ER stress in ethanol-induced injury to hepatocytes

 描述（由申请人提供）：酒精消费导致酒精性肝病（ALD），包括从脂肪肝到肝硬化的一系列肝损伤。酒精账户 肝硬化导致的死亡占所有死亡的40-50%，是肝脏相关死亡的最常见原因。内质网（ER）应激，由酒精损伤的蛋白质在ER中的积累引起，最近被确定为是导致ALD的重要机制。然而，两个事件的严重程度，管理ER的压力，包括频谱的蛋白质损伤的ER由酒精和内在的保护机制，消除受损的蛋白质，是未知的。本申请的目的是1）通过新开发的蛋白质组学方法鉴定酒精诱导的ER蛋白损伤，旨在揭示ALD的新分子理解; 2）阐明消除这些受损蛋白并提供保护的机制。使用原代肝细胞作为模型，将使用空间限制酶标记技术结合质谱法鉴定酒精损伤的ER蛋白。ER腔中的所有蛋白质将首先用生物素标记。基于ERAD的机制，生物素化的受损ER蛋白将在蛋白酶体抑制后从胞质溶胶中回收，并将通过质谱法鉴定。蛋白质组学结果将通过生物化学和细胞生物学分析进行验证。除了诱导ER应激外，还将通过途径分析评估受损ER蛋白在ALD发展中的参与。有了这项新的创新技术，我们将能够首次系统地识别被酒精损伤的ER蛋白，这将导致更清晰的分子图像，以了解酒精如何导致ER应激和ALD。ERAD通过促进错误折叠或受损蛋白从ER的降解，是对抗ER应激的主要防御。斑马鱼是研究ALD的新兴模型。我们的初步研究表明，gp 78，一个主要的E3泛素连接酶在ERAD中发挥作用，防止酒精诱导的ER应激。因此，我们将研究ALD的发展，在转基因斑马鱼系特异性过表达野生型gp 78和其显性负突变体，gp 78 R2 m，在肝脏中，我们最近已经建立。我们还将验证在酒精处理的斑马鱼肝脏中的肝细胞中获得的蛋白质组学结果。总之，这项研究将首次阐明酒精损伤的ER蛋白和gp 78对酒精诱导的ER应激和ALD的保护作用。这不仅将为ALD发病机制的分子机制提供新的见解，也可能为ALD的治疗干预提供新的保护机制。

 DESCRIPTION (provided by applicant): Alcohol consumption causes alcoholic liver disease (ALD) that includes an array of liver injury ranging from fatty liver to cirrhosis. Alcohol account for 40-50% of all the death due to cirrhosis and is the most common cause of liver-related mortality. Endoplasmic reticulum (ER) stress, caused by accumulation of alcohol-damaged proteins in the ER, has recently been established as a significant mechanism contributing to ALD. However, two events that govern the severity of ER stress, including the spectrum of proteins damaged in the ER by alcohol and the intrinsic protective mechanisms that remove the damaged proteins, are not known. The goal of this application is to 1) identify alcohol-induced damage of ER proteins by a newly developed proteomic approach with an aim to uncover novel molecular understanding of ALD; 2) elucidate mechanisms that eliminate these damaged proteins and offer protection. Using primary hepatocytes as a model, alcohol- damaged ER proteins will be identified using the spatially restricted enzymatic tagging technique in combination with mass spectrometry. All proteins in the ER lumen will first be tagged with biotin. Based on the mechanism of ERAD, biotinylated damaged ER proteins will be recovered from the cytosol following proteasome inhibition and will be identified by mass spectrometry. The proteomic results will be validated by biochemical and cell biological analysis. The involvement of the damaged ER proteins in ALD development in addition to inducing ER stress will be assessed by pathway analysis. With this new and innovative technology, we will be able to systematically identify, for the first time, the ER proteins that are damaged by alcohol, which wil lead to a clearer molecular picture as to how alcohol causes ER stress and ALD. ERAD is the major defense against ER stress by facilitating degradation of misfolded or damaged proteins from the ER. Zebrafish is an emerging model for studying ALD. Our preliminary studies suggest that gp78, one of the major E3 ubiquitin ligases functioning in ERAD, protects against alcohol-induced ER stress. Accordingly, we will study ALD development in transgenic zebrafish lines specifically overexpressing wt gp78 and its dominant negative mutant, gp78R2m, in liver, which we have established recently. We will also validate the proteomic results obtained in hepatocytes in alcohol-treated zebrafish liver. Together, this study will, for the first time, idenify alcohol-damaged ER proteins and the protective role of gp78 against alcohol-induced ER stress and ALD. It will not only provide new insight into the molecular mechanisms of ALD pathogenesis and may also identify novel protective mechanisms for therapeutic intervention to ALD.