Chemical Biology of the Visual Pigments

视觉颜料的化学生物学

基本信息

批准号：
10566896
负责人：
Philip David Kiser
金额：
$ 48.08万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-03-01 至 2027-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10566896
关键词：
ARRB1 gene Acceleration Address Affect Agonist Antibodies Architecture Binding Biochemical Biological Assay Biology Chemicals Chemistry Complex Cone Crystallography Dark Adaptation Dependence Detergents Deuterium Development Disease Gases Glean Heart Human Hydrolysis In Vitro Isomerism Isotopes Kinetics Knockout Mice Knowledge Libraries Light Light Adaptations Lipids Mass Spectrum Analysis Membrane Membrane Proteins Methodology Methods Micelles Modeling Molecular Molecular Conformation Mus Mutagenesis Natural regeneration Nature Ocular Physiology Opsin Penetration Pharmacodynamics Phase Phospholipids Photobleaching Photochemistry Photons Phototransduction Physiological Physiology Pigments Play Process Property Protein Conformation Proteins Reaction Research Retina Retinal Cone Retinal Diseases Retinal Pigments Retinaldehyde Rhodopsin Rod Outer Segments Role Sampling Schiff Bases Series Signal Transduction Stimulus Structure Structure-Activity Relationship Surface Techniques Testing Time Vertebrate Photoreceptors Vision Visual absorption chromophore cis trans isomerization experience experimental study extracellular in vivo insight interest metarhodopsin mouse model mutant nanobodies novel novel strategies pharmacokinetics and pharmacodynamics pharmacologic photoactivation receptor regenerative response sensor small molecule small molecule therapeutics structural biology therapeutic candidate tool visual cycle

项目摘要

ABSTRACT: Visual pigments initiate the human visual experience, making them of great physiological interest, and also are affected in retinal diseases. Accordingly, numerous research efforts have been devoted to characterizing their structure-function relationships. Despite these efforts, critical gaps remain in our understanding of visual pigment photochemistry and signaling properties. Knowledge of this fundamental visual physiology is necessary to make accelerated progress in developing treatments for associated retinopathies. At the heart of all visual pigments is a retinaldehyde chromophore that undergoes a cis-trans isomerization upon absorption of a photon of a suitable wavelength. This complex reaction, which proceeds through several photointermediates, triggers the conformational changes necessary for the propagation of a light stimulus into a biochemical response. This photoactivation process ends with the hydrolysis and release of retinaldehyde, which is required for renewal of the receptor light-sensitive state and hence continuous visual function. Fundamental questions remain regarding receptor structure, mechanisms and modulators of hydrolysis of the retinaldehyde Schiff base, and the modes of interaction of small molecule therapeutic candidates. Here, we will pursue four specific aims that employ newly developed tools and approaches that we believe will overcome previously insurmountable experimental challenges. 1) Elucidate structures of rhodopsin photointermediates stabilized by nanobodies. Using a novel series of camelid antibodies that arrest the rhodopsin photocycle, we will perform a detailed structure-function characterization of metarhodopsin intermediates. 2) Define the kinetics of hydrolysis of the retinaldehyde chromophores of rhodopsin and cone opsin pigments in native membranes. We have developed a novel mass spectrometry-based method that can, for the first time, directly detect the retinal conjugation state of visual pigments in native membranes; we will use this method to determine key rate constants necessary to model the interplay between visual pigment bleaching cycles and the regenerative visual cycles. 3) Assess the influence of cytosolic effectors and visual cycle components on the rate of hydrolysis of rhodopsin chromophore in knockout mouse models. Using the methods described in Aim 2, we will characterize the rate of Schiff base hydrolysis in Arr1-/-, Grk1-/-, Abca4-/-, and Rdh8-/- mice, providing new insights into how light and dark adaptation are modulated by phototransduction and visual cycle proteins. 4) Characterize the molecular architecture of rhodopsin complexes with lipids and small molecules using native mass spectrometry. Using the native MS technique, we will quantify phospholipids that associate with rhodopsin in its various activation states. We will also validate the pharmacodynamics and pharmacokinetics of small molecule therapeutic candidates in vivo. We believe the information gleaned from these studies will enhance our understanding of retinal diseases at the molecular level and enable the development of novel strategies for their treatment.

摘要：视觉颜料启动人类的视觉体验，使它们具有极大的生理兴趣，并且在视网膜疾病中也受到影响。因此，许多研究工作已致力于表征他们的结构功能关系。尽管做出了这些努力，但仍在我们的了解视觉色素光化学和信号传导特性。了解这种基本视觉生理学对于在开发相关视网膜病的治疗方面加速进展是必要的。在所有视觉颜料的心脏都是视网膜甲醛发色团，它在顺式trans trans上进行异构化。合适波长的光子吸收。这种复杂的反应，通过几个光插图，触发光刺激繁殖到一个的构象变化生化反应。这种光激活过程以视网膜水的水解和释放结尾更新受体光敏态并因此是连续的视觉功能所必需的。基本的关于视网膜醛水解的受体结构，机制和调节剂的问题仍然存在。 Schiff基础，以及小分子治疗候选物的相互作用模式。在这里，我们将追求四个采用新开发的工具和方法的特定目标克服以前无法克服的实验挑战。 1）阐明视紫红质的结构通过纳米稳定的光介体中间体。使用一系列新型的骆驼抗体，这些抗体阻碍 Rhodopsin光循环，我们将执行metarhopopsin的详细结构功能表征中间人。 2）定义视紫红蛋白和锥体的视网膜水解水解的动力学原生膜中的Opsin色素。我们已经开发了一种基于质谱的新型方法，可以首次直接检测天然膜中视觉色素的视网膜缀合状态；我们将使用此方法确定对视觉色素之间相互作用进行建模所需的关键率常数漂白周期和再生视觉周期。 3）评估胞质效应子和视觉的影响敲除小鼠模型中视紫红质发色团水解速率的循环成分。使用 AIM 2中描述的方法，我们将表征ARR1 - / - ，GRK1 - / - ，ABCA4 - / - ，中心的Schiff碱水解速率和rdh8 - / - 鼠标，提供有关光和黑暗适应如何调节的新见解光转导和视觉循环蛋白。 4）表征视紫红质的分子结构使用天然质谱法与脂质和小分子的复合物。使用本机MS技术，我们将量化与视紫红质在其各种激活状态下相关的磷脂。我们还将验证体内小分子治疗候选物的药效和药代动力学。我们相信这些研究收集的信息将增强我们对分子视网膜疾病的理解水平并使他们的治疗策略发展。