Molecular Sites of Neurosteroid Binding

神经类固醇结合的分子位点

• 批准号: 10357845
• 负责人: ALEX S. EVERS
• 金额: $ 58.98万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10357845
• 关键词: ART protein; Affect; Affinity; Allopregnanolone; Amino Acids; Anesthetics; Antidepressive Agents; Antiepileptic Agents; Baculoviruses; Behavior; Binding; Binding Sites; Biochemical; Biological Assay; Biology; Brain; Cell surface; Cells; Chloride Channels; Data; Development; Docking; GABA Receptor; Goals; Label; Laboratories; Libraries; Ligand Binding; Ligands; Mass Spectrum Analysis; Measures; Mediating; Methods; Molecular; Molecular Target; Muscimol; Mutate; Mutation; Neuroprotective Agents; Nootropic Agents; Outcome; Pharmaceutical Chemistry; Pharmaceutical Preparations; Photoaffinity Labels; Positioning Attribute; Pregnanolone; Production; Protein Chemistry; Protein Isoforms; Proteins; Reagent; Research; Site; Sterols; Surface; Synapses; System; Techniques; Therapeutic; Therapeutic Agents; Transferase; Transmembrane Domain; Vertebral column; Viral; Work; analog; gamma-Aminobutyric Acid; glycosylation; insight; molecular modeling; molecular site; mutant; nervous system development; neuronal excitability; neurosteroids; novel; novel therapeutics; pharmacophore; positive allosteric modulator; pregnenolone sulfate; prevent; protein expression; receptor; receptor expression; receptor function; sedative; simulation; stoichiometry; trafficking

Neurosteroids are important modulators of neuronal excitability and nervous system development with enormous therapeutic potential as anti-depressants, anesthetics and neuro-protectants. The principal molecular target of neurosteroids is the GABAA receptor. We have shown that there are multiple, subunit- specific binding sites for neurosteroids on GABAA receptors, each of which contributes to the effects of neurosteroids on receptor expression and function. This project will use photolabeling techniques to define the precise sites of neurosteroid binding on the most abundant forms of synaptic and extrasynaptic GABAA receptors. Novel neurosteroid analogue photolabeling reagents will be developed and used to determine the number of neurosteroid binding sites on each subunit, to identify the binding sites and to determine the orientation of the neurosteroids in these sites. To achieve these goals we will utilize state-of-the-art protein chemistry and expression techniques in conjunction with cutting edge mass spectrometry (MS) methods, including middle-down, intact protein and native MS. We will then mutate amino acids in each of the identified binding sites and use functional readouts to determine which binding sites mediate the various effects of neurosteroids on GABAA receptor expression and function. The Project has two specific aims: In Aim 1, a viral expression system (BacMam) will be used to express large quantities of synaptic (α1β2γ2) and extrasynaptic (α4β3δ) GABAA receptors and a suite of neurosteroid analogue photolabeling reagents will be synthesized in which the photolabeling moieties are placed at various positions around the neurosteroid backbone. The expressed receptors will be used in conjunction with the photolabeling reagents to determine the number of labeling sites on each subunit using intact protein MS and on each pentameric receptor using native MS. The specific amino acids modified by photolabeling will be identified using middle-down MS. The photolabeling data will then be used in conjunction with molecular modeling and docking to determine the preferred orientation of neurosteroids in each of the binding pockets and to identify critical residues which may be necessary for neurosteroid binding or effect. In Aim 2 receptors will be expressed in which these critical residues are mutagenized. The mutant receptors will be used in functional assays (channel gating, trafficking of receptors to the surface and modulation of orthosteric ligand binding) to determine the contribution of each binding site to neurosteroid action(s). Neurosteroid labeling and orientation will be then be assessed in the mutated receptors (as in Aim 1) to determine how the mutations alter neurosteroid binding and/or effect. The data from these studies will provide insight into the specific molecular interactions underlying neurosteroid actions at each of its binding sites on GABAA receptors. These data will provide a structural template for development of subunit- and function-specific neurosteroid analogues as well as novel insights into neurosteroid biology.

神经类固醇是神经兴奋性和神经系统发育的重要调节剂

项目成果

Click Chemistry Reagent for Identification of Sites of Covalent Ligand Incorporation in Integral Membrane Proteins.

• DOI: 10.1021/acs.analchem.6b05003
• 发表时间: 2017-02-21
• 期刊: Analytical chemistry
• 影响因子: 7.4
• 作者: Budelier MM;Cheng WW;Bergdoll L;Chen ZW;Abramson J;Krishnan K;Qian M;Covey DF;Janetka JW;Evers AS
• 通讯作者: Evers AS