Structure, function, and regulation of Thrombin-Activatable Fibrinolysis Inhibitor (TAFI)

凝血酶激活纤溶抑制剂 (TAFI) 的结构、功能和调节

• 批准号: RGPIN-2017-05571
• 负责人: Boffa, Michael
• 金额: $ 1.89万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=711730
• 关键词: Structure; function; regulation; Thrombin; Activatable

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a protein found in blood plasma that regulates blood clot breakdown, a process known as fibrinolysis. Our proposal seeks to investigate the mechanisms of how TAFI is activated and how its activity is turned off. Together, these events control the function of TAFI in normal physiology (such as prevention of bleeding) and in disease (such as the development of heart attack and strokes). TAFI is the precursor of an enzyme, and requires cleavage by another enzyme to form activated TAFI (TAFIa). The enzymes that are capable of activating TAFI are thrombin or plasmin, although both of these enzymes are very inefficient in this role. However, when thrombin interacts with a cell-surface cofactor called thrombomodulin (TM), the efficiency with which it activates TAFI increases by more than a thousand fold. While previous studies have defined particular parts of TM that are necessary to accelerate TAFI activation, it is not known how these parts contribute to the remarkable degree of acceleration that is observed. We believe that some parts of TM interact with thrombin, and other parts interact with TAFI. We plan to focus on each of these parts of TM. We will make mutations in both TM and in TAFI to define the exact amino acid residues in the respective proteins participate in the TAFI-TM interaction. We will assess the effect of the mutations on the rate of TAFI activation and on the ability of TM to bind to TAFI. In addition, we will mutate a key residue in TM that influences how TM interacts with thrombin and that appears to discriminate between the two substrates of thrombin-TM: TAFI and Protein C. These structure-function studies will be allied with molecular modeling analyses in order to corroborate the roles of the various amino acid residues we believe to be important. Most enzymes formed in the blood have matching inhibitors that bind rapidly and tightly to control the amount of enzyme that remains. TAFIa is unusual in that it lacks such an inhibitor. Instead, TAFIa activity rapidly decays with a half-life of only 8 15 minutes at body temperature. Previous findings have shown that TAFIa undergoes a large structural change as it loses activity. Some regions of the protein that may participate in this structural change have been identified, but their exact role remains a mystery. In addition, there are other regions that likely contribute, and the details of the structural change remain to be determined. We will identify all of the amino acids in TAFIa that control the rate of its inactivation by mutating particular amino acids or groups of amino acids. We will use a technique known as hydrogen-deuterium exchange (a form of structural mass spectrometry) in order to track, in real time, structural changes in TAFIa as it loses activity. These highly innovative investigations will establish new paradigms for the understanding of the structural basis for enzyme instability.

凝血酶激活纤溶抑制剂 (TAFI) 是血浆中发现的一种蛋白质，可调节血凝块分解，这一过程称为纤溶。我们的提案旨在研究 TAFI 如何激活以及如何关闭其活动的机制。这些事件共同控制 TAFI 在正常生理（如预防出血）和疾病（如心脏病和中风的发生）中的功能。 TAFI 是一种酶的前体，需要被另一种酶裂解才能形成活化的 TAFI (TAFIa)。能够激活 TAFI 的酶是凝血酶或纤溶酶，尽管这两种酶在该作用中效率非常低。然而，当凝血酶与称为血栓调节蛋白 (TM) 的细胞表面辅因子相互作用时，其激活 TAFI 的效率会增加一千倍以上。虽然之前的研究已经定义了 TM 的特定部分，这些部分是加速 TAFI 激活所必需的，但尚不清楚这些部分如何促成所观察到的显着加速程度。我们认为TM的某些部分与凝血酶相互作用，而其他部分与TAFI相互作用。我们计划重点关注 TM 的每个部分。我们将在 TM 和 TAFI 中进行突变，以定义参与 TAFI-TM 相互作用的各个蛋白质中的确切氨基酸残基。我们将评估突变对 TAFI 激活速率以及 TM 与 TAFI 结合能力的影响。此外，我们将突变 TM 中的一个关键残基，该残基影响 TM 与凝血酶的相互作用，并且似乎可以区分凝血酶-TM 的两种底物：TAFI 和蛋白 C。这些结构功能研究将与分子模型分析相结合，以证实我们认为重要的各种氨基酸残基的作用。 血液中形成的大多数酶都有匹配的抑制剂，它们可以快速而紧密地结合，以控制残留的酶量。 TAFIa 的不同寻常之处在于它缺乏这样的抑制剂。相反，TAFIa 活性迅速衰减，在体温下半衰期仅为 8 15 分钟。先前的研究结果表明，TAFIa 在失去活性时经历了巨大的结构变化。已经确定了可能参与这种结构变化的蛋白质的一些区域，但它们的确切作用仍然是个谜。此外，还有其他地区也可能做出贡献，但结构变化的细节仍有待确定。我们将鉴定 TAFIa 中通过突变特定氨基酸或氨基酸组来控制其失活速率的所有氨基酸。我们将使用一种称为氢-氘交换（结构质谱的一种形式）的技术来实时跟踪 TAFIa 失去活性时的结构变化。 这些高度创新的研究将为理解酶不稳定性的结构基础建立新的范例。