Wilson Disease: Regulation of Steatosis by Methionine Metabolism

描述（由申请人提供）：本提案描述了一个为期5年的培训计划，旨在发展肝病学的学术生涯，以基础科学研究为导向。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。该计划将促进对威尔逊病中铜超载与脂肪变性相关的分子机制的理解，最终目标是基于这些异常潜在过程的修改来探索这种罕见疾病的新疗法。CH Halsted博士和JC拉特利奇博士将指导主要研究者的科学和职业发展。他们是蛋氨酸和脂质代谢领域公认的领导者，他们培养了许多博士后研究员和研究生。研究将集中在铜积累对S-腺苷高半胱氨酸（SAH）水解酶的抑制作用。初步数据表明，SAH水解酶抑制后，肝S-腺苷甲硫氨酸（SAM）：SAH的比例，甲基化状态的标志物显着减少，后来的时间依赖性同型半胱氨酸增加。所提出的实验将需要使用铜螯合剂来降低肝铜浓度并改善SAH水解酶活性，从而证明铜对该酶的直接作用，以及在体内（用DZA）和体外（用慢病毒携带的shRNA）进一步抑制SAH水解酶，并提供甲基作为补充甜菜碱，甜菜碱是已知纠正异常甲硫氨酸代谢的化合物。生物化学，分子和细胞技术，包括表观遗传学方法的分类将用于研究蛋氨酸和脂质代谢。具体目标包括：1）建立铜积累、肝脂肪变性和异常甲硫氨酸代谢之间的关系，2）确定甲基供体甜菜碱是否可以改善来自相同动物模型的tx-j小鼠和原代肝细胞中的脂质代谢，和3）确定威尔逊病中的脂肪变性是否由与脂质合成相关的基因的表达的表观遗传调节介导。这将是第一次详细分析肝脂肪变性的机制的基础上代谢蛋氨酸在这种罕见的疾病。加州戴维斯大学通过将来自不同资源的专业知识融入定制课程，为培训医生-科学家提供了理想的环境。这样的环境最大限度地发挥了主要研究者的潜力，以建立一个科学的利基，从学术生涯可以构建。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，威尔逊病是一种孤儿病（包括在NIDDK肝病研究行动计划中），其遗传背景已得到充分证实，但其代谢发病机制尚不清楚。该研究还将深入了解铜和蛋氨酸代谢在更常见疾病中的作用，如非酒精性脂肪肝。 公共卫生相关性：肝豆状核变性是一种常染色体隐性遗传性疾病，由于肝铜过量，经常以脂肪堆积在肝脏。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。该研究还将深入了解铜和蛋氨酸代谢在更常见疾病中的作用，如非酒精性脂肪肝。

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.