Wilson Disease: Regulation of Steatosis by Methionine Metabolism

描述(由申请者提供)：这份建议书描述了一项为期5年的肝病学术生涯发展培训计划，以基础科学研究为导向。这位首席研究员具有威尔逊病的科学背景，现在将通过独特的资源整合来扩展她的科学技能。该计划将促进对肝豆状核变性中铜超载与脂肪变性相关的分子机制的理解，最终目的是探索基于这些异常潜在过程的改变来治疗这种罕见疾病的新方法。霍尔斯特德博士和拉特利奇博士将指导首席研究员的科学和职业发展。他们是蛋氨酸和脂类代谢领域公认的领导者，培养了许多博士后研究员和研究生。研究的重点是铜积累对S-同型半胱氨酸腺苷水解酶的抑制作用。初步数据显示，抑制SAH水解酶后，肝脏S-腺苷甲硫氨酸：SAH比值(甲基化状态的标志)显著降低，随后同型半胱氨酸随时间增加。建议的实验将需要使用铜络合剂来降低肝脏中的铜浓度并提高SAH水解酶的活性，从而证明铜对该酶的直接影响，并进一步抑制体内(用DZA)和体外(用慢病毒携带的shRNA)的SAH水解酶，并提供甲基作为补充甜菜碱，一种已知纠正蛋氨酸代谢异常的化合物。将使用各种生化、分子和细胞技术，包括表观遗传学方法来研究蛋氨酸和脂肪代谢。其具体目的包括：1)建立铜蓄积、肝脏脂肪变性和蛋氨酸代谢异常之间的关系；2)确定甲基供体甜菜碱是否可以改善TX-j小鼠和同一动物模型的原代肝细胞的脂肪代谢；3)确定肝豆状核变性是否由脂质合成相关基因表达的表观遗传调控所介导。这将是首次详细分析这种罕见疾病中基于蛋氨酸代谢的肝脏脂肪变性的机制。加州大学戴维斯分校通过将来自不同资源的专业知识整合到定制的课程中，为培训内科科学家提供了理想的环境。这样的环境最大限度地增加了首席研究人员建立一个科学利基的潜力，可以在这个利基上建立学术生涯。拟议实验的成功结果将促进对威尔逊病脂肪变性发病机制的代谢过程的理解，这是一种孤儿疾病(包括在NIDDK肝病研究行动计划中)，其遗传背景已确立，但其代谢病因尚不清楚。这项研究还将为铜和蛋氨酸代谢在更常见的疾病中的作用提供见解，例如非酒精性脂肪性肝病。 公共卫生相关性：肝豆状核变性是一种常染色体隐性遗传病，由肝脏铜过量引起，常以肝脏脂肪堆积为特征。我们将研究铜和肝脏蛋氨酸代谢之间的相互作用，最终目的是探索基于这些潜在过程的修改的新疗法。这项研究还将为铜和蛋氨酸代谢在更常见的疾病中的作用提供见解，例如非酒精性脂肪性肝病。

DESCRIPTION (provided by applicant): This proposal describes a 5 year training program for the development of an academic career in Hepatology, oriented to basic science research. The principal investigator has scientific background on Wilson Disease and now will expand upon her scientific skills through a unique integration of resources. This program will promote the understanding of the molecular mechanisms relating copper overload to steatosis in Wilson Disease, with the ultimate objective to explore new therapies for this rare disease based on the modification of these aberrant underlying processes. Dr. CH Halsted and Dr. JC Rutledge will mentor the principal investigator's scientific and career development. They are recognized leaders in the fields of methionine and lipid metabolism and they have trained numerous postdoctoral fellows and graduate students. The research will focus on the inhibitory effect exerted by copper accumulation on the enzyme S-adenosylhomocysteine (SAH) hydrolase. Preliminary data demonstrate that SAH hydrolase inhibition is followed by significant reduction of the hepatic S-adenosylmethionine (SAM):SAH ratio, a marker of methylation status, and later time-dependent homocysteine increase. The proposed experiments will entail the use of a copper chelator to reduce hepatic copper concentration and improve SAH hydrolase activity, thereby demonstrating the direct effect of copper on this enzyme, and further inhibition of SAH hydrolase both in vivo (with DZA) and in vitro (with shRNA carried by lentivirus) and provision of methyl groups as supplemental betaine, a compound known to correct aberrant methionine metabolism. An assortment of biochemical, molecular, and cellular techniques, including epigenetic methods will be used to study methionine and lipid metabolism. The specific aims include: 1) Establishing relationships among copper accumulation, hepatic steatosis, and aberrant methionine metabolism, 2) Determining if the methyl donor betaine can improve lipid metabolism in tx-j mice and in primary hepatocytes from the same animal model, and 3) Determining if steatosis in Wilson disease is mediated by epigenetic regulation of expressions of genes relevant to lipid synthesis. This will be the first detailed analysis of the mechanisms of hepatic steatosis based on the metabolism of methionine in this rare disease. The University of California Davis provides an ideal setting for training physician- scientists by incorporating expertise from diverse resources into customized programs. Such an environment maximizes the potential for the principal investigator to establish a scientific niche from which an academic career can be constructed. The successful outcome of the proposed experiments will promote an understanding of the metabolic processes underlying the pathogenesis of steatosis in Wilson disease, an orphan disease (included in the NIDDK action plan for Liver Disease Research) whose genetic background is well-established but whose metabolic etiopathogenesis is unclear. The study will also provide insights into the role of copper and methionine metabolism in more common diseases, such as non-alcoholic fatty liver disease. PUBLIC HEALTH RELEVANCE: Wilson disease, an autosomic recessive disorder due to hepatic copper excess, is frequently characterized by fat accumulation in the liver. We will study the interaction between copper and hepatic methionine metabolism with the ultimate objective to explore new therapies based on modification of these underlying processes. The study will also provide insights into the role of copper and methionine metabolism in more common diseases, such as non-alcoholic fatty liver disease.