Global Characterization of Lysine Acetylation in Diabetic Retina

描述（由申请方提供）：本拟定研究的目的是定量表征糖尿病动物视网膜核组分中的赖氨酸N5-乙酰化（LysAc）事件（乙酰组），并确定组蛋白乙酰转移酶p300如何调节单个LysAc位点。糖尿病视网膜病变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，最终导致神经节细胞、水平细胞、无长突细胞和光感受器的逐渐丧失。不幸的是，目前没有有效的治疗方法来预防或减缓疾病的进展。因此，我们必须继续寻找新的治疗靶点。最近对LysAc的蛋白质组学研究表明，LysAc是一种常见的和动态的翻译后修饰，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）来调节糖尿病动物中的赖氨酸乙酰化事件已显示对视网膜病变的发展具有有益作用。因此，我们推测赖氨酸乙酰化事件的失调参与了糖尿病视网膜病变的发展。在这个R21的建议，我们将定量表征乙酰组在视网膜核馏分。我们将采用尖端的质谱定量蛋白质组学技术，结合亲和富集技术的乙酰赖氨酸含有肽。将使用基于生物信息学的工具分析获得的乙酰组数据集，以绘制参与糖尿病视网膜病变发展的生物过程。我们还将通过质谱法以及使用生物化学方法来验证所识别的LysAc位点和生物过程。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该疾病的病理生理学至关重要，并为糖尿病视网膜病变的新治疗策略的开发提供基础。提出了以下具体目标。具体目标1：确定参与糖尿病视网膜病变发展的LysAc位点和生物过程/途径，具体目标2：验证目标1中确定的LysAc位点和生物过程/途径。 公共卫生相关性：糖尿病是20-74岁成人新发失明病例的主要原因。然而，没有有效的治疗方法来预防或减缓疾病的进展。差异乙酰化位点的鉴定将为糖尿病视网膜病变新的治疗策略的开发提供基础。

DESCRIPTION (provided by applicant): The goal of this proposed study is to quantitatively characterize the lysine N5-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 is involved in the development of diabetic retinopathy and Specific Aim 2: To verify the LysAc sites and biological processes/pathways identified in Aim 1. PUBLIC HEALTH RELEVANCE: Diabetes is the leading cause of new cases of blindness among adults aged 20-74 years. However, no effective therapies to prevent or slow the progress of the disease are available. The identifications of differentially acetylated sites will provide a foundation for the development of novel therapeutic strategies for diabetic retinopathy.