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

基本信息

批准号：
10428556
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
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10428556
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

项目摘要

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.

抽象的血红素蛋白在生物学上是基础的，但我们不了解调节其调节的机制细胞中的成熟和功能，或它们在疾病中的功能失调。我们的实验室正在发现新角色用于控制这些过程的细胞伴侣和一氧化氮（NO）。可溶性鸟苷酸环化酶（SGC），肌红蛋白（MB）和NO合酶（NOS）将在我们的提案中充当模型血红素蛋白。 SGC 在NO产生较高的炎症性疾病中变得功能失调。我们的工作表明细胞伴侣（HSP90）控制着所有三种血蛋白的成熟，但是驱动其血红素的机制插入，而在SGC的情况下，驱动其失活的插入尚不清楚。我们假设：（i）健康，HSP90 通过与每个客户蛋白进行直接相互作用来支持SGC，MB和NOS的成熟启用其血红素插入，可能与共伴侣蛋白一起运行。（ii）在疾病中，较高的NO 通过引起其血红素的氧化和其蛋白质Cys组（SNO）的S-硝化作用来使SGC失活。 sno 修改反过使用HSP90。（iii）细胞根硝基酶（硫氧还蛋白1，TRX1）和SGC血红素还原（细胞色素B5R）酶可以保护和/或维修SGC。要了解这些事件的分子基础，我们的目标是协调的生化，生物物理和基于细胞的方法。目标1。伴侣如何驱动血红素插入？确定启用Apo-MB和Apo-NOS中的区域与HSP90的复合形成，建立复合物的结构模型，测试是否按模型预测的相互作用使复合物的形成和血红素插入发生在哺乳动物细胞中。识别副伴侣，在血红素插入SGC，MB和NOS客户端时可能有助于HSP90的蛋白质。创建定义研究其血红素插入的蛋白质系统。表征HSP90-Apo-脱脂蛋白客户群络合物的结构由HXD MS和EM。 AIM 2。如何灭活SGC？如何进行恢复？在细胞和纯化系统中，确定：（i）SGCβ血红素占用和血红素氧化态在催化特定SNO中的重要性 SGC/的修改，（ii）SNO修饰与驾驶SGC Heterodimer Break的血红素氧化或损失＆将SGCβ重新固定为HSP90；（iii）保护/恢复机制，包括TRX1，HSP90-的SNO去除介导的血红素再插入，通过细胞色素B5R降低SGCβ血红素，并结合占据的药物 SGCβ血红素位点（60湾）。通过定义SGC，MB和NOS期间链酮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，我们的研究将使基本对我们对消息蛋白成熟和功能的理解的贡献，并将揭示优化的新方法血蛋白在健康和疾病中起作用。