Functional domains in melanopsin: natural variants and molecular engineering

视杆细胞和视锥细胞是视网膜中对光敏感的细胞，负责弱光视觉和色觉。最近，在视网膜中发现了第三种类型的光敏细胞，它表达一种名为黑素的蓝光敏感蛋白。这些细胞被称为光敏性视网膜神经节细胞(PRGC)，它们与大脑内的许多结构相连，控制一系列对光的非视觉反应，包括诱导睡眠、瞳孔光反射和对日常光周期的夹带。最近，这些细胞被证明在视觉通路、情绪和抑郁中发挥作用，并正在成为调节人类生理和健康的关键生物通路。视杆细胞和视锥细胞光敏分子的突变会导致一系列视网膜疾病，是导致失明的主要原因。在人类群体中也检测到黑色素的突变和多态，导致对光的生理反应发生变化，并与季节性情感障碍有关。了解突变对黑素特性的影响是预测其对人类生理和健康影响的基础。与所有生物分子一样，黑素的结构是其功能的关键。然而，黑素分子的不同区域发挥的功能作用还没有得到很好的确定。在这项研究中，我们将产生一些在蛋白质特定区域内含有靶向性变化的黑素分子，作为一种手段来确定这些区域在一些关键功能中所扮演的角色。我们主要将专注于对已知存在于人类群体中的突变进行建模，并确定发现这些突变的分子区域所发挥的一般作用。此外，我们还将确定影响黑素蛋白对光的敏感性的区域，特别是该蛋白质对光的颜色做出反应的区域(称为光谱调谐位置)。在视杆细胞和视锥细胞中发现的对光敏感的视蛋白分子也进行了类似的研究，但黑素视蛋白代表了一类功能不同的视蛋白，具有重要的功能和结构差异。到目前为止，还没有研究过这类非视觉视光的光谱调谐。作为这项研究的结果，我们将产生一系列对不同颜色的光产生反应的黑色素变体。这些变体在研究应用方面具有巨大的潜力，其中黑素的表达可以用来增强对光的敏感性，并允许在体外和体内选择性地激活感兴趣的细胞类型，以及用于治疗视网膜疾病和恢复视力的基于基因治疗的方法。这项工作计划将从根本上增加我们对黑素蛋白的了解，这是一种在人类生理和健康中发挥重要作用的蛋白质。这项研究还将提供一系列适用于广泛生物学研究领域的新研究工具，并将开发用于治疗人类疾病的新工具。

Rods and cones are light sensitive cells in the retina responsible for dim light vision and colour vision. More recently a third type of light sensitive cell has been found in the retina, which express the blue-light sensitive protein called melanopsin. These cells, termed photosensitive retinal ganglion cells (pRGCs), send connections to numerous structures within the brain and control a range of non visual responses to light including the induction of sleep, pupil light reflexes and entrainment to daily light cycles. More recently these cells have been shown to perform roles in visual pathways, mood and depression, and are emerging as a key biological pathway regulating human physiology and health.Mutations in the light sensitive molecules of rods and cones lead to a range of retinal diseases and are a leading cause of blindness. Mutation and polymorphisms in melanopsin have also been detected in the human population, resulting in changes in physiological responses to light and have been associated with seasonal affective disorder. Understanding the consequences of mutations on the properties of melanopsin is fundamental to predicting their effects on human physiology and health. As with all biological molecules, the structure of melanopsin is key to its function. However the functional roles performed by the different regions of the melanopsin molecule are not well established. In this study we will produce a number of melanopsin molecules containing targeted changes within specific regions of the protein, as a means to determining the roles performed by these regions in a number of key functions. Principally we will focus on modelling the mutations known to be present in the human population, and determine the general roles performed by areas of the molecule in which these mutations are found. In addition, we will determine the regions of the melanopsin protein that influence sensitivity to light and specifically the colour of light to which this protein responds (termed spectral tuning sites). Similar studies have been conducted for the light sensitive 'opsin' molecules found in rods and cones, yet melanopsin represents a functionally different class of opsin with important functional and structural differences. To date no study has investigated the spectral tuning of this class of 'non-visual' opsin. As a consequence of this study we will generate a range of melanopsin variants that respond to different colours of light. Such variants have huge potential for research applications, where the expression of melanopsin can be used to confer sensitivity to light and allow the selective activation of cell types of interest both in vitro and in vivo, and also for gene therapy based approaches aimed at treating retinal disease and restoring vision. This programme of work will lead to a fundamental increase in our understanding of the melanopsin protein, a protein that performs important roles in human physiology and health. This study will also provide a range of new research tools that are applicable to a wide range of biological research fields and also the development of novel tools for the treatment of human disease.