HK活性的重要特征，并使用溶液状态分析来定义HK和HK-铁蛋白复合物中的关键分子内和分子间接触。总的来说，这些实验将首次定义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异性靶点。简单总结：血管生成是从现有血管形成新血管。这一过程必须仔细调节，因为过度的血管形成会导致糖尿病和癌症等疾病。另一方面，血管生成不足也会产生负面影响，导致伤口愈合不良。我们已经发现，铁蛋白，一种天然存在的蛋白质，可能在调节血管形成中发挥以前未被怀疑的作用。我们建议测试这是如何发生的。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.