Regulation and mechanism of nitric oxide synthase isozymes

一氧化氮（NO）是哺乳动物体内分布最广泛的信号分子之一，对控制体内几乎所有细胞和器官的功能都很重要。由于NO在调节多种器官功能方面发挥着多种生理作用，因此NO的产生缺陷也参与了许多病理生理状态，包括动脉粥样硬化、高血压、中风和糖尿病血管并发症。一氧化氮在体内由一氧化氮合酶（NOS）合成。NOS酶是迄今发现的最复杂的酶之一。NOS酶的精确控制具有潜在的治疗价值，因为NO具有多种生物学功能，也是几种严重疾病状态的关键部分。在我们能够充分利用这种靶酶的治疗潜力之前，需要回答关于NOS的大量基本问题。我们的长期研究目标是了解NOS酶的调控和机制，以及NO在细胞功能，信号传导和分化中的作用。在我们的研究中，我们使用了三种不同哺乳动物NOS酶的重组形式。位点定向诱变用于破译酶的重要区域的作用。我们通过对天然和变异形式的酶进行实验来了解它们的基本特性。例如，尽管这三种酶在与一种叫做钙调蛋白的钙感应蛋白结合时都被激活，但每种酶的激活过程却存在显著差异。我们的研究计划将提供钙调素结合和NOS酶活化的重要机制的理解。我们还将使用哺乳动物细胞培养研究进行体内研究，以了解细胞功能，稳态和分化以及酶活性如何受到诱导NOS酶翻译后加工的影响。更好地了解这些酶的功能可能有助于开发各种疾病的治疗方法。

Nitric oxide (NO) is one of the most widespread signaling molecules in mammals and important in controlling nearly every cellular and organ function in the body.    Since NO plays multiple physiological roles in regulating varied and diverse organ functions, defects in NO production are also involved in a number of pathophysiological states including atherosclerosis, hypertension, stroke and vascular complications in diabetes.  NO is synthesized in the body by enzymes named nitric oxide synthases or NOS.  NOS enzymes are some of the most complex enzymes ever discovered.  Precise control of NOS enzymes is of potential therapeutic value since NO has diverse biological functions and is also a key part of several serious disease states. A significant number of fundamental questions about NOS need to be answered before we can take full advantage of the therapeutic potential of this target enzyme.  The long-term goals of our research program are to understand the regulation and mechanism of NOS enzymes and the role of NO in cell function, signalling and differentiation.  We use the recombinant forms of the three different mammalian NOS enzymes in our investigations.  Site-directed mutagenesis is used to decipher the role of important regions of the enzymes.  We learn about their fundamental properties by performing experiments on native and mutated forms of the enzymes.  For example, although all three enzymes are activated when bound to a calcium sensing protein called calmodulin, there are significant differences in the processes leading to the activation of each enzyme.  Our research program will provide an understanding of the important mechanism of calmodulin binding and activation of NOS enzymes.  We will also perform in vivo investigations using mammalian cell culture studies to understand how cell function, homeostasis and differentiation  as well as enzyme activity and are affected by the post-translational processing of the inducible NOS enzyme.  Gaining a better understanding of how these enzymes function may help in the development of therapies for a wide variety of diseases.