Isoform specific effects of the autoinhibitory element and the C-terminus of nitr

自抑制元件和硝基 C 末端的亚型特异性效应

• 批准号: 7934276
• 负责人: JOHN C SALERNO
• 金额: $ 7.59万
• 依托单位: KENNESAW STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7934276
• 关键词: Active Sites; Aplysia; Apoptosis; Autoimmune Process; Binding; Biochemistry; Bioinformatics; Biology; Blood Vessels; C-terminal; Calcium; Calmodulin; Carbon; Cattle; Chimera organism; Electron Transport; Electrons; Elements; Environment; Enzymatic Biochemistry; Enzymes; Flavin Mononucleotide; Genes; Goals; Holoenzymes; Human; Hypertension; Immune response; Indium; Influentials; Insulin; Kinetics; Knowledge; Laboratories; Location; Molecular Genetics; NADP; Neuraxis; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Non-Insulin-Dependent Diabetes Mellitus; Organism; Oxidoreductase; Oxygenases; Pathology; Physiological; Physiological Processes; Platelet aggregation; Play; Procedures; Production; Protein Isoforms; Rattus; Regulation; Regulatory Element; Research; Role; Signal Transduction; Signaling Molecule; Specificity; Structure of beta Cell of islet; Students; Tail; Testing; Vascular Diseases; Work; base; constriction; coral; design; enzyme activity; experience; heme a; human NOS2A protein; human NOS3 protein; insight; mutant; novel; programs; protein protein interaction; research study; sea slug

DESCRIPTION (provided by applicant): Nitric oxide synthases play multiple roles in such diverse physiological processes as control of vascular tone, signal transduction in the central nervous system, and immune response. Temporally and/or spatially inappropriate production of nitric oxide (NO) leads to several different pathologies. The endothelial isoform of NOS (eNOS) controls vascular constriction and dilation and has effects on platelet aggregation, with pathologies including hypertension and other vascular diseases. Autoimmune mechanisms trigger NO production by iNOS, leading to apoptosis of pancreatic beta cells in both insulin-dependent and non-insulin-dependent diabetes mellitus. NOS is a large modular enzyme with a heme containing oxygenase domain and a three domain reductase component. Primary control of eNOS and nNOS is exerted through regulation of electron flux from NADPH to the oxygenase active site. The most important of several inputs is calcium/calmodulin (Ca+2/CaM), but NO synthesis is also influenced by phosphorylation and protein-protein interactions. Ca+2/CaM control requires the participation of control elements located in the reductase region. The most important of these is the autoinhibitory insertion in the FMN binding domain, but the C terminal extension is also influential. The proposed work is organized around five hypotheses: 1. The autoinhibitory element of constitutively expressed NOS (cNOS) has isoform-specific effects in the absence of the C-terminal tail 2. The C-terminal tail has isoform-specific effects in the absence of the AI. 3. The C-terminal tail has isoform-specific effects in the presence of the AI 4. The autoinhibitory element and the C-terminal tail modulate each other's effects 5. Autoinhibitory element components have isoform-specific effect(s). The project will examine these hypotheses by creating novel chimeral genes in which control elements will be exchanged with cognates in distantly related NOS enzymes. Related chimera have been produced in a number of laboratories, establishing the feasibility of the approach. By using a larger section of the naturally occurring variability of the control sequences, we hope to greatly extend our knowledge of the control mechanism. Experiments suitable for undergraduate participation will be used to evaluate control of NO synthesis and electron transfer in mutants. More sophisticated kinetic analysis will allow us to obtain deeper insights than previous experiments with control element chimera.

描述（由申请人提供）：一氧化氮合酶在多种生理过程中发挥多种作用，如控制血管张力、中枢神经系统中的信号转导和免疫应答。一氧化氮（NO）的时间和/或空间不适当的产生导致几种不同的病理。NOS的内皮同种型（eNOS）控制血管收缩和扩张，并对血小板聚集具有影响，病理学包括高血压和其他血管疾病。自身免疫机制通过iNOS触发NO产生，导致胰岛素依赖型和非胰岛素依赖型糖尿病中胰腺β细胞的凋亡。NOS是一个大的模块化酶，具有含血红素的加氧酶结构域和三结构域还原酶组分。eNOS和nNOS的主要控制通过调节从NADPH到加氧酶活性位点的电子通量来实现。几种输入中最重要的是钙/钙调蛋白（Ca+2/CaM），但NO的合成也受到磷酸化和蛋白质-蛋白质相互作用的影响。Ca+2/CaM控制需要位于还原酶区域的控制元件的参与。其中最重要的是FMN结合结构域中的自抑制插入，但C末端延伸也有影响。拟议的工作是围绕五个假设：1。组成型表达的NOS（cNOS）的自抑制元件在C-末端尾2不存在的情况下具有亚型特异性作用。C-末端尾在不存在AI的情况下具有同种型特异性作用。3. C-末端尾在AI 4存在下具有同种型特异性作用。自抑制元件和C-末端尾调节彼此的作用5。自身抑制元件组分具有同种型特异性作用。该项目将通过创造新的嵌合基因来检验这些假设，其中控制元件将与远缘NOS酶中的同源物交换。相关的嵌合体已经在一些实验室中产生，建立了该方法的可行性。通过使用更大的控制序列的自然发生的可变性，我们希望大大扩展我们的知识的控制机制。适合本科生参与的实验将用于评估突变体中NO合成和电子转移的控制。更复杂的动力学分析将使我们获得比以前的控制元件嵌合体实验更深入的见解。