Aspartate beta-hydroxylase and DNA damage in chronic liver diseases

项目摘要 慢性饮酒与酒精性肝病（ALD）的发展和进展有关， 损害细胞DNA的手段，导致肝细胞死亡，导致酒精中毒。 脂肪性肝炎、肝纤维化和潜在的肝癌。然而，目前尚不清楚酒精如何促进 ALD发展中的DNA损伤反应和了解潜在的机制可能会产生洞察力 用于治疗酒精性肝病在初步研究中，证明天冬氨酸β- 羟化酶（ASPH）在来自乙醇喂养组的啮齿动物肝脏中下调。此外，DNA 损伤标志物，包括γ-H2 aX（H2 aX的磷酸化形式）和GADD 45 α，被发现升高， 与对照饮食喂养组相比。肝脏主要由肝细胞组成，其功能是 对维持其体内平衡至关重要。因此，我们研究了是否存在相关性， 人肝细胞中ASPH和DNA损伤。我们异位表达ASPH并激发肝细胞 DNA损伤诱导剂有趣的是，ASPH的表达几乎消除了γ-H2 aX的表达;然而， ASPH的敲低实质上促进了DNA损伤。这些观察结果表明， ASPH在DNA损伤反应中基于这些发现，我们假设长期饮酒 消耗导致肝脏中ASPH下调，从而导致DNA损伤反应， 慢性肝病进展中肝细胞死亡。我们的长期目标是了解ASPH是如何 参与乙醇诱导的肝细胞DNA损伤以及对ALD进展的后续影响。到 为了验证我们的假设并实现这一目标，我们提出了两个具体目标。在目标1中，我们将研究如何 ASPH的表达改变了体内ALD的进展。我们将确定是否使用靶向肝ASPH AAV策略将加剧乙醇喂养小鼠肝细胞中的DNA损伤和细胞死亡。此外，委员会认为， 我们研究了在ALD模型中ASPH的过表达是否保护肝细胞。在aim 2中，我们将破译 ASPH保护肝细胞免受DNA损伤诱导的细胞死亡的机制。我们将 通过使用CRISPR/Cas9敲除ASPH来确定ASPH如何参与乙醇介导的DNA损伤 或通过慢病毒系统异位表达ASPH。为了描述这种调节所涉及的机制， 我们将确定ASPH的酶功能如何与ASPH介导的DNA损伤相关 反应和细胞死亡。使用蛋白质组学方法，我们还将说明创新的ASPH下游 乙醇处理的目标。这些结果将大大促进我们对分子生物学的理解。 肝细胞死亡机制和ALD进展的病理生理机制。我们也 预计这将拓宽我们对ASPH表达及其与肝细胞功能关系的认识， 将军

Project summary Chronic alcohol drinking has been linked to the development and progression of alcoholic liver disease (ALD) by means of damaging cellular DNA, which results in cell death of hepatocytes, contributing to alcoholic steatohepatitis, liver fibrosis, and potentially liver cancers. However, it remains unclear how alcohol promotes DNA damage responses in ALD development and understanding the underlying mechanisms may yield insight into therapeutic approaches toward treating ALD. In the pilot studies, it was demonstrated that aspartate beta- hydroxylase (ASPH) is down-regulated in the rodent livers derived from the ethanol-fed group. In addition, DNA damage markers, including γ-H2aX (a phosphorylated form of H2aX) and GADD45α, were found elevated when compared to the control-diet fed group. The liver is predominantly composed of hepatocytes whose function is critical in the maintenance of homeostasis thereof. Thus, we investigated whether there is a correlation between ASPH and DNA damage in human hepatocytes. We ectopically expressed ASPH and challenged hepatocytes with DNA damage inducers. Intriguingly, the expression of ASPH almost eliminated γ-H2aX expression; however, knockdown of ASPH substantially promoted DNA damage. These observations suggest a protective role of ASPH in the DNA damage response. Based on these findings, we hypothesize that chronic alcohol consumption leads to ASPH down-regulation in the liver contributing to DNA damage responses and hepatocyte cell death in chronic liver disease progression. Our long-term goal is to understand how ASPH is involved in ethanol-induced hepatocyte DNA damage and the consequent impacts on ALD progression. To test our hypothesis and achieve this goal, two specific aims are proposed. In aim 1, we will investigate how the expression of ASPH alters ALD progression in vivo. We will determine if targeting hepatic ASPH using an AAV strategy will exacerbate DNA damage and cell death in the hepatocytes of ethanol-fed mice. Furthermore, we investigate if overexpression of ASPH protects hepatocytes in an ALD model. In aim 2, we will decipher the mechanism by which ASPH protects hepatocytes from DNA damage induced cell death. We will determine how ASPH is involved in ethanol-mediated DNA damage by using CRISPR/Cas9 to knock out ASPH or ectopically expressing ASPH via a lentiviral system. To delineate the mechanisms involved in this regulation, we will determine how the enzymatic functions of ASPH are associated with the ASPH-mediated DNA damage responses and cell death. Using the proteomic approach, we will also illustrate innovative ASPH downstream targets upon ethanol treatment. The results will significantly advance our understanding of the molecular mechanisms in hepatocyte cell death and pathophysiological mechanisms underlying ALD progression. We also anticipate that it will broaden our knowledge of ASPH expression and its relationship to hepatocyte function in general.