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：视觉色素启动了人类的视觉体验，使其具有巨大的生理兴趣，并且也受到视网膜疾病的影响。因此，人们投入了大量的研究工作表征它们的结构-功能关系。尽管做出了这些努力，但我们在了解视觉色素光化学和信号特性。了解这一基本视觉生理学对于加速开发相关视网膜病的治疗方法是必要的。在所有视觉色素的核心是视黄醛发色团，它会发生顺反异构化吸收适当波长的光子。这个复杂的反应要经过几个过程光中间体，触发光刺激传播到光中所需的构象变化生化反应。该光活化过程以视黄醛的水解和释放而结束，是更新受体光敏状态以及因此持续视觉功能所必需的。基本的关于视黄醛水解的受体结构、机制和调节剂仍然存在疑问希夫碱，以及小分子治疗候选物的相互作用模式。在这里，我们将追求四个具体目标，采用新开发的工具和方法，我们相信这些目标将克服以前无法克服的实验挑战。 1) 阐明视紫红质的结构由纳米抗体稳定的光中间体。使用一系列新颖的骆驼抗体来阻止视紫红质光循环，我们将对变视紫红质进行详细的结构功能表征中间体。 2) 定义视紫红质和视锥细胞视黄醛发色团水解的动力学天然膜中的视蛋白色素。我们开发了一种新颖的基于质谱的方法，可以，首次直接检测视网膜原生膜视色素的结合状态；我们将使用此方法来确定模拟视色素之间相互作用所需的关键速率常数漂白周期和再生视觉周期。 3) 评估胞质效应器和视觉的影响循环成分对敲除小鼠模型中视紫红质发色团水解速率的影响。使用目标 2 中描述的方法，我们将表征 Arr1-/-、Grk1-/-、Abca4-/- 中席夫碱水解的速率，和 Rdh8-/- 小鼠，提供了关于光和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形成复合物。使用原生 MS 技术，我们将量化与各种激活状态下的视紫红质相关的磷脂。我们还将验证小分子候选治疗药物的体内药效学和药代动力学。我们相信从这些研究中收集的信息将增强我们对视网膜疾病分子水平的理解水平并能够制定新的治疗策略。