Investigation of Nitric Oxide Synthase Structure, Function and Inhibition

一氧化氮合酶结构、功能及抑制作用的研究

• 批准号: RGPIN-2017-04007
• 负责人: Guillemette, Joseph
• 金额: $ 1.89万
• 依托单位: University of Waterloo
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=710637
• 关键词: Investigation; Nitric; Oxide; Synthase; Structure

Nitric oxide (NO) is an important endogenous messenger in a variety of physiological and pathophysiological processes. NO is synthesized by the three isoforms of nitric oxide synthase (NOS) that are encoded by different genes, neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). The dual role of NO in the body as a second messenger or cytotoxic agent shows the importance for tight regulation of the different NOS isoforms. Each subunit of NOS is composed of a reductase domain that contains the binding sites for NADPH, and two flavins as well as an oxygenase domain that contains the binding sites for heme, L-arginine and tetrahydrobiopterin. The two domains are linked by a calmodulin (CaM) binding domain that plays an important role in the activation of the enzyme. CaM is the primary protein mediator of the calcium cation that is also an important secondary messenger. CaM undergoes calcium-dependent conformational changes that allow it to bind and activate scores of target proteins including all three NOS isozymes. Two of the long-term goals of this research program are to: 1) understand the mechanism(s) of CaM binding and control for all three NOS isozymes; 2) study the selective ligand binding and inhibition of human NOS isozymes. A critical structural determinant of NOS activity is the CaM controlled electron transfer from NADPH to the heme via two flavin cofactors. How CaM controls electron transfer is not fully understood and several differences have been noted between the three isoforms of NOS. Due to the large size and dynamic properties of the NOS enzymes, it is very difficult to get information on their full length protein structure. Structural information on NOS isozymes is limited to truncations encompassing their heme domains, reductase domains and the iNOS FMN subdomain bound to CaM. This is most likely due to the large conformational shift of the FMN subdomain believed to occur due to electron transfer during catalysis. As part of our proposed research program, we will perform NMR investigations on full-length as well as judiciously truncated forms of the enzymes to decipher the role of CaM and the different domains in the regulatory mechanism of the enzyme. These NMR experiments when combined with other biophysical studies and enzymes assays will provide a better understanding of the CaM dependent activation of NOS enzymes. The second major goal of our research program is to study selective binding and inhibition of human NOS isozymes. While considerable research has been performed on mammalian NOS enzymes, there is limited information on the human isozymes. We propose to use the human isozymes in our investigation and compare the results with previous studies performed on the corresponding mammalian enzymes. Our rational is based on the possibility that small differences between human and mammalian enzymes may be important in our search for isoform selective binding and enzyme inhibition.

一氧化氮（NO）是一种重要的内源性信使，参与多种生理和病理生理过程。NO是由一氧化氮合酶（NOS）的三种亚型合成的，它们由不同的基因编码，即神经元型（nNOS）、内皮型（eNOS）和诱导型（iNOS）。NO在体内作为第二信使或细胞毒性剂的双重作用显示了对不同NOS亚型的严格调节的重要性。NOS的每个亚基由含有NADPH结合位点的还原酶结构域和两个黄素以及含有血红素、L-精氨酸和四氢生物蝶呤结合位点的加氧酶结构域组成。这两个结构域通过钙调蛋白（CaM）结合结构域连接，该结构域在酶的活化中起重要作用。CaM是钙离子的主要蛋白质介体，也是重要的第二信使。钙调素经历钙依赖的构象变化，使其能够结合和激活包括所有三种NOS同工酶的靶蛋白的分数。本研究计划的两个长期目标是：1）了解CaM结合和控制所有三种NOS同工酶的机制; 2）研究人NOS同工酶的选择性配体结合和抑制。 一氧化氮合酶活性的一个关键结构决定因素是钙调素控制的电子转移从NADPH血红素通过两个黄素辅因子。钙调素如何控制电子传递还没有完全了解，已经注意到NOS的三种亚型之间的一些差异。由于NOS酶的大尺寸和动态特性，很难获得其全长蛋白质结构的信息。NOS同工酶的结构信息仅限于涵盖其血红素结构域、还原酶结构域和与CaM结合的iNOS FMN亚结构域的截短。这很可能是由于FMN亚结构域的大构象转变，据信是由于催化过程中的电子转移而发生的。作为我们提出的研究计划的一部分，我们将对全长以及明智的截断形式的酶进行NMR研究，以破译钙调素的作用和不同的结构域在酶的调节机制。 这些NMR实验与其他生物物理学研究和酶分析相结合时，将提供一个更好的理解钙调素依赖激活NOS酶。 我们研究计划的第二个主要目标是研究人NOS同工酶的选择性结合和抑制。虽然对哺乳动物NOS酶进行了大量研究，但对人类同工酶的信息有限。我们建议在我们的调查中使用人类同工酶，并与以前的研究进行相应的哺乳动物酶的结果进行比较。我们的理由是基于人类和哺乳动物酶之间的微小差异可能在我们寻找同种型选择性结合和酶抑制中很重要的可能性。