Global Characterization of Lysine Acetylation in Diabetic Retina

项目摘要 这项研究的目的是定量表征赖氨酸N <$-乙酰化（LysAc） 事件（乙酰组）在视网膜核部分糖尿病动物，并确定如何 单个LysAc位点由组蛋白乙酰转移酶p300调节。糖尿病视网膜病变是 糖尿病是最常见的糖尿病眼病，也是美国成年人失明的主要原因。它 是由视网膜血管的变化引起的，这最终导致了 神经节细胞、水平细胞、无长突细胞和光感受器的进行性损失。 不幸的是，目前没有预防或减缓疾病进展的有效疗法。 available.因此，我们必须继续寻找新的治疗靶点。近期蛋白质组学 对LysAc的研究表明，LysAc是一种常见的动态翻译后酶， 修饰存在于组蛋白和非组蛋白蛋白中，这表明LysAc被用作 类似于蛋白质磷酸化的调节修饰。有趣的是， 通过抑制组蛋白乙酰转移酶（p300） 或激活NAD+依赖性蛋白脱乙酰酶（Sirt 1）已被证明具有有益的 对视网膜病变的发展有影响。因此，我们假设赖氨酸的失调 乙酰化事件参与糖尿病视网膜病变的发展。在R21提案中， 我们将定量表征视网膜核部分中的乙酰组。我们会委聘 尖端的基于质谱的定量蛋白质组学技术， 含有乙酰赖氨酸的肽的亲和富集技术。获得的乙酰组数据 将使用基于生物信息学的工具进行分析，以绘制生物过程， 参与糖尿病视网膜病变的发展。我们还将验证已确定的LysAc位点 和生物过程的质谱分析以及使用生物化学方法。的 差异乙酰化位点的鉴定将在该领域产生重大影响，因为 这些知识对于理解疾病的病理生理学和提供 糖尿病视网膜病变的新的治疗策略的发展的基础。的 提出了以下具体目标。具体目标1：鉴定LysAc位点和生物活性 参与糖尿病视网膜病变发展的过程/途径， 目的2：验证目的1中确定的LysAc位点和生物过程/途径。

Project Summary The goal of this proposed study is to quantitatively characterize the lysine N¿-acetylation (LysAc) events (acetylome) in the retinal nuclear fraction from diabetic animals, and determine how the individual LysAc sites are modulated by histone acetyltransferase, p300. Diabetic retinopathy is the most common diabetic eye disease and a leading cause of blindness in American adults. It is caused by changes in the blood vessels of the retina, which eventually leads to the progressive loss of ganglion cells, horizontal cells, amacrine cells and photoreceptors. Unfortunately, currently no effective therapies to prevent or slow the progress of the disease are available. Therefore, we must continue searching new therapeutic targets. Recent proteomic studies on LysAc have revealed that LysAc is a common and dynamic post-translational modification present in both histone and non-histone proteins, suggesting that LysAc is used as a regulatory modification similar to that of protein phosphorylation. Interestingly, modulation of lysine acetylation events in diabetic animals by either inhibiting histone acetyltransferase (p300) or activating NAD+-dependent protein deacetylase (Sirt1) has been shown to have beneficial effect on the development of retinopathy. Thus, we hypothesize that dysregulation of lysine acetylation events are involved in the development of diabetic retinopathy. In this R21 proposal, we will quantitatively characterize the acetylome in the retinal nuclear fraction. We will employ cutting-edge mass spectrometry based quantitative proteomic techniques, in conjunction with an affinity enrichment technique for acetyl lysine containing peptides. The obtained acetylome data set will be analyzed using bioinformatics-based tools to map the biological processes that are involved in the development of diabetic retinopathy. We will also verify the identified LysAc sites and biological processes by mass spectrometry as well as using biochemical approaches. The identifications of differentially acetylated sites will provide significant impact in the field, because such knowledge is essential to understand the pathophysiology of the disease and provide a foundation for the development of novel therapeutic strategies for diabetic retinopathy. The following specific aims are proposed. Specific Aim 1: To identify LysAc sites and biological processes/pathways that are involved in the development of diabetic retinopathy and Specific Aim 2: To verify the LysAc sites and biological processes/pathways identified in Aim 1.