Role of SAMe on UBC9 and sumolyation in liver cancer and alcoholic liver injury

SAMe 对 UBC9 的作用以及肝癌和酒精性肝损伤中的求和作用

• 批准号: 8147672
• 负责人: Maria Lauda Tomasi
• 金额: $ 5.16万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2013-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8147672
• 关键词: APEX1 gene; Acute; Address; Affect; Africa; Age-Months; Alcoholic Liver Cirrhosis; Alcoholic Liver Diseases; Alcohols; Anabolism; Antioxidants; Apoptosis; Apoptotic; Area; Asia; Attention; Biological; Breast Cancer Cell; Cancer Etiology; Catalytic Domain; Cell Line; Cell Transplants; Cells; Cellular Stress Response; Cessation of life; Chemicals; Chronic; Cirrhosis; Country; DNA Damage; DNA Repair; Development; Down-Regulation; Enzymes; Ethanol; Exhibits; Family member; Fatty Liver; Gene Proteins; Genes; Genome Stability; Genotoxic Stress; Glutathione; Growth; Half-Life; Hepatic; Hepatitis C; Hepatocarcinogenesis; Hepatocyte; Human; Incidence; Inflammation; Injury; Injury to Liver; Isoenzymes; Knock-out; Knockout Mice; Lead; Ligation; Lipid Peroxidation; Liver; Liver diseases; Lung Adenocarcinoma; MCF7 cell; Malignant Neoplasms; Malignant neoplasm of liver; Mammalian Cell; Mammals; Mediating; Messenger RNA; Methionine; Methionine Metabolism Pathway; Modeling; Mus; Mutate; Mutation; NF-kappa B; Nuclear; Nude Mice; Ovarian Carcinoma; Oxidative Stress; Pathway interactions; Patients; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Play; Polyamines; Post-Translational Protein Processing; Precancerous Conditions; Prevalence; Primary carcinoma of the liver cells; Process; Proteins; RNA Interference; Reporting; Rodent; Role; S-Adenosylmethionine; Signal Pathway; Site; Steatohepatitis; Substrate Specificity; System; Testing; Translational Repression; Ubiquitin; Ubiquitin Like Proteins; Ubiquitin-Conjugating Enzymes; Up-Regulation; Wild Type Mouse; alcohol response; cancer cell; cell growth; choline deficient diet; falls; feeding; hepatoma cell; improved; in vivo; inhibitor/antagonist; insight; knock-down; melanoma; methionine adenosyltransferase; novel; overexpression; polypeptide; prevent; protein expression; protein protein interaction; public health relevance; response; therapy development; tumor progression; tumorigenesis; vector

DESCRIPTION (provided by applicant): Posttranslational modifications mediated by ubiquitin-like proteins regulate a variety of cellular pathways. Although small ubiquitin-like modifier (SUMO) is a new member of this family, it has attracted a great deal of attention recently because of its novel and distinct functions. The sumoylation cycle is a multistep process, involving maturation, activation, conjugation and ligation; it is catalyzed by multiple enzymes, including E1, E2 and E3 enzymes. Ubiquitin-conjugating enzyme 9 (UBC9) is the sole E2 conjugating enzyme and is the best characterized enzyme in the sumoylation cycle. UBC9 transfers the activated SUMO to the target protein and is believed to play an important role in regulating the substrate specificity and enhancing the efficiency of sumoylation in vivo. Genotoxic stress induces sumoylation of a broad number of proteins involved in nuclear function as well as proteins involved in important signaling pathways, such as the nuclear factor kappa B (NF- :B). UBC9 and SUMO are highly expressed in human premalignant conditions in response to low-grade, long- term genotoxic stress, implying that upregulation of sumoylation may be an adaptive process to genotoxic stress. Furthermore, UBC9 is overexpressed in several malignancies, such as lung adenocarcinoma, ovarian carcinoma, and melanoma. Antagonizing UBC9 function in MCF-7 breast cancer cells transplanted in nude mice inhibited cell growth and increased apoptosis via Bcl-2 dependent mechanisms. Inactivating mutations of UBC9's SUMO conjugating activity enhances sensitivity to DNA damaging agents. UBC9 may be fundamental for tumorigenesis and tumor progression by preventing activation of apoptotic pathways and by minimizing the acute cellular stress response associated with the accumulating DNA damage of tumor progression. Whether UBC9 is deregulated in hepatocellular carcinoma (HCC) is unknown. How UBC9 protein stability is regulated post-translationally is also unknown. Finally, whether UBC9 expression is altered in response to alcohol is also unknown. S-adenosylmethionine (SAMe) is the principle biological methyl donor that is made in all mammalian cells as the first product of methionine metabolism, catalyzed by methionine adenosylmethionine (MAT). Besides being a methyl donor, accumulating evidence show SAMe regulates many critical cellular pathways that control growth and apoptosis. In mammals two MAT genes, MAT1A and MAT2A, encode for two catalytic subunits of MAT, 11 and 12, respectively. Patients with chronic liver disease including alcohol have impaired hepatic SAMe biosynthesis due to decreased MAT1A mRNA levels and post-translational inhibition of the MAT1A-encoded isoenzymes. Chronic hepatic SAMe deficiency occurs in MAT1A knockout (KO) mice, which exhibit increased propensity to choline-deficient diet induced fatty liver, higher level of lipid peroxidation, spontaneous development of steatohepatitis and HCC. We have recently shown that there is increased genotoxic stress in this model as early as at one month of age. Consistently, we found UBC9 expression is increased in the MAT1A KO livers and importantly administration of SAMe lowered UBC9 expression at the protein level. Treatment of HepG2 and HuH-7 cells, two human hepatoma cell lines, with SAMe also lowered the UBC9 protein level. This is a surprise finding as we reported recently that SAMe inhibits proteosomal activity. Since alcohol feeding lowers hepatic SAMe level, we also examined whether UBC9 expression is altered in this model and found that like the MAT1A KO livers, hepatic UBC9 protein level is increased in response to alcohol feeding. In this proposal we are testing several novel hypotheses, 1) UBC9 protein half-life is affected by phosphorylation so that phosphorylation at critical site(s) protects against degradation, 2) SAMe and its metabolite methylthioadenosine (MTA) can lower the UBC9 protein half-life by lowering its phosphorylation at these critical sites, 3) SAMe and MTA, known to be pro-apoptotic in liver cancer cells, cause apoptosis by lowering UBC9 expression, 4) UBC9 expression and hence sumoylation are increased during the development of alcoholic liver injury and they may contribute to many of the abnormalities seen. Three aims are proposed in this application to test these hypotheses and elucidate how SAMe affects signaling pathways that are important in hepatocarcinogenesis and development of alcoholic liver injury. PUBLIC HEALTH RELEVANCE: S-adenosylmethionine (SAMe) is an important biological molecule that controls growth, death and anti-oxidant response in cells. SAMe is synthesized by methionine adenosyltransferase (MAT) proteins. SAMe levels fall during alcoholic liver injury and cancer. This project aims to understand how SAMe controls UBC9, an important protein that is induced in cancer development. Given that liver cancer is a leading cause of cancer death worldwide, if the proposed studies are successfully accomplished, they may uncover very novel insights into the development and therapy of liver cancer and alcoholic liver disease and as such they have very high public health relevance.

描述（由申请人提供）：由泛素样蛋白介导的翻译后修饰调节多种细胞途径。尽管小泛素样修饰剂（SUMO）是该家族的新成员，但由于其新颖且独特的功能，近年来引起了广泛的关注。 sumoylation 循环是一个多步骤过程，涉及成熟、激活、缀合和连接；它由多种酶催化，包括E1、E2和E3酶。泛素结合酶 9 (UBC9) 是唯一的 E2 结合酶，也是苏酰化循环中特征最明确的酶。 UBC9将活化的SUMO转移到靶蛋白上，被认为在调节底物特异性和增强体内SUMO化效率方面发挥重要作用。基因毒性应激会诱导大量参与核功能的蛋白质以及参与重要信号传导途径的蛋白质发生苏酰化，例如核因子 kappa B (NF-:B)。 UBC9 和 SUMO 在人类癌前病变中高表达，以应对低度、长期的基因毒性应激，这意味着 SUMO 化的上调可能是对基因毒性应激的适应性过程。此外，UBC9在多种恶性肿瘤中过度表达，例如肺腺癌、卵巢癌和黑色素瘤。在裸鼠移植的 MCF-7 乳腺癌细胞中拮抗 UBC9 功能，可通过 Bcl-2 依赖性机制抑制细胞生长并增加细胞凋亡。 UBC9 SUMO 结合活性的失活突变可增强对 DNA 损伤剂的敏感性。 UBC9可能通过阻止细胞凋亡途径的激活以及最大限度地减少与肿瘤进展中累积的DNA损伤相关的急性细胞应激反应而对于肿瘤发生和肿瘤进展至关重要。 UBC9 在肝细胞癌 (HCC) 中是否失调尚不清楚。 UBC9 蛋白稳定性如何在翻译后调节尚不清楚。最后，UBC9 表达是否会因酒精而改变尚不清楚。 S-腺苷甲硫氨酸 (SAMe) 是主要的生物甲基供体，在所有哺乳动物细胞中产生，作为甲硫氨酸代谢的第一个产物，由甲硫氨酸腺苷甲硫氨酸 (MAT) 催化。除了作为甲基供体之外，越来越多的证据表明 SAMe 还调节许多控制生长和凋亡的关键细胞途径。在哺乳动物中，两个 MAT 基因 MAT1A 和 MAT2A 分别编码 MAT 的两个催化亚基 11 和 12。患有慢性肝病（包括酒精）的患者由于 MAT1A mRNA 水平降低和 MAT1A 编码同工酶的翻译后抑制而导致肝脏 SAMe 生物合成受损。慢性肝 SAMe 缺乏症发生在 MAT1A 敲除 (KO) 小鼠中，这些小鼠表现出缺乏胆碱饮食诱导的脂肪肝的倾向增加、脂质过氧化水平升高、脂肪性肝炎和肝癌的自发发展。我们最近表明，早在一个月龄时，该模型中的遗传毒性应激就增加了。一致地，我们发现 MAT1A KO 肝脏中的 UBC9 表达增加，重要的是，SAMe 的施用降低了蛋白质水平上的 UBC9 表达。用 SAMe 处理两种人类肝癌细胞系 HepG2 和 HuH-7 细胞也降低了 UBC9 蛋白水平。这是一个令人惊讶的发现，因为我们最近报道 SAMe 抑制蛋白体活性。由于饮酒会降低肝脏 SAMe 水平，我们还检查了该模型中 UBC9 表达是否发生改变，发现与 MAT1A KO 肝脏一样，肝脏 UBC9 蛋白水平因饮酒而增加。在本提案中，我们正在测试几个新的假设，1) UBC9 蛋白半衰期受到磷酸化的影响，因此关键位点的磷酸化可以防止降解，2) SAMe 及其代谢物甲硫腺苷 (MTA) 可以降低 UBC9 蛋白半衰期-通过降低这些关键位点的磷酸化来维持生命，3) SAMe 和 MTA（已知在肝癌细胞中具有促凋亡作用）通过降低 UBC9 导致细胞凋亡表达，4) UBC9 表达以及因此的苏酰化在酒精性肝损伤的发展过程中增加，并且它们可能导致许多所见的异常。本申请提出了三个目标来测试这些假设并阐明 SAMe 如何影响在肝癌发生和酒精性肝损伤发展中重要的信号通路。 公共健康相关性：S-腺苷甲硫氨酸 (SAMe) 是一种重要的生物分子，可控制细胞的生长、死亡和抗氧化反应。 SAMe 由甲硫氨酸腺苷转移酶 (MAT) 蛋白合成。 SAMe 水平在酒精性肝损伤和癌症期间下降。该项目旨在了解 SAMe 如何控制 UBC9（一种在癌症发展过程中诱导的重要蛋白质）。鉴于肝癌是全世界癌症死亡的主要原因，如果拟议的研究成功完成，它们可能会发现关于肝癌和酒精性肝病的发展和治疗的非常新颖的见解，因此它们具有非常高的公共卫生相关性。