IDENTIFICATION OF INTERACTIONS BETWEEN NITRIC OXIDE SYNTHASE AND CALMODULIN

一氧化氮合酶和钙调蛋白之间相互作用的鉴定

• 批准号: 7359143
• 负责人: REGINA STEVENS-TRUSS
• 金额: $ 4.07万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7359143
• 关键词: IDENTIFICATION; INTERACTIONS; BETWEEN; NITRIC; OXIDE

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Nitric oxide synthase (NOS) plays a role in a variety of physiological functions from smooth muscle relaxation to cell-cell signaling in neurons. This enzyme catalyzes the oxidation of arginine to generate the multifunctional small molecule nitric oxide (¿NO). The unregulated production of ¿NO can lead to a number of pathological conditions such as stroke and Alzheimer¿s disease. There are three distinct isoforms of NOS that are differentially regulated by calmodulin (CaM). The isoforms found in neuronal and endothelial cells are regulated by the reversible binding of Ca2+/CaM, while the form found in macrophages (inducible) binds CaM tight enough to be seemingly irreversible.1 Previous research has demonstrated that the neuronal and the inducible NOS have very different requirements for which Ca2+ binding site of CaM must be Ca2+ bound in order to achieve activation, and suggests that the interactions between CaM and these two NOS isoforms is in opposite orientation.2,3 In lieu of molecular structures of these proteins, understanding this phenomenon requires studies geared at the gradual unraveling of the interactions and then piecing the information back together. The long-term objective of Dr. Stevens-Truss¿ laboratory is to understand the mechanisms underlying CaM¿s binding to and regulation of NOS. Mass spectrometry offers a tool that can be used to unravel these interactions. Traditional protein fingerprinting methodologies coupled with mass spectrometry are planned for use in this project, in an attempt to develop ways to study the interactions between CaM and the various NOS isoforms. This research is important because (i) it will increase our understanding of the overall folded structure of NOS as it relates to CaM binding and activation, (ii) it will shed light on the still enigmatic role of CaM in binding and activating over 30 different proteins, and (iii) it will advance efforts to design NOS isoform selective inhibitors. Moreover, these studies will aid in our understanding of factors that disrupt protein-protein interactions such as light energy collisions. Nitric oxide synthase (NOS) plays a role in a variety of physiological functions from smooth muscle relaxation to cell-cell signaling in neurons. This enzyme catalyzes the oxidation of arginine to generate the multifunctional small molecule nitric oxide (¿NO). The unregulated production of ¿NO can lead to a number of pathological conditions such as stroke and Alzheimer¿s disease. There are three distinct isoforms of NOS that are differentially regulated by calmodulin (CaM). The isoforms found in neuronal and endothelial cells are regulated by the reversible binding of Ca2+/CaM, while the form found in macrophages (inducible) binds CaM tight enough to be seemingly irreversible.1 Previous research has demonstrated that the neuronal and the inducible NOS have very different requirements for which Ca2+ binding site of CaM must be Ca2+ bound in order to achieve activation, and suggests that the interactions between CaM and these two NOS isoforms is in opposite orientation.2,3 In lieu of molecular structures of these proteins, understanding this phenomenon requires studies geared at the gradual unraveling of the interactions and then piecing the information back together. The long-term objective of Dr. Stevens-Truss¿ laboratory is to understand the mechanisms underlying CaM¿s binding to and regulation of NOS. Mass spectrometry offers a tool that can be used to unravel these interactions. Traditional protein fingerprinting methodologies coupled with mass spectrometry are planned for use in this project, in an attempt to develop ways to study the interactions between CaM and the various NOS isoforms. This research is important because (i) it will increase our understanding of the overall folded structure of NOS as it relates to CaM binding and activation, (ii) it will shed light on the still enigmatic role of CaM in binding and activating over 30 different proteins, and (iii) it will advance efforts to design NOS isoform selective inhibitors. Moreover, these studies will aid in our understanding of factors that disrupt protein-protein interactions such as light energy collisions.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。一氧化氮合酶 (NOS) 在从平滑肌松弛到神经元细胞间信号传导等多种生理功能中发挥着重要作用。该酶催化精氨酸氧化生成多功能小分子一氧化氮 (¿NO)。一氧化氮的不受控制的产生会导致许多病理状况，例如中风和阿尔茨海默氏病。 NOS 存在三种不同的亚型，它们受到钙调蛋白 (CaM) 的差异调节。神经元和内皮细胞中发现的亚型受 Ca2+/CaM 可逆结合的调节，而巨噬细胞（诱导型）中发现的亚型与 CaM 结合得足够紧密，看似不可逆。1 先前的研究表明，神经元和诱导型 NOS 有非常不同的要求，即 CaM 的 Ca2+ 结合位点必须与 Ca2+ 结合才能实现激活，并表明 CaM 和 CaM 之间的相互作用 这两种 NOS 亚型的方向相反。2,3 为了理解这种现象，我们需要研究逐渐阐明相互作用，然后将信息重新拼凑在一起，而不是这些蛋白质的分子结构。 Stevens-Truss 博士实验室的长期目标是了解 CaM 与 NOS 结合和调节的机制。质谱分析提供了一种可用于揭示这些相互作用的工具。该项目计划使用传统的蛋白质指纹分析方法与质谱分析相结合，试图开发研究 CaM 与各种 NOS 亚型之间相互作用的方法。这项研究很重要，因为（i）它将增加我们对 NOS 整体折叠结构的理解，因为它与 CaM 结合和激活有关，（ii）它将揭示 CaM 在结合和激活 30 多种不同蛋白质中仍然神秘的作用，（iii）它将推进设计 NOS 异构体选择性抑制剂的努力。此外，这些研究将有助于我们理解破坏蛋白质-蛋白质相互作用的因素，例如光能碰撞。一氧化氮合酶 (NOS) 在从平滑肌松弛到神经元细胞间信号传导等多种生理功能中发挥着重要作用。该酶催化精氨酸氧化生成多功能小分子一氧化氮 (¿NO)。一氧化氮的不受控制的产生会导致许多病理状况，例如中风和阿尔茨海默氏病。 NOS 存在三种不同的亚型，它们受到钙调蛋白 (CaM) 的差异调节。神经元和内皮细胞中发现的亚型受 Ca2+/CaM 可逆结合的调节，而巨噬细胞（诱导型）中发现的亚型与 CaM 结合得足够紧密，看似不可逆。1 先前的研究表明，神经元和诱导型 NOS 有非常不同的要求，即 CaM 的 Ca2+ 结合位点必须与 Ca2+ 结合才能实现激活，并表明 CaM 和 CaM 之间的相互作用 这两种 NOS 亚型的方向相反。2,3 为了理解这种现象，我们需要研究逐渐阐明相互作用，然后将信息重新拼凑在一起，而不是这些蛋白质的分子结构。 Stevens-Truss 博士实验室的长期目标是了解 CaM 与 NOS 结合和调节的机制。质谱分析提供了一种可用于揭示这些相互作用的工具。该项目计划使用传统的蛋白质指纹分析方法与质谱分析相结合，试图开发研究 CaM 与各种 NOS 亚型之间相互作用的方法。这项研究很重要，因为（i）它将增加我们对 NOS 整体折叠结构的理解，因为它与 CaM 结合和激活有关，（ii）它将揭示 CaM 在结合和激活 30 多种不同蛋白质中仍然神秘的作用，（iii）它将推进设计 NOS 异构体选择性抑制剂的努力。此外，这些研究将有助于我们理解破坏蛋白质-蛋白质相互作用的因素，例如光能碰撞。