NMR STUDIES OF THE PLASMINOGEN SYSTEM

纤溶酶原系统的核磁共振研究

• 批准号: 2838906
• 负责人: MIGUEL LLINAS
• 金额: $ 31.82万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-07-01 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2838906
• 关键词: angiostatins; antifibrinolytic agents; chemical association; chemical binding; conformation; endopeptidases; intermolecular interaction; molecular cloning; molecular dynamics; nuclear magnetic resonance spectroscopy; plasminogen; plasminogen activator; protein structure function; recombinant proteins; site directed mutagenesis; urokinase

Removal of blood clots (thrombi) is an important aspect of hemostasis, the regulation of the physiological steady state. As exemplified by infarct or brain stroke, thrombolytic failure can lead to fatal outcomes. The main protein structuring the clot meshwork is fibrin, a nicked, crosslinked derivative of fibrinogen (Mr approximately 350K). Thus, defradation of the fibrin polymer, or fibrinolysis, is a key component of the hemostatic response. Fibrinolysis involves a cascade of enzyme- catalyzed reactions whereby plasminogen (Pgn), a glycoprotein of Mr approximately 93K, becomes activated to plasmin, a proteinase which effectively digests the clot fibrin matrix. Two physiological Pgn activators are known, the tissue-type plasminogen activator (tPA; Mr approximately 70K) and the kidney-type plasminogen activator (urokinase, uPA; Mr approximately 54K). TPA is secreted by the vessel endothelial cells in response to fibrin deposition, and its activity is highly fibrin-dependent. UPA, in contrast, is not fibrin-dependent but activates Pgn wit 100-fold higher efficiency when complexed to a receptor, uPAR (Mr approximately 58K). Pgn activation via the uPA- uPAR complex is important for cell migration and metastasis as the generated plasmin plays an important role in degrading tissue matrix proteins.Pgn, tPA, uPA are mosaic proteins composed of various modules known as finger, growth factor (GF), kringle (K) and protease. Similarily, the uPAR extracellular fragment contains three repeats homologous to snake venom neurotoxins, which bind the uPA/GF unit. This proposal addresses the questions of the structure of (a) Pgn/K2+3 and how kringles interact with alpha2-antiplasmin, its physiological inhibitor, and with thePgn N-terminal peptide domain (NTP), with particular focus on the NTP-K1 construct; (b); the Pgn K1+2+3 and K1+2+3+4 tandem arrays ('angiostatin') which have been proposed to inhibit vascularization of tumors; ' tPA/K1, protease and a K2 protease consruct (BM 06.022) and how the latter interact with the Pgn activation loop, (d) uPA/K and how it interacts with the Pgn kringles and heparin-like polyanions, (e) uPAR and how its various modules interact with the uPA/GF domain. We also propose to investigate the way Pgn kringles interact with fibrinopeptides and lysine containing model peptides. Except for the protease (Mr approximately 28K), the modules are rather small (Mr approximately 10K). Our approach is to generate the units via proteolytic fragmentation of the parent proteins and/or microbial expression of the recombinant polypeptides, and analysis of the various constructs via heternuclear, multidimensional NMR spectroscopy. By studying how the modular arrays interact with their substrates, effectors and drugs of clinical interest we expect to gain understanding of the way the Pgn molecular system works while helping generate models of use in fibrinolytic therapy, the control of tissue vascularization and the regulation of malignant cell proliferation.

清除血栓（血栓）是止血的一个重要方面， 生理稳态的调节。 举例来说 梗塞或脑中风、溶栓失败可能导致致命的后果。 构成血块网络的主要蛋白质是纤维蛋白，一种有切口的、 纤维蛋白原的交联衍生物（Mr约350K）。 因此， 纤维蛋白聚合物的破坏或纤维蛋白溶解是关键组成部分 的止血反应。 纤维蛋白溶解涉及一系列酶- 纤溶酶原 (Pgn)（Mr 的一种糖蛋白）催化的反应 大约 93K，被激活为纤溶酶，一种蛋白酶， 有效消化凝块纤维蛋白基质。 两个生理Pgn 激活剂是已知的，组织型纤溶酶原激活剂（tPA；Mr 约70K）和肾型纤溶酶原激活剂 （尿激酶、uPA；先生约 54K）。 TPA 是由 血管内皮细胞对纤维蛋白沉积的反应及其活性 具有高度纤维蛋白依赖性。 相比之下，UPA 不依赖于纤维蛋白 但当与 a 复合时，Pgn 的激活效率提高 100 倍 受体，uPAR（Mr约58K）。 通过 uPA 激活 Pgn uPAR 复合物对于细胞迁移和转移很重要，因为 产生的纤溶酶在降解组织基质中起重要作用 蛋白质。Pgn、tPA、uPA 是由多种成分组成的嵌合蛋白 称为手指、生长因子 (GF)、kringle (K) 和 蛋白酶。 类似地，uPAR胞外片段包含三个 重复与蛇毒神经毒素同源，它结合 uPA/GF 单位。 该提案解决了结构问题 (a) Pgn/K2+3 以及 kringles 如何与 alpha2-抗纤溶酶相互作用，其 生理抑制剂，并具有Pgn N端肽结构域 (NTP)，特别关注 NTP-K1 结构； （二）； PGN K1+2+3 和 K1+2+3+4 串联阵列（“血管抑制素”）具有 被提议抑制肿瘤的血管形成； ' tPA/K1、蛋白酶 和 K2 蛋白酶结构 (BM 06.022) 以及后者如何相互作用 与 Pgn 激活环路，(d) u​​PA/K 以及它如何与 Pgn kringles 和类肝素聚阴离子，(e) uPAR 及其如何 各种模块与 uPA/GF 域相互作用。 我们还建议 研究 Pgn kringles 与纤维蛋白肽相互作用的方式 含有赖氨酸的模型肽。除了蛋白酶（先生 大约28K），模块相当小（先生大约 10K）。我们的方法是通过蛋白水解生成单元 亲本蛋白的片段化和/或微生物表达 重组多肽，并通过以下方式分析各种构建体 异核、多维核磁共振波谱。 通过研究如何 模块化阵列与其基底、效应物和药物相互作用 临床兴趣，我们希望了解 Pgn 的方式 分子系统在帮助生成使用模型的同时发挥作用 纤溶治疗、组织血管化的控制和 恶性细胞增殖的调节。