Acylation of Lens Proteins

胞质蛋白质的N-酰化在组织中是常见的，并且其调节蛋白质结构和功能。 功能细胞中主要的N-酰化类型是乙酰化、丙酰化、琥珀酰化和丙二酰化， 其通过乙酰辅酶A、丙酰辅酶A、琥珀酰辅酶A 和丙二酰辅酶A分别与赖氨酸残基连接。透镜蛋白质寿命长，很少或没有周转， 因此，翻译后修饰（PTM）随着年龄的增长而积累。缺乏酶活性蛋白质， 特别是在透镜的芯中，阻止了PTM的反转。因此，如果脱酰基酶在透镜中较弱， 酰化主要是导致透镜蛋白永久性修饰的单向反应， （根据我们的初步数据）。酰化对透镜蛋白的影响仍然很大 未知如我们先前的研究所示，乙酰化使α-晶状体蛋白成为更好的伴侣。此外，本发明还提供了一种方法， 我们的初步数据显示透镜蛋白也是丙酰化的、琥珀酰化的和丙二酰化的， 乙酰化和琥珀酰化增加了蛋白质的热稳定性和溶解性。因此我们 假设透镜蛋白的酰化是一种有益的PTM，有助于维持α-晶状体蛋白的伴侣活性， 晶状体蛋白以及透镜蛋白在老化过程中的稳定性和溶解性。我们将测试这个假设， 三个目标。目的1：通过质量分析确定人透镜α-晶状体蛋白的主要酰化位点 光谱分析，并确定年龄对这些修改的影响。在目标2中，我们将确定 酰化对α-晶状体蛋白结构和功能的影响。在目标3中，我们将评估 酰化对透镜蛋白的稳定性和溶解性的影响，并确定酰化是否可用于 改善老化的人类晶状体的受损弹性。

N-acylation of cytosolic proteins is a common occurrence in tissues, and it regulates protein structure and function. The major N-acylation types in cells are acetylation, propionylation, succinylation and malonylation, which are initiated by the enzymatic or non-enzymatic transfer of acetyl CoA, propionyl CoA, succinyl CoA and malonyl CoA to lysine residues, respectively. Lens proteins are long lived with little or no turnover, and thus, post-translational modifications (PTMs) accumulate with age. The lack of enzymatically active proteins, particularly in the core of the lens, prohibits the reversal of PTMs. Thus, if deacylases are weak in the lens, acylation is mostly a one-way reaction that leads to permanent modifications of lens proteins, which appears to be the case (based on our preliminary data). The effects of acylation on lens proteins remain largely unknown. As shown in our previous study, the acetylation makes α-crystallin a better chaperone. In addition, our preliminary data show that lens proteins are also propionylated, succinylated and malonylated and that acetylation and succinylation increase the thermal stability and solubility of the proteins. Thus, we hypothesize that acylation of lens proteins is a beneficial PTM that helps maintain the chaperone activity of α- crystallin as well as the stability and solubility of lens proteins during aging. We will test this hypothesis in three aims. In Aim 1, we will determine the major acylation sites in human lens α-crystallin by mass spectrometric analyses and determine the effect of age on these modifications. In Aim 2, we will determine effects of acylation on the structure and function of α-crystallin. In Aim 3, we will evaluate the effects of acylation on the stability and solubility of lens proteins and determine whether acylation can be used to improve the compromised resilience of aged human lenses.