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: 7366892
• 负责人: JOHN C SALERNO
• 金额: $ 20.1万
• 依托单位: KENNESAW STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-15 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7366892
• 关键词: Active Sites; Aplysia; Apoptosis; Autoimmune Process; Binding; Biochemistry; Bioinformatics; Biology; Blood Vessels; Bos taurus; C-terminal; Calcium; Calmodulin; Carbon; Cattle; Chimera organism; Constriction procedure; Depth; 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; NOS1 protein, human; Neuraxis; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Non-Insulin-Dependent Diabetes Mellitus; Numbers; Organism; Oxidoreductase; Oxygenases; Pathology; Personal Satisfaction; Physiological; Physiological Processes; Platelet aggregation; Play; Procedures; Production; Protein Isoforms; Range; Rattus; Regulation; Regulatory Element; Research; Role; Signal Transduction; Signaling Molecule; Specificity; Structure of beta Cell of islet; Students; Tail; Testing; Vascular Diseases; Work; base; 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）：一氧化氮合酶在控制血管张力，中枢神经系统中信号转导和免疫反应等生理过程中起多种作用。一氧化氮（NO）在时间和/或空间上不适当产生几种不同的病理。 NOS（ENOS）的内皮同工型控制血管收缩和扩张，并对血小板聚集产生影响，包括高血压和其他血管疾病在内。自身免疫机制触发了iNOS的无生产，导致胰岛素依赖性和非胰岛素依赖性糖尿病的胰腺β细胞凋亡。 NOS是一种大型模块化酶，具有含有氧合酶结构域的血红素和三个域还原酶成分。通过调节从NADPH到氧合酶活性位点的电子通量来施加eNOS和NNOS的主要控制。几个输入中最重要的是钙/钙调蛋白（CA+2/CAM），但没有合成也受磷酸化和蛋白质 - 蛋白质相互作用的影响。 CA+2/CAM控制需要参与还原酶区域中的控制元件。其中最重要的是在FMN结合域中的自抑制性插入，但C末端扩展也具有影响力。提出的工作是在五个假设的左右组织的：1。在缺乏C端尾巴2的情况下，组成型表达的NOS（CNO）具有同工型特异性效应。在没有AI的情况下，C端尾部具有同工型特异性效应。 3。在存在AI 4的情况下，C末端尾部具有同工型特异性效应。自抑制元件和C末端尾部调节彼此的效果5。自抑制性元件成分具有同种型特异性效应（S）。该项目将通过创建新型的Chimert基因来检查这些假设，其中控制元素将与远距离相关的NOS酶中的认知交换。相关的嵌合体是在许多实验室中生产的，建立了该方法的可行性。通过使用控制序列的自然变异性的较大部分，我们希望大大扩展我们对控制机制的了解。适合本科参与的实验将用于评估对突变体中无合成和电子转移的控制。比以前具有控制元件嵌合体的实验，更复杂的动力学分析将使我们获得更深入的见解。