STUDIES ON RHODOPSIN & AGE-RELATED MACULAR DEGENERATION

视紫红质的研究

• 批准号: 6855133
• 负责人: KOJI NAKANISHI
• 金额: $ 35.32万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6855133
• 关键词: DNA damage; Drosophilidae; antioxidants; apoptosis; circular dichroism; conformation; crosslink; epoxides; fluorescence resonance energy transfer; fluorescent dye /probe; high performance liquid chromatography; infrared spectrometry; interferometry; ionophores; macular degeneration; nuclear magnetic resonance spectroscopy; opsonin; protein protein interaction; protein structure function; retinoids; rhodopsin; site directed mutagenesis; transducin; visual phototransduction; vitamin biosynthesis

DESCRIPTION (provided by applicant): GPCRs (G protein coupled receptors) are involved in about half of the drugs studied by pharmaceutical companies. Absorption of light by the visual pigment, rhodopsin (Rh), the prototypical GPCR leads to cis--> trans isomerization of its 11-cis-retinal chromophore, followed by thermal relaxation of Rh through intermediates that can be sequestered at low temperatures: photo-Rh --> batho--> lumi--> meta-I--> meta-IIRh. Meta-II Rh, the key intermediate, activates the G protein (or transducin, Gt), which initiates by a cascade of enzymatic reactions resulting in a neural signal transmitted to the brain. Despite the availability of the Rh dark state X-ray structure, the conformational changes of Rh upon activation, the interactions between meta-II and Gt and the determining factors of their circular dichroism (CD) and ultra-violet (UV) spectra remain to be clarified. In this proposal the following will be studied: (i) structural aspects of Rh activation by photoaffinity crosslinking of a Gt peptide segment to Rh extramembrane loops; (ii) fluorescence resonance energy transfer (FRET)studies of Rh conformations and meta-II/Gt interactions by linking FRET donors and acceptors to the cytoplasmic loops; (iii) origin of the CD maxima of Rh by incorporation of a retinoid into single and double site-specific mutants of Rh; (iv) origin of Drosophila UV vision. Evidence is mounting that two major fiuorophores, A2E and iso-A2E, wedge-shaped amphiphilic bisretinoids that accumulate in the retinal pigment epithelium (RPE) are involved in the etiology of AMD. The following subjects will be studied in this proposal: i) further clarification of the bisretinoid biosynthetic route, especially full characterization of a key hypothetical dihydropyridinium intermediate; ii) sodium NMR and fluorescence studies of the mechanism by which A2E perturbs membranes iii) prevention of blue-light induced A2E epoxidation by antioxidants iv) perturbation of Rh function by the A2E precursor (A2-PE) which forms in photoreceptor outer segments v) examination of ability of A2E-epoxides (induced by 480 nm illumination of A2E) to damage DNA by binding covalently to nucleic acids; vi) chemical and cellular studies of the all-trans-retinal dimer, a fluorophore newly isolated from rod outer segments, the same source as the A2E pigments.

描述（由申请人提供）：GPCR（G蛋白偶联受体）参与制药公司研究的大约一半的药物。 Absorption of light by the visual pigment, rhodopsin (Rh), the prototypical GPCR leads to cis--> trans isomerization of its 11-cis-retinal chromophore, followed by thermal relaxation of Rh through intermediates that can be sequestered at low temperatures: photo-Rh --> batho--> lumi--> meta-I--> meta-IIRh.元II RH（关键中间体）激活G蛋白（或转杜氏度，GT），该蛋白通过一系列酶促反应引发，导致传播到大脑的神经信号。尽管RH暗状态X射线结构可用，但RH激活后RH的构象变化，Meta-II和GT之间的相互作用以及其圆形二科主义（CD）和Ultra-Violet（UV）光谱的确定因素仍然有待澄清。在此提案中，将研究以下内容：（i）通过将GT肽段的光性交联到RH的结构方面； （ii）通过将FRET供体和受体与细胞质环连接到RH构象和元II/GT相互作用的荧光共振能量转移（FRET）研究； （iii）RH的Cd最大值的起源是通过将视网膜定性掺入RH的单位和双位点特异性突变体中； （iv）果蝇紫外线的起源。有证据表明，两个主要的fiuorophores A2E和ISO-A2E，楔形的两亲性双蛋白素会累积在视网膜色素上皮（RPE）中，与AMD病因有关。该提案将研究以下主题：i）进一步阐明双肾上腺素生物合成途径，尤其是对主要假设二氢吡啶中间体的全面表征； ii）NMR钠和荧光研究的A2E伴有膜的机制III）预防蓝光诱导的抗氧化剂IV诱导的A2E环氧化IV）通过A2E前体（A2-PE）扰动RH功能，该功能在光段v）中的A2E前体（A2-PE）均形成了A2E-EPECTIEDS A2E-EPECTIEDS A2E-EPECIDES A2E-EPOXIDES A2E-EPOXIDES A2E-EPOXIDES A2 E2E-EPOXIDES A2 E2EIDEDS（A2E）。 A2E）通过共价与核酸结合来损害DNA； vi）全反视网膜二聚体的化学和细胞研究，一种与A2E色素相同的来源，是一种新近分离的荧光团新近分离的荧光团。