Novel Role of Corneal Crystallins as Modulators of Cell Growth and Transparency

摘要 该项目的长期目标是了解角膜晶体蛋白在角膜生理和眼球发育中的生物学作用。 病理生理学。根据定义，角膜晶状体蛋白是高丰度表达的胞浆酶/蛋白质。 角膜中分类群特有的方式。第一个被鉴定为角膜晶体蛋白的角膜蛋白是乙醛。 脱氢酶3A1(ALDH3A1)，以及其他几种蛋白质，包括转酮醇酶、ALDH1A1、异柠檬酸脱氢酶、 和谷胱甘肽S转移酶，后来也被相似地归类。我们和其他人已经证明了角膜 在发育和分化过程中，特别是当细胞退出细胞周期时，晶状体蛋白的表达显著上调。 相反，角膜损伤(触发细胞增殖)与角膜晶体蛋白和 与角膜雾霾有关的光散射增加。我们发现，角膜ALDH3A1的过度表达导致 显著抑制细胞增殖，减少体外光散射，保护细胞免受氧化应激。我们的 新的初步数据表明，ALDH3A1通过酶和非酶途径影响细胞的增殖和分化 酶机制。我们最近开发的同源基因Aldh3a1基因敲除小鼠表现出角膜混浊或云雾， 类似于晶状体白内障，这一观察结果证实了我们长期以来的假设，即晶体蛋白对 保持蜂窝的透明度。这是第一个由细胞引起的角膜“雾霾”的遗传动物模型。我们的工作 假说保持不变，认为角膜晶体蛋白(特别是ALDHs)调节细胞的生长、分化 以及通过代谢(酶)和/或结构功能实现的细胞透明度。我们现在提出一种系统生物学 在我们的新的动物模型中，我们采用了一种新的方法来确定调节角膜透明度的分子机制。我们 将使用最先进的方法来量化与ALDH3A1相关的组织结构和功能的变化 表达，包括：(A)RNA测序，(B)基质辅助激光解吸电离成像质谱仪(TO 直接原位测量代谢物和蛋白质)，(C)整合途径分析，和(D)免疫荧光断层扫描 (开发角膜内生物分子的三维生物图谱)。

ABSTRACT The long-range goal of this project is to understand the biological role of corneal crystallins in corneal physiology and pathophysiology. By definition, corneal crystallins are cytosolic enzymes/proteins expressed in high abundance and in a taxon-specific manner in the cornea. The first corneal protein to be identified as a corneal crystallin was aldehyde dehydrogenase 3A1 (ALDH3A1), and several other proteins, including transketolase, ALDH1A1, isocitrate dehydrogenase, and glutathione S-transferase, have subsequently been similarly categorized. We and others have shown that corneal crystallin expression is markedly up-regulated during development and differentiation, specifically as cells exit the cell cycle. Conversely, corneal injury (which triggers cell proliferation) is associated with a loss in expression of corneal crystallins and an increase in light scattering related to corneal haze. We have found that the over-expression of corneal ALDH3A1 causes a profound retardation in cell proliferation, decreased light scattering in vitro and protection against oxidative stress. Our new preliminary data indicate that ALDH3A1 affects cell proliferation and differentiation through both enzymatic and non- enzymatic mechanisms. Our recently-developed congenic Aldh3a1 knockout mice exhibit corneal haze or clouding, analogous to lens cataracts, an observation that confirms our long-standing hypothesis that crystallins are critical to the maintenance of cellular transparency. This is the first genetic animal model of cellular-induced corneal 'haze'. Our working hypothesis remains the same and contends that corneal crystallins (specifically, ALDHs) regulate cell growth, differentiation and cellular transparency through metabolic (enzymatic) and/or structural functions. We now propose a systems biology approach in our novel animal models to identify the molecular mechanism involved in regulating corneal transparency. We will employ state-of-the-art methods in order to quantify changes in tissue structure and function associated with ALDH3A1 expression, including: (a) RNA-sequencing, (b) matrix-assisted laser desorption ionization imaging mass spectrometry (to directly measure metabolites and proteins in situ), (c) integrated pathway analysis, and (d) immunofluorescent tomography (to develop three-dimensional biological mapping of biomolecules within the cornea).