HOMOCYSTEINE, NITRIC OXIDE AND HEME: ROLE IN THROMBOSIS

同型半胱氨酸、一氧化氮和血红素：在血栓形成中的作用

• 批准号: 6390800
• 负责人: Judith N Burstyn
• 金额: $ 23.82万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-05 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6390800
• 关键词: binding sites; circular magnetic dichroism; cystathionine beta synthase; electron spin resonance spectroscopy; enzyme activity; enzyme induction /repression; enzyme structure; heme; homocysteine; nitric oxide; structural biology; thrombosis

Homocysteine stimulates vascular smooth muscle and endothelial nitric oxide (NO) production and impairs NO anticoagulant and vasodilatory functions, raising he possibility of a feed back loop, whereby NO regulates the levels of homocysteine through cystathionine beta-synthase (CBS). Dysfunction in this putative feedback loop may constitute a novel mechanism underlying certain types of thromboembolic disease. The studies described herein will provide insight into the role of heme in homocysteine metabolism, and reveal whether there is heme-related cross-talk between the trans-sulfuration and NO/cGMP pathways. Our studies will focus on CBS, a heme-containing enzyme that is critical to the control of physiological homocysteine levels. There are two overarching goals of this sub-project within the collaborative effort described in the accompanying proposal from Jan P. Kraus entitled "Homocysteine and Thrombosis"> The goal of this project are: 1) to elucidate the role of heme in CBS, and 2) to determine whether there is interaction between homocysteine and NO in vascular pathology. The biochemical studies described herein will be complemented by in vivo studies carried out in the Kraus laboratory. The primary hypothesis of this proposal is that heme in CBS is serving a regulatory function, similar to the roles now attributed to the heme moiety in the NO receptor, soluble guanylyl cyclase. We hypothesize that the heme is regulatory and that NO, carbon monoxide or another physiologically relevant is the regulator in vivo. The goal of the research proposed herein is to test this hypothesis. Our laboratory will test the biochemical feasibility, focusing primarily on characterizing the purified CBS protein and its interaction with exogenous ligands through spectroscopic and enzymological studies. The Kraus laboratory will explore the physiological feasibility through studies in tissue culture and knockout mouse models. There are three Specific Aims that we will address: 1) we will determine CBS is regulated by No or other heme ligands by correlating changes in the heme absorption spectrum on ligand binding with changes in enzyme activity, 2) we will identify structural changes at the CBS heme site relevant to enzyme function, characterizing the active core heme site and its ligand adducts by EPR and MCD spectroscopy, 3) we will apply the methods of Aim 1 and 2 to identify differences between the CBS active core and the full length polypeptide, correlating regulation by heme and regulation by Adomet.

同型半胱氨酸刺激血管平滑肌和内皮一氧化氮 (NO) 的产生，并损害 NO 的抗凝和血管舒张功能，从而增加了反馈循环的可能性，其中 NO 通过胱硫醚β-合酶 (CBS) 调节同型半胱氨酸的水平。这种假定的反馈环路的功能障碍可能构成某些类型的血栓栓塞性疾病的潜在新机制。本文描述的研究将深入了解血红素在同型半胱氨酸代谢中的作用，并揭示反式硫酸化和 NO/cGMP 途径之间是否存在与血红素相关的串扰。我们的研究将集中在 CBS，这是一种含血红素的酶，对于控制生理同型半胱氨酸水平至关重要。 Jan P. Kraus 题为“同型半胱氨酸和血栓形成”的随附提案中描述了该子项目的两个总体目标。该项目的目标是：1) 阐明血红素在 CBS 中的作用，以及2)确定同型半胱氨酸和NO在血管病理学中是否存在相互作用。本文描述的生化研究将由克劳斯实验室进行的体内研究补充。该提议的主要假设是，CBS 中的血红素具有调节功能，类似于现在归因于 NO 受体（可溶性鸟苷酸环化酶）中血红素部分的作用。我们假设血红素是调节性的，而一氧化氮、一氧化碳或其他生理相关的物质是体内的调节器。本文提出的研究的目的是检验这一假设。我们的实验室将测试生化可行性，主要侧重于通过光谱和酶学研究表征纯化的 CBS 蛋白及其与外源配体的相互作用。克劳斯实验室将通过组织培养和基因敲除小鼠模型的研究来探索生理可行性。我们将解决三个具体目标：1）我们将通过将配体结合的血红素吸收光谱的变化与酶活性的变化相关联，确定 CBS 是否受 1 或其他血红素配体调节，2）我们将确定 CBS 的结构变化与酶功能相关的 CBS 血红素位点，通过 EPR 和 MCD 光谱表征活性核心血红素位点及其配体加合物，3) 我们将应用目标 1 和 2 的方法来识别 CBS 活性之间的差异核心和全长多肽，将血红素的调节与 Adomet 的调节相关联。