STUDIES ON RHODOPSIN & AGE-RELATED MACULAR DEGENERATION

描述（由申请人提供）：gpcr （G蛋白偶联受体）与制药公司研究的大约一半的药物有关。视觉色素视紫红质（Rh）吸收光，典型的GPCR导致其11-顺式视网膜发色团的顺式->反式异构化，随后Rh通过可在低温下被隔离的中间体热松弛：光-Rh -> batho- > lumi- > meta-I- > meta-IIRh。Meta-II Rh是关键的中间体，激活G蛋白（或转导蛋白，Gt），通过一系列酶促反应启动，导致神经信号传递到大脑。尽管存在Rh暗态x射线结构，但活化后Rh的构象变化、meta-II和Gt之间的相互作用以及它们的圆二色性（CD）和紫外线（UV）光谱的决定因素仍有待阐明。在本提案中，将研究以下内容：(i)通过Gt肽段与Rh膜外环的光亲和交联激活Rh的结构方面；（ii）荧光共振能量转移（FRET）研究Rh构象和meta-II/Gt相互作用，通过将FRET供体和受体连接到细胞质环；（iii）通过将类维生素a掺入Rh的单位点和双位点特异性突变体中，导致Rh的CD最大值的来源；（四）果蝇紫外线视觉的起源。越来越多的证据表明，积聚在视网膜色素上皮（RPE）中的两种主要的荧光团，A2E和isoa2e，楔形两亲性双维甲酸参与了AMD的病因。本提案将研究以下主题：i)进一步阐明类双维甲酸生物合成路线，特别是充分表征一个关键的假设的二氢吡啶中间体；ii)钠核磁共振和荧光研究A2E扰乱膜的机制iii)抗氧化剂预防蓝光诱导的A2E环氧化iv)在光感受器外段形成的A2E前体（A2-PE）对Rh功能的扰动v) A2E环氧化物（由480nm A2E照射诱导）通过与核酸共价结合而破坏DNA的能力的检验；vi)全反式视网膜二聚体的化学和细胞研究，这是一种新从杆状细胞外节分离出来的荧光团，与A2E色素的来源相同。

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.