PHPT1 knockout for investigation of ethanol-induced hepatic steatosis

项目总结 尽管组氨酸在生物学上具有重要意义，但在哺乳动物细胞信号转导中的组氨酸磷酸化在很大程度上是未知的 其中识别和验证的磷酸组氨酸(PHI)靶标很少。这些关键的生物靶点 包括与脂肪酸生物合成、细胞信号、基因转录调控和离子有关蛋白质 运输。鉴于这些基本细胞过程的重要性以及组氨酸的相关影响 蛋白质功能上的磷酸化，PHIs水平的失调被认为是 包括肝脏疾病在内的疾病的病理生理学，它是世界上最主要的15种死亡原因之一 美国。酒精性肝病(ALD)约占这些死亡病例的50%。澄清： 肌萎缩侧索硬化症的病理生理机制是最终开发新的临床的关键 介入治疗，尤其是在早期发病时。 我们的研究小组一直在积极研究哺乳动物磷酸组氨酸磷酸酶的作用， PHPT1，在乙醇诱导的肝细胞功能障碍中，我们的数据显示了以下关键结果。 首先，我们已经确定PHPT1显著下调，细胞质PHIs水平在 长期接触乙醇的小鼠的肝脏。其次，我们的体内数据表明，转基因交付 PHPT1抑制酒精喂养小鼠的肝脏脂肪变性。综上所述，我们假设慢性乙醇 暴露诱导PHPT1下调和随后肝脏PHIs信号的变化，改变 导致肝脏脂肪变性发生的关键促脂肪靶点的表达和活性。至 针对这一点，我们产生了一种新型的PHPT1基因敲除小鼠。我们的初步证据表明 PHPT1在胚胎上是致命的。我们的杂合子PHPT1小鼠的PHPT1大约减少了50%， 特别是在肝脏中，但还需要进一步的鉴定。因此，这款R03的目的是为了充分 检测PHPT1基因敲除的脂肪变性表型与PHPT1抑制乙醇诱导的关系 肝脏脂肪变性。解决我们假设的核心是，PHPT1 KO将暴露在NIAAA慢性 暴饮暴食模型有两个特定目的：(1)确定PHPT1的降低是否促进了乙醇诱导的肝脏 PHPT1基因敲除(PHPT1KO)小鼠的脂肪变性和(2)确定PHPT1的肝脏特异性表达 可挽救PHPT1+/-中的肝脏脂肪变性。拟议的研究将大大有助于理解 阿尔茨海默病病理生理学。我们这项提案的主要科学目标非常适合NIAAA的使命 和R03小额赠款计划。我们的自给自足的研究项目已经建立了 基本动物模型(PHPT1KO)和其他工具(Ad-PHPT1)可快速用于直接解决 PHPT1在酒精性肝脂肪变性中的作用

PROJECT SUMMARY Histidine phosphorylation in mammalian cell signaling is largely unexplored despite the biological importance where few phosphohistidine (pHis) targets have been identified and validated. These critical biological targets include proteins involved in fatty acid biosynthesis, cell signaling, regulation of gene transcription and ion transport. Given the importance of these fundamental cellular processes and related impact of histidine phosphorylation on protein function, dysregulation of pHis levels has been implicated as a mechanism underlying the pathophysiology of disease including liver disease, which is among the top 15 leading causes of death in the United States. Alcoholic liver disease (ALD) accounts for approximately 50% of these deaths. Elucidation of the mechanisms underlying the pathophysiology of ALD is critical to ultimately developing novel clinical interventional therapy, particularly at early onset. Our research group has been actively investigating the role of a mammalian phosphohistidine phosphatase, PHPT1, in ethanol-induced hepatocyte dysfunction and our data has demonstrated the following critical results. First, we have identified that PHPT1 is significantly downregulated and cytoplasmic pHis levels are increased in the livers of chronic ethanol-exposed mice. Second, our in vivo data has demonstrated that transgenic delivery of PHPT1 inhibits hepatic steatosis in ethanol-fed mice. Taken together, we hypothesize that chronic ethanol exposure induces downregulation of PHPT1 and subsequent changes in hepatic pHis signaling, altering expression and activity of critical hepatic lipogenic targets contributing to the onset of hepatic steatosis. To address this, we have generated a novel PHPT1 knockout mouse. Our initial evidence indicates disruption of PHPT1 is embryonically lethal. Our heterozygote PHPT1 mice have an approximate 50% reduction of PHPT1, particularly in the liver but further characterization is needed. Therefore, the purpose of this R03 is to fully examine the steatotic phenotype of the PHPT1 knockout in relation to PHPT1 inhibition of ethanol-induced hepatic steatosis. Central to addressing our hypothesis, the PHPT1 KO will be exposed to the NIAAA chronic binge model with 2 specific aims: (1) Determine if the reduction of PHPT1 promotes ethanol-induced hepatic steatosis in the PHPT1 Knockout (PHPT1KO) mouse and (2) Determine if liver-specific expression of PHPT1 can rescue hepatic steatosis in PHPT1+/-. The proposed studies will significantly contribute to the understanding of ALD pathophysiology. Our primary scientific goals of this proposal are strongly suited to the mission of NIAAA and that of the R03 small grant program. Our self-contained research project already has both an established essential animal model (PHPT1KO) and additional tools (Ad-PHPT1) to rapidly employ to directly address the role of PHPT1 in ethanol-induced hepatic steatosis.