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）是多种生理和病理生理过程中重要的内源性信使。 NO 由一氧化氮合酶 (NOS) 的三种亚型合成，这三种亚型由不同基因编码：神经型 (nNOS)、内皮型 (eNOS) 和诱导型 (iNOS)。 NO 在体内作为第二信使或细胞毒剂的双重作用表明了严格调节不同 NOS 亚型的重要性。 NOS 的每个亚基均由一个包含 NADPH 结合位点的还原酶结构域、两个黄素以及一个包含血红素、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，该结合结构域在酶的激活中发挥重要作用。 CaM 是钙阳离子的主要蛋白质介体，也是重要的第二信使。 CaM 经历钙依赖性构象变化，使其能够结合并激活多种靶蛋白，包括所有三种 NOS 同工酶。该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。 NOS 活性的一个关键结构决定因素是 CaM 通过两种黄素辅助因子控制从 NADPH 到血红素的电子转移。 CaM 如何控制电子转移尚不完全清楚，并且已经注意到 NOS 的三种异构体之间存在一些差异。由于 NOS 酶的尺寸较大且具有动态特性，因此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包含其血红素结构域、还原酶结构域和与 CaM 结合的 iNOS FMN 子结构域的截短。这很可能是由于 FMN 子结构域的巨大构象转变，据信这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，我们将对酶的全长以及明智的截短形式进行 NMR 研究，以破译 CaM 的作用以及酶调节机制中的不同结构域。 这些 NMR 实验与其他生物物理研究和酶测定相结合，将更好地了解 NOS 酶的 CaM 依赖性激活。 我们研究计划的第二个主要目标是研究人类 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，但有关人类同工酶的信息有限。我们建议在我们的研究中使用人类同工酶，并将结果与​​之前对相应哺乳动物酶进行的研究进行比较。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对我们寻找异构体选择性结合和酶抑制很重要的可能性。