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.

去除血块（血栓）是止血的重要方面， 生理稳态的调节。 例如 梗塞或脑部，溶栓衰竭会导致致命的结果。 凝块网状结构的主要蛋白质是纤维蛋白，一个成昵称， 纤维蛋白原的交联衍生物（MR约350K）。 因此， 纤维蛋白聚合物或纤维蛋白溶解的脱位是关键成分 止血反应。 纤维蛋白溶解涉及一系列酶 - 催化反应，纤溶酶原（PGN），MR的糖蛋白 大约93K，被激活为纤溶酶，纤溶酶，一种蛋白酶，该蛋白酶 有效消化凝块纤维蛋白基质。 两个生理PGN 已知激活剂，组织型纤溶酶原激活剂（TPA； MR） 大约70K）和肾型纤溶酶原激活剂 （尿蛋白酶，UPA；先生大约54k）。 TPA由 血管内皮细胞响应纤维蛋白沉积及其活性 高度依赖于纤维蛋白。 相反，UPA不依赖于纤维蛋白 但是，当复合到A时，激活PGN WIT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 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是由各种组成的镶嵌蛋白 模块称为手指，生长因子（GF），Kringle（K）和 蛋白酶。 类似地，UPAR细胞外碎片包含三个 重复与蛇毒神经毒素同源的，该神经毒素结合 UPA/GF单元。 该建议解决了结构的问题 （a）pgn/k2+3以及kringles如何与alpha2-antiplasmin相互作用 生理抑制剂，以及PGN N末端肽结构域 （NTP），特别关注NTP-K1构建体； （b）; PGN K1+2+3和K1+2+3+4串联阵列（'Angiostatin'） 提议抑制肿瘤的血管化； 'tpa/k1，蛋白酶 和K2蛋白酶浓缩（BM 06.022），后者如何相互作用 使用PGN激活环，（d）UPA/K及其如何与 pgn kringles和类似肝素的聚元，（e）UPAR以及它如何 各种模块与UPA/GF域相互作用。 我们也建议 研究PGN Kringles与纤维蛋白肽的相互作用和 含有模型肽的赖氨酸。除了蛋白酶（MR 大约28K），模块很小（MR大约 10k）。我们的方法是通过蛋白水解产生单位 母体蛋白质的破碎和/或微生物表达 重组多肽以及通过 核，多维NMR光谱。 通过研究如何 模块化阵列与其底物，效应子和药物相互作用 我们期望了解PGN的方式 分子系统在帮助生成使用模型的同时起作用 纤维蛋白水解疗法，组织血管的控制和 调节恶性细胞增殖。