Regulation of Nox Enzymes by Calcium and Novel Subunits

描述(申请人提供)：NOx/DUOX酶-NADPH-产生超氧化物和次级活性氧物种(ROS)的氧化酶-参与正常生理，包括细胞信号、先天免疫、甲状腺激素合成和重力感知。由这些酶过量产生的ROS与分子损伤和信号异常有关，这些疾病包括增生性疾病(如癌症、高血压、动脉粥样硬化)、纤维性疾病(肺纤维化、肝硬变、糖尿病肾病)、炎性疾病(ARDS、关节炎、动脉粥样硬化)和再灌注损伤(中风、心肌梗死、器官移植)。人类的7种同工酶反映了三种调节模式：1)结构性激活(NOX4)；2)通过与调节亚基组装激活(NOX1、NOX2和NOX3)；以及3)钙激活(NOX5、DUOX1和DUOX2)。我们将以NOX2作为亚基调控的NOx的代表，NOX4作为结构性活性的NOx，NOX5作为钙离子调节的NOx，来研究催化亚基调控的分子机制。有待探索的基本假设是，所有三种激活机制都在催化部分诱导相同的活性构象，允许电子从NADPH流动形成ROS。将识别和表征与钙或亚基有关的催化亚基上的区域，并将使用新开发的NOx催化亚基的同源结构模型整合信息。我们将探索催化必需二聚的可能性，并将通过对多个物种中100多种NOx酶的进化比较来研究关键保守蛋白质区域的作用。对NOx/DUOX酶调控的分子理解将提供关键信息，这些信息将是防止过量或不适当的ROS产生和缓解这些疾病进程的关键。 与公共健康相关：NOx/DUOX酶会产生一种被称为活性氧物种(ROS)的自由基，它在正常的生物过程中被用来调节许多类型的细胞，并在白细胞抵抗感染的能力、甲状腺产生激素和许多其他正常功能中发挥作用。然而，在疾病中，这些酶过度产生ROS会导致组织损伤和基本细胞功能的异常，并在一些癌症、糖尿病并发症、中风、心血管疾病和许多其他疾病中发挥关键作用。为了防止这些疾病的发展，了解这些酶产生过多ROS的分子变化是至关重要的。这一提议的核心是理解这一过程的基本原理，并具有直接的影响，例如，我们有能力设计出通过靶向NOX/DUOX酶来治疗这些疾病的新型药物。

DESCRIPTION (provided by applicant): Nox/Duox enzymes - NADPH-oxidases that generate superoxide and secondary reactive oxygen species (ROS) - participate in normal physiology including cell signaling, innate immunity, thyroid hormone synthesis, and gravity perception. Over-production of ROS by these enzymes is associated with molecular damage and aberrant signaling in disease classes such as hyperproliferative disorders (e.g., cancer, hypertension, atherosclerosis), fibrotic disease (pulmonary fibrosis, cirrhosis, diabetic nephropathy), inflammatory disorders (ARDS, arthritis, atherosclerosis), and reperfusion injury (stroke, myocardial infarction, organ transplantation). The seven human Nox isoenzymes reflect three modes of regulation: 1) constitutively active (Nox4); 2) activation by assembly with regulatory subunits (Nox1, Nox2 and Nox3); and 3) calcium-activated (Nox5, Duox1 and Duox2). We will study the molecular mechanisms of regulation of the catalytic subunits, using Nox2 as representative of subunit-regulated Noxes, Nox4 as a constitutively active Nox, and Nox5 as a Ca2+regulated Nox. The underlying hypothesis to be explored is that all three activation mechanisms induce the same active conformation in the catalytic moiety, allowing electron flow from NADPH to form ROS. Regions on the catalytic subunit involved in responding to calcium or subunits will be identified and characterized, and information will be integrated using a newly developed homology structural model of the Nox catalytic subunit. We will explore the possibility of catalytically essential dimerization, and will investigate the roles of key conserved protein regions identified by an evolutionary comparison of more than 100 Nox enzymes in multiple species. A molecular understanding of the regulation of Nox/Duox enzymes will provide key information that will be key to preventing excess or inappropriate ROS generation and mitigating the course of these diseases. PUBLIC HEALTH RELEVANCE: Nox/Duox enzymes generate a form of free radical referred to as reactive oxygen species (ROS), which is used in normal biological processes to regulate many types of cells and to play a role in the ability of white blood cells to fight infections, the thyroid to produce hormones, and many other normal functions. However, in disease, overproduction of ROS by these enzymes causes both tissue damage and abnormalities in basic cellular functions, and plays a key role in some cancers, complications of diabetes, stroke, cardiovascular diseases, and many other diseases. In order to prevent the progression of these diseases, it is essential to understand the molecular changes that turn on these enzymes to produce too much ROS. This proposal centers on understanding the fundamentals of this process, and has direct implications, for example in our ability to design new classes of drugs that treat these diseases by targeting Nox/Duox enzymes.