Characterizing the role of peroxiredoxin 6 in Alcoholic Liver Disease.

描述(由申请人提供)：这项建议的长期目标是研究过氧化还蛋白6(PRX6)在肝脏中的抗氧化作用，以及这种蛋白质的活性可能如何受到慢性酒精摄入造成的氧化应激的影响。这个项目有三个拟议的具体目标。第一个具体目的是鉴定和表征重组PRX6的4-羟基壬烯醛和4-氧酮烯醛修饰的位置和生物学效应。加合物将在体外用液相色谱和串联质谱仪进行鉴定和表征。分子模拟将被用来研究由于醛修饰引起的任何构象变化，生化分析将被用来评估修饰的生物学意义。第二个具体目标是使用不同的啮齿动物模型评估PRX6在酒精性肝病进展中的作用。野生型小鼠将长期喂食含乙醇的食物，并将通过双向电泳和Western blotts在不同的时间点评估醛-蛋白质加合物的数量。此外，PRX6/-基因敲除小鼠将被长期喂食含酒精的饮食，以研究PRX6是否是肝脏中的重要抗氧化剂。最后，将利用PRX6过表达转基因小鼠来评价这种过表达对酒精性肝脏的保护作用。最后一个具体目标是评估野生型和PrX6/-基因敲除小鼠肝脏中慢性乙醇摄入所造成的任何可能的抗氧化补偿。使用分离的肝细胞和肝匀浆，通过定量RT-PCR的mRNA表达，通过Western blotting的蛋白表达和主要的细胞抗氧化蛋白，即过氧化氢酶，谷胱甘肽过氧化物酶和超氧化物歧化酶的酶活性将被评估。在美国，长期大量饮酒是导致肝病和死亡的主要原因。因此，酒精性肝病(ALD)是一个重大的公共卫生问题。ALD是一种多因素疾病，氧化应激是已知的致病因素。因此，通过彻底研究PRX6在ALD进展机制中的作用，科学界可以向更好地了解疾病更近一步，并更接近于设计预防ALD进展的治疗策略。

DESCRIPTION (provided by applicant): 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.