IDENTIFICATION OF INTERACTIONS BETWEEN NITRIC OXIDE SYNTHASE AND CALMODULIN

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。一氧化氮合酶(NOS)在神经元的多种生理功能中发挥作用，从平滑肌松弛到细胞-细胞信号转导。该酶催化精氨酸氧化生成多功能小分子一氧化氮(？NO)。NO的不受控制的产生会导致许多病理情况，如中风和阿尔茨海默病S病。有三种不同的一氧化氮合酶亚型受钙调蛋白(CaM)的不同调节。在神经元和内皮细胞中发现的异构体受钙/CaM可逆结合的调节，而在巨噬细胞中发现的形式(可诱导的)与CaM结合得足够紧密，以至于似乎是不可逆转的。1先前的研究表明，神经元和可诱导的NOS对CaM的钙结合部位有非常不同的要求才能实现激活，并表明CaM和这两种NOS异构体之间的相互作用是相反的方向。2，3要了解这种现象，需要研究逐步解开相互作用，然后将信息拼接在一起。史蒂文斯-特拉斯博士实验室的长期目标是了解钙调素S与一氧化氮合酶结合和调控的机制。质谱学提供了一种工具，可以用来解开这些相互作用。这个项目计划使用传统的蛋白质指纹图谱方法和质谱学相结合的方法，试图开发研究CaM和各种NOS亚型之间相互作用的方法。这项研究之所以重要，是因为(I)它将增加我们对与CaM结合和激活有关的NOS整体折叠结构的理解，(Ii)它将揭示CaM在结合和激活30多种不同蛋白质中的神秘作用，以及(Iii)它将推动设计NOS亚型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，如光能碰撞。一氧化氮合酶(NOS)在神经元的多种生理功能中发挥作用，从平滑肌松弛到细胞-细胞信号转导。该酶催化精氨酸氧化生成多功能小分子一氧化氮(？NO)。NO的不受控制的产生会导致许多病理情况，如中风和阿尔茨海默病S病。有三种不同的一氧化氮合酶亚型受钙调蛋白(CaM)的不同调节。在神经元和内皮细胞中发现的异构体受钙/CaM可逆结合的调节，而在巨噬细胞中发现的形式(可诱导的)与CaM结合得足够紧密，以至于似乎是不可逆转的。1先前的研究表明，神经元和可诱导的NOS对CaM的钙结合部位有非常不同的要求才能实现激活，并表明CaM和这两种NOS异构体之间的相互作用是相反的方向。2，3要了解这种现象，需要研究逐步解开相互作用，然后将信息拼接在一起。史蒂文斯-特拉斯博士实验室的长期目标是了解钙调素S与一氧化氮合酶结合和调控的机制。质谱学提供了一种工具，可以用来解开这些相互作用。这个项目计划使用传统的蛋白质指纹图谱方法和质谱学相结合的方法，试图开发研究CaM和各种NOS亚型之间相互作用的方法。这项研究之所以重要，是因为(I)它将增加我们对与CaM结合和激活有关的NOS整体折叠结构的理解，(Ii)它将揭示CaM在结合和激活30多种不同蛋白质中的神秘作用，以及(Iii)它将推动设计NOS亚型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，如光能碰撞。

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.