Structure of DREAM a Calcium Sensor in Pain Control

DREAM 疼痛控制钙传感器的结构

• 批准号: 7166041
• 负责人: JAMES B AMES
• 金额: $ 20万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7166041
• 关键词: Absence of pain sensation; Active Sites; Amino Acids; Analgesics; Behavioral; Binding; Binding Sites; Biological Assay; Brain; C-terminal; Calcium; Calcium-Binding Domain; Calorimetry; Cells; Complex; Condition; Crystallization; Crystallography; DNA; DNA Binding; DNA Sequence; Data; Detergents; Development; Drug Delivery Systems; Drug Design; Dyes; EF Hand Motifs; Electrostatics; Employee Strikes; Equilibrium; Exhibits; FOS gene; Family; Fluorescence; Genetic Transcription; Growth; Hybrids; Injury; Kinetics; Knockout Mice; Length; Maps; Measurement; Measures; Microscopic; Monitor; Motor; Mus; Mutagenesis; Mutation; Names; Neurons; Nuclear Magnetic Resonance; Numbers; Pain; Pain management; Phenotype; Promoter Regions; Proteins; Regulatory Element; Repressor Proteins; Research Personnel; Resolution; Screening procedure; Site; Site-Directed Mutagenesis; Solutions; Solvents; Speed; Structural Models; Structure; System; Testing; Therapeutic; Thermodynamics; Titrations; Transcription Repressor/Corepressor; Tryptophan; Yeasts; calsenilin; design; member; microcalorimetry; mutant; prodynorphin; programs; protein phosphatase inhibitor-2; protein structure; sensor; stoichiometry; three dimensional structure; transmission process; yeast two hybrid system

DESCRIPTION (provided by applicant): The overall objectives are to elucidate the structure and mechanism of action of a Ca2+-sensitive transcriptional repressor protein named, DREAM (Downstream Regulatory Element Antagonist Modulator). DREAM, a member of the neuronal calcium sensor subclass of the EF-hand super family, is expressed in the brain where it binds to specific DNA sequences downstream of the promoter region (DRE, Downstream Regulatory Element) and blocks transcription of downstream targets only at low Ca2+ levels. DREAM has been shown to suppress the transcription of prodynorphin and c-fos genes at basal Ca2+ levels, but does not suppress transcription at elevated Ca2+ levels caused by cell stimulation or injury. The importance of DREAM as a calcium-sensitive transcriptional repressor has been demonstrated in DREAM-deficient mice that exhibit the very striking phenotype of ongoing analgesia due to upregulated expression of prodynorphin. Importantly, the DREAM knockout mice do not exhibit any motor or behavioral abnormalities. Hence, DREAM regulates pain transmission by controlling prodynorphin expression and represents an attractive therapeutic opportunity for managing pain. The atomic-level structural characterization of DREAM and its Ca2+-regulated interaction with target DNA are crucial for the development of therapeutics designed to eliminate pain by specifically targeting DREAM. The specific aims are 4-fold: The first aim is to determine the structure of the C-terminal calcium-binding domain of DREAM in solution by nuclear magnetic resonance (NMR). The structure is important for understanding how calcium-induced conformational changes within the EF-hand motifs control the binding of DNA targets. The second aim is to measure the energetics and kinetics of DREAM binding to calcium and DNA targets. The thermodynamics and kinetics of binding will augment the structural data in the rational design of drugs that target DREAM and block pain. The third aim is to determine the active-site amino acid residues of DREAM that control binding of DNA targets. Site directed mutagenesis studies will identify important amino acid residues and structural interactions of the target complex and may reveal structural determinants important for drug design. The fourth aim is to obtain high quality protein crystals of DREAM that will then be used to solve the atomic resolution structure of the protein-DNA complex by x-ray crystallography.

描述（由申请人提供）：总体目标是阐明Ca2+敏感的转录抑制剂蛋白的作用结构和机理，称为Dream（下游调节元件拮抗剂调节器）。 Dream是EF手超级家族的神经元钙传感器子类的成员，它在大脑中表达，它与启动子区域下游的特定DNA序列（DRE，下游调节元件）结合，并阻止仅在低Ca2+水平下下游靶标的转录。梦已证明可以抑制基底Ca2+水平下的肾上飞和C-FOS基因的转录，但不会抑制由细胞刺激或损伤引起的Ca2+水平升高的转录。在梦中的小鼠中证明了梦想作为钙敏感的转录阻遏物的重要性，这些小鼠表现出由于prodynorphin的上调表达而表现出持续镇痛的惊人表型。重要的是，梦想中的淘汰小鼠不会表现出任何运动或行为异常。因此，梦想通过控制脑力表达来调节疼痛的传播，并代表了控制疼痛的有吸引力的治疗机会。梦想的原子水平结构表征及其与靶DNA的CA2+调节的相互作用对于开发旨在通过专门针对梦的疼痛的疗法的发展至关重要。具体目的是4倍：第一个目的是确定通过核磁共振（NMR）在溶液中的C末端钙结合结构域的结构。该结构对于理解钙诱导的构象如何控制DNA靶标的结合很重要。第二个目的是测量梦想与钙和DNA靶标的能量和动力学。结合的热力学和动力学将增加针对梦和阻止疼痛的药物的合理设计中的结构数据。第三个目的是确定控制DNA靶标结合的梦想中的活跃位点氨基酸残基。位置定向诱变研究将确定重要的氨基酸残基和靶复合物的结构相互作用，并可能揭示对药物设计重要的结构决定因素。第四个目的是获得梦想中的高质量蛋白质晶体，然后通过X射线晶体学来解决蛋白质-DNA复合物的原子分辨率结构。