Characterizing the role of peroxiredoxin 6 in Alcoholic Liver Disease.

本提案的长期目标是研究过氧化物氧还蛋白6（PRX 6）的抗氧化作用， 肝脏以及这种蛋白质的活性如何受到慢性乙醇引起的氧化应激的影响 消费该项目有三个拟议的具体目标。第一个具体目标是确定 并表征了4-羟基壬烯醛和4-氧代壬烯醛修饰的位置和生物学效应。 重组PRX 6。将使用液相色谱法在体外鉴定和表征加合物， 串联质谱法分子建模将用于研究任何构象变化 由于醛修饰，将进行生物化学测定以评估 的修改。第二个具体目标将评估PRX 6在酒精性糖尿病进展中的作用。 肝脏疾病使用各种啮齿动物模型。野生型小鼠将长期喂食含乙醇的饮食 并且将通过二维图像在不同时间点评估蛋白质-蛋白质加合物的数目。 电泳和蛋白质印迹。此外，PRX 6-/-敲除小鼠将长期喂食乙醇 为了研究PRX 6是否是肝脏中重要的抗氧化剂，最后， 将使用转基因PRX 6过表达小鼠来评估酒精性肝中的保护作用 由于这种过度表达。最后一个具体目标是评估任何可能的抗氧化剂 在野生型和PRX 6-/-敲除小鼠的肝脏中，由于慢性乙醇消耗而引起的补偿。 使用分离的肝细胞和肝匀浆，通过定量RT-PCR，蛋白质表达， 将通过蛋白质印迹法评估主要细胞抗氧化剂的表达和酶活性 蛋白质，即过氧化氢酶、谷胱甘肽过氧化物酶和超氧化物歧化酶。长期大量饮酒 在美国肝病是导致疾病和死亡的主要原因。因此，酒精性肝病 (ALD)这是一个重大的公共卫生问题。ALD是一种多因素疾病，其中氧化应激是 一个已知的影响因素。因此，通过深入研究PRX 6在细胞凋亡机制中的作用， ALD进展科学界可以向更好地了解这种疾病迈进一步 并更接近于设计预防ALD进展的治疗策略。

The long term objective of this proposal is to investigate the antioxidant role of peroxiredoxin 6 (PRX6) in the liver and how the activity of this protein might be affected by oxidative stress due to chronic ethanol consumption. There are three proposed specific aims for this project. The first specific aim is to identify and characterize the location and biological effect of 4-hydroxynonenal and 4-oxononenal modification of recombinant PRX6. Adducts will be identified and characterized in vitro using liquid chromatography and tandem mass spectrometry. Molecular modeling will be used to investigate any conformational changes due to aldehyde modification and biochemical assays will be performed to assess biological significance of the modification. The second specific aim will evaluate the role of PRX6 in the progression of alcoholic liver disease using various rodent models. Wild type mice will be chronically fed an ethanol containing diet and the number of aldehyde-protein adducts will be assessed at various time points via two dimensional electrophoresis and Western blots. Also, PRX6 -/- knockout mice will be chronically fed an ethanol containing diet in order to investigate whether or not PRX6 is an important antioxidant in the liver. Lastly, transgenic, PRX6 over-expressing mice will be used to evaluate the protective effects in the alcoholic liver due to this over-expression. The last specific aim is designed to evaluate any possible antioxidant compensation due to chronic ethanol consumption in the liver of wild type and PRX6 -/- knockout mice. Using isolated hepatocytes and liver homogenates, mRNA expression via quantitative RT-PCR, protein expression via Western blotting and enzymatic activity will be assessed for the major cellular antioxidant proteins, i.e. catalase, glutathione peroxidase, and superoxide dismutase. Long-term, heavy alcohol use is a leading cause of illness and death from liver diease in the United States. As such, alcoholic liver disease (ALD) represents a major public health concern. ALD is a multifactorial disease in which oxidative stress is a known contributing factor. Therefore, by thoroughly investigating the role of PRX6 in the mechanism of ALD progression the scientific community can move a step closer to a better understanding of the disease and closer to designing treatment strategies for preventing the advancement of ALD.