Chemical Biology of the Visual Pigments

视觉颜料的化学生物学

• 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
• 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

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：视觉色素开启了人类的视觉体验，使其具有极大的生理价值， 在视网膜疾病中也会受到影响。因此，许多研究工作都致力于 表征它们的结构-功能关系。尽管作出了这些努力，但我们的 了解视觉色素的光化学和信号特性。对这种基本视觉的了解 在开发相关视网膜疾病的治疗方法方面，生理学是必要的。在… 所有视觉色素的核心是视黄醛生色团，它在 吸收适当波长的光子。这个复杂的反应，通过几个 光中间体，触发光刺激传播到 生化反应。这个光活化过程以视黄醛的水解和释放而结束，这是 是受体光敏状态更新所必需的，因此是持续的视觉功能。基本原理 关于视黄醛的受体结构、机制和调节剂的问题仍然存在。 席夫碱，以及小分子治疗候选药物的相互作用模式。 在这里，我们将追求四个具体目标，这些目标采用了我们相信将 克服以前难以克服的实验挑战。1)阐明视紫红质的结构 纳米体稳定的光中间体。使用一系列新的骆驼抗体来阻止 视紫红质的光周期，我们将进行详细的结构和功能的表征 中间体。2)确定视紫红质和视锥细胞视黄醛生色团的水解动力学。 天然膜中的视黄素色素。我们开发了一种新的基于质谱学的方法， 首次直接检测天然膜中视色素的视网膜结合状态；我们将 使用这种方法来确定对视觉色素之间的相互作用进行建模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 基因敲除小鼠模型中循环成分对视紫红质发色团水解率的影响。使用 方法在目标2中描述，我们将表征Arr1-/-，Grk1-/-，ABCa4-/-， 和Rdh8-/-小鼠，为光和暗适应是如何通过 光转导和视觉循环蛋白。4)表征视紫红质的分子结构 天然质谱法测定与脂类和小分子的络合物。使用原生MS技术，我们 将量化与视紫红质相关的各种激活状态的磷脂。我们还将验证 小分子候选药物在体内的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从分子上增强我们对视网膜疾病的理解 这一水平的提高，使人们能够制定新的治疗策略。