Regulation of PTEN in chronic alcohol mediated liver disease

本提案的目的是确定脂质磷酸酶PTEN参与酒精性肝病（ALD）期间脂肪变性和肝损伤的形成。尽管有关于PTEN在ALD中的作用的出版物，但这些报道在流体模型中使用静态时间点，并且没有解决在酒精暴露期间通过醛修饰的PTEN失活和PTEN调节/表达变化的可能性。我们的工作假设表明，在慢性乙醇处理的小鼠的肝脏中产生的反应性醛4-HNE共价加合PTEN，从而降低酶活性，导致PTEN下游信号传导的失调。4-HNE已经涉及在肝脏中慢性乙醇暴露期间的蛋白质失活。还假设增加的PTEN表达导致下游途径的变化，最终导致ALD中观察到的脂肪变性增加。为了实现本研究提案的目标，将使用质谱法进行实验，以确定PTEN上4-HNE修饰的位点，这些加合物抑制酶活性、膜结合和Trx 1结合的能力。此外，使用蛋白质印迹法，免疫组织化学和mRNA表达，可变的PTEN表达对下游信号通路的影响将在9周的慢性乙醇暴露后进行检查。

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.