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

摘要 血红素蛋白是生物学中的基础，但我们不了解调节它们的机制。 细胞的成熟和功能，或者它们如何在疾病中变得功能失调。我们的实验室正在发现新的角色 细胞伴侣和一氧化氮（NO）在调节这些过程。可溶性鸟苷酸环化酶（sGC）， 肌红蛋白（Mb）和一氧化氮合酶（NOS）将作为模型血红素蛋白。SGC 在NO产生较高的炎症性疾病中变得功能失调。我们的研究表明细胞 伴侣蛋白（hsp90）控制所有三种血红素蛋白的成熟，但驱动它们的血红素的机制 插入，以及在sGC的情况下，驱动其失活的原因尚不清楚。我们假设：（一）在健康，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， 使得它们能够插入血红素，可能与辅助分子伴侣蛋白协同作用。(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基团（SNO）的S-亚硝化使sGC失活。SNO 修饰反过来导致sGC异二聚体断裂，sGC重排可能导致血红素丢失，以及sGC重排 HSP 90 (iii)细胞脱硝酶（硫氧还蛋白1，Trx 1）和sGC血红素还原酶（细胞色素b5r） 可以保护和/或修复sGC。为了了解这些事件的分子基础，我们的目标协调 生物化学、生物物理和基于细胞的方法。 AIM 1.伴侣如何驱动血红素插入？确定apo-Mb和apo-NOS中能够 与热休克蛋白90形成复合物，建立复合物的结构模型，测试是否与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。确定共同监护人， 在血红素插入sGC底物、Mb和NOS过程中可能协助hsp90的蛋白质。创建定义的 蛋白质系统来研究血红素插入。表征HSP 90-脱辅基血红素蛋白客户端复合物的结构 通过HXD MS和EM。 AIM 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， 确定的重要性：（i）sGC β血红素占用和血红素氧化态催化特定的SNO在 （ii）SNO修饰与驱动sGC异二聚体分解中的血红素氧化或损失 sGC β与hsp90的再结合;（iii）保护/恢复机制，包括Trx 1、hsp90- 介导的血红素再插入，细胞色素b5r还原sGC β血红素，以及占据 sGC β血红素位点（BAY 60）。 通过定义sGC、Mb和NOS过程中伴侣驱动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，我们的研究将使基础 有助于我们理解血红素蛋白的成熟和功能，并将揭示新的方法来优化 血红素蛋白在健康和疾病中起作用。

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.