Regulation of Nox Enzymes by Calcium and Novel Subunits

描述（由申请人提供）：Nox/Duox酶-产生超氧化物和次级活性氧（ROS）的NADPH氧化酶-参与正常生理学，包括细胞信号传导、先天免疫、甲状腺激素合成和重力感知。这些酶的ROS过度产生与疾病类别如过度增殖性疾病（例如，癌症、高血压、动脉粥样硬化）、纤维化疾病（肺纤维化、肝硬化、糖尿病肾病）、炎性病症（ARDS、关节炎、动脉粥样硬化）和再灌注损伤（中风、心肌梗塞、器官移植）。七种人类Nox同工酶反映了三种调节模式：1）组成型活性（Nox 4）; 2）通过与调节亚基组装而活化（Nox 1、Nox 2和Nox 3）;和3）钙激活（Nox 5、Duox 1和Duox 2）。我们将研究催化亚基的调节的分子机制，使用Nox 2作为亚基调节的Nox的代表，Nox 4作为组成型活性Nox，Nox 5作为Ca 2+调节的Nox。待探索的基本假设是，所有三种活化机制在催化部分中诱导相同的活性构象，允许电子从NADPH流动形成ROS。参与响应钙或亚基的催化亚基上的区域将被识别和表征，并将使用新开发的Nox催化亚基的同源结构模型来整合信息。我们将探索催化必需的二聚化的可能性，并将调查的关键保守的蛋白质区域的作用，通过进化比较100多个物种中的Nox酶。对Nox/Duox酶调节的分子理解将提供关键信息，这将是防止过量或不适当的ROS产生和减轻这些疾病的过程的关键。 公共卫生相关性：Nox/Duox酶产生一种称为活性氧（ROS）的自由基，其在正常生物过程中用于调节多种类型的细胞，并在白色血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，这些酶过量产生ROS会导致组织损伤和基本细胞功能异常，并在某些癌症、糖尿病并发症、中风、心血管疾病和许多其他疾病中起关键作用。为了防止这些疾病的进展，有必要了解这些酶产生过多ROS的分子变化。这项提案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通过靶向Nox/Duox酶治疗这些疾病的新药物。

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.