STUDIES ON RHODOPSIN & AGE-RELATED MACULAR DEGENERATION

描述（由申请人提供）：GPCR（G蛋白偶联受体）涉及制药公司研究的约一半药物。光被视色素视紫红质（Rh）吸收，原型GPCR导致其11-顺式-视网膜发色团的顺式->反式异构化，然后Rh通过可在低温下螯合的中间体热弛豫：光-Rh->浴->光->间-I->间-IIRh。关键中间体Meta-II Rh激活G蛋白（或转导素，Gt），其通过级联酶促反应启动，导致神经信号传递到大脑。尽管Rh暗态X-射线结构的可用性，活化后的Rh的构象变化，meta-II和GT之间的相互作用和它们的圆二色性（CD）和紫外（UV）光谱的决定因素仍有待澄清。本论文的研究内容包括：（i）通过Gt肽段与Rh膜外环的光亲和交联活化Rh的结构方面;（ii）通过将FRET供体和受体连接到胞质环来研究Rh构象和meta-II/Gt相互作用;（iii）起源的CD最大的Rh的类维生素A纳入单和双位点特异性突变体的Rh;（iv）起源的果蝇紫外线视觉。越来越多的证据表明，两种主要的荧光团A2 E和iso-A2 E，即在视网膜色素上皮（RPE）中积累的楔形两亲性双类视色素参与AMD的病因学。本提案将研究以下主题：i）进一步阐明双视黄酸的生物合成路线，特别是对一个关键的假设二氢吡啶中间体的全面表征; ii）A2 E扰动膜的机制的钠NMR和荧光研究iii）通过抗氧化剂防止蓝光诱导的A2 E环氧化iv）通过A2 E前体（A2-PE）扰动Rh功能其在光感受器外节中形成; v）检测A2 E-环氧化物（由A2 E的480 nm照射诱导）通过共价结合核酸而损伤DNA的能力; vi）全反式视网膜二聚体的化学和细胞研究，所述全反式视网膜二聚体是一种新分离自视杆外节的荧光团，与A2 E色素来源相同。

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.