Regulation of PTEN in chronic alcohol mediated liver disease

本研究的目的是确定脂肪磷酸酶PTEN在酒精性肝病(ALD)脂肪变性和肝损伤形成中的作用。虽然有关于PTEN在ALD中的作用的文献，但这些报告使用流体模型中的静态时间点，没有讨论通过醛修饰使PTEN失活的可能性以及在酒精暴露期间PTEN调节/表达的变化。我们的工作假设是，慢性乙醇处理的小鼠肝脏中产生的反应醛4-HNE与PTEN共价加合，从而降低了酶活性，导致PTEN下游信号的失调。4-HNE已经被认为与肝脏慢性酒精暴露过程中蛋白质的失活有关。还假设PTEN表达增加会导致下游通路的改变，最终导致ALD中脂肪变性的增加。为了实现本研究计划的目标，将利用质谱仪进行实验，以确定PTEN上4-HNE修饰的位置、这些加合物抑制酶活性的能力、膜结合和Trx1结合。此外，通过蛋白质印迹、免疫组织化学和mRNA表达，我们将研究慢性酒精暴露9周后，不同的PTEN表达对下游信号通路的影响。

The objective of this proposal is to determine the involvement of the lipid phosphatase PTEN in the formation of steatosis and liver damage during alcoholic liver disease (ALD). Although there are publications concerning the role of PTEN in ALD, these reports are using a static time point in a fluid model and do not address the possibility of both PTEN inactivation via aldehyde modification and changes in PTEN regulation/expression over a period of alcohol exposure. Our working hypothesis states the reactive aldehyde 4-HNE, produced in the liver of chronic ethanol treated mice covalently adducts PTEN thereby reducing enzymatic activity leading to disregulation in PTEN downstream signaling. 4-HNE has already been implicated in the inactivation of proteins during chronic ethanol exposure in the liver. It is also hypothesized that increased PTEN expression leads to changes in downstream pathways ultimately leading to increased steatosis seen in ALD. In order to fulfill the objectives of this research proposal, experiments will be performed using mass spectrometry to identify the sites of 4-HNE modification on PTEN, the ability of these adducts to inhibit enzymatic activity, membrane association and Trx1 association. In addition, using western blotting, immunohistochemistry and mRNA expression, the effects of variable PTEN expression on downstream signaling pathways will be examined following 9-weeks of chronic ethanol exposure.