Coordinate control of hemeprotein maturation and function by cell chaperones, heme, and nitric oxide

细胞伴侣、血红素和一氧化氮协调控制血红素蛋白的成熟和功能

• 批准号: 10207671
• 负责人: DENNIS J STUEHR
• 金额: $ 52.12万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10207671
• 关键词: Apoproteins; Asthma; Autoimmune Diseases; Automobile Driving; Bacterial Infections; Binding; Biochemical; Biology; Biophysics; Blood Vessels; Cardiovascular Diseases; Cardiovascular system; Cell Culture Techniques; Cell model; Cell physiology; Cells; Client; Complex; Cytochrome P450; Cytochromes; Disease; Dissociation; Enzymes; Event; Excision; Health; Heme; Hemeproteins; Hemoglobin; Human; Infection; Inflammation; Inflammatory; Learning; Ligands; Lung; Mammalian Cell; Mammals; Mediating; Medical; Modeling; Modification; Molecular; Molecular Chaperones; Myoglobin; Nitric Oxide; Nitric Oxide Synthase; Nitrosation; Oxidoreductase; Oxygen; Pharmaceutical Preparations; Play; Process; Production; Proteins; Reaction; Recovery; Regulation; Research; Role; Signal Transduction; Site; Soluble Guanylate Cyclase; Structural Models; Structure; System; TXN gene; Testing; Tissues; Transport Process; Work; base; catalase; dimer; fighting; novel strategies; oxidation; predictive modeling; preservation; prevent; reconstitution; repaired; treatment strategy

ABSTRACT Heme proteins are fundamental in biology yet we do not understand the mechanisms that regulate their maturation and function in cells, or how they become dysfunctional in disease. Our lab is discovering new roles for cell chaperones and nitric oxide (NO) in regulating these processes. Soluble guanylate cyclase (sGC), myoglobin (Mb), and NO synthase (NOS) will serve as the model heme proteins in our proposal. sGC becomes dysfunctional in inflammatory diseases with higher NO production. Our work reveals that cells chaperones (hsp90) govern the maturation of all three hemeproteins, but the mechanisms driving their heme insertion, and in the case of sGC, that drive its inactivation, are unclear. We hypothesize: (i) In health, hsp90 supports maturation of sGC, Mb, and NOS through making a direct interaction with each client protein that enables their heme insertion, likely operating in concert with co-chaperone proteins. (ii) In disease, higher NO inactivates sGC by causing oxidation of its heme and S-nitrosation of its protein Cys groups (SNO). The SNO modifications in turn causes sGC heterodimer breakup, possible heme loss from sGC, and sGC re-association with hsp90. (iii) Cell denitrosylase (thioredoxin 1, Trx1) and sGC heme reductase (cytochrome b5r) enzymes may protect and/or repair sGC. To understand the molecular basis for these events, our Aims coordinate biochemical, biophysical, & cell-based approaches. AIM 1. How do chaperones drive heme insertion? Determine regions in apo-Mb and apo-NOS that enable complex formation with hsp90, build structural model of the complexes, test if interaction as predicted by models enable complex formation and heme insertion to occur in mammalian cells. Identify the co-chaperones and proteins that may assist hsp90 during heme insertions into the sGC, Mb, and NOS clients. Create defined protein systems to study their heme insertion. Characterize structure of hsp90-apo-hemeprotein client complexes by HxD MS and EM. AIM 2. How is sGC inactivated & how might its recovery take place? In cells and in a purified system, determine the importance of: (i) sGCβ heme occupancy & heme oxidation state in catalyzing specific SNO in modifications in sGC/, (ii) SNO modifications vs heme oxidation or loss in driving sGC heterodimer breakup & the rebinding of sGCβ to hsp90; (iii) protective/recovery mechanisms, including SNO removal by Trx1, hsp90- mediated heme reinsertion, sGCβ heme reduction by cytochrome b5r, & binding of a drug that occupies the sGCβ heme site (BAY 60). By defining the molecular & cellular mechanisms of chaperone-driven heme insertion during sGC, Mb, and NOS maturation, and the mechanisms causing sGC inactivation and recovery, our study will make fundamental contributions to our understanding of hemeprotein maturation and function, and will reveal new ways to optimize hemeprotein functions in health and disease.

摘要 血红素蛋白在生物学中是基本的，但我们并不了解调节它们的机制 细胞的成熟和功能，或它们在疾病中如何变得功能失调。我们的实验室正在发现新的角色 调节这些过程中的细胞伴侣和一氧化氮(NO)。可溶性鸟苷环化酶(SGC)， 肌红蛋白(Mb)和一氧化氮合酶(NOS)将作为我们提案中的模型血红素蛋白。SGC 在炎症性疾病中变得功能失调，产生较高的NO。我们的研究表明，细胞 伴侣蛋白(HSP90)控制着所有三种血红素的成熟，但驱动它们的血红素的机制 插入，以及在sGC的情况下，驱动其失活的因素尚不清楚。我们假设：(I)在健康方面，热休克蛋白90 通过与每个客户蛋白直接相互作用，支持sGC、Mb和NOS的成熟 使它们能够插入血红素，很可能与辅助伴侣蛋白协同工作。(Ii)在疾病方面，较高的编号 通过引起其血红素的氧化和其蛋白质半胱氨酸基(SNO)的S亚硝化而使sGC失活。SNO 修饰反过来会导致sGC异二聚体断裂、sGC可能丢失的血红素以及sGC重新结合 使用HSP90。(Iii)细胞脱氮酶(硫氧还蛋白1，Trx1)和sGC血红素还原酶(细胞色素b5r)酶 可以保护和/或修复sGC。为了了解这些事件的分子基础，我们的目标是协调 生物化学、生物物理学和基于细胞的方法。 目的1.伴侣如何驱动血红素插入？确定apo-Mb和apo-NOS中启用 用HSP90形成络合物，建立络合物的结构模型，检验模型预测的相互作用是否 能够在哺乳动物细胞中形成复合体和插入血红素。确定共同监护人和 在将血红素插入sGC、Mb和nos客户端期间可能帮助热休克蛋白90的蛋白质。创建已定义的 蛋白质系统来研究它们的血红素插入。HSP90-载脂蛋白-血红蛋白客户复合体的结构表征 经HXD MS和EM鉴定。 目标2.sgc是如何失活的，它的恢复是如何发生的？在细胞和纯化系统中， 确定：(I)sGCβ血红素占有率和血红素氧化态在催化特定SNO中的重要性 SGC/中的修饰，(Ii)sNO修饰与血红素氧化或丢失在驱动sGC杂二聚体断裂中的作用 &sGCβ与热休克蛋白90的重新结合；(Iii)保护/恢复机制，包括Trx1、热休克蛋白90- 介导的血红素重新插入，细胞色素b5r对sGCβ血红素的还原，以及占据 SGCβ血红素站点(海湾60)。 通过确定在sGC、Mb和NOS期间伴侣驱动的血红素插入的分子和细胞机制 成熟，以及导致sGC失活和恢复的机制，我们的研究将为 有助于我们理解血红蛋白的成熟和功能，并将揭示优化的新方法 血红蛋白在健康和疾病中的作用。