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: 8320778
• 负责人: Maria Lauda Tomasi
• 金额: $ 5.42万
• 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/8320778
• 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; Health; Hepatic; Hepatitis C; Hepatocarcinogenesis; Hepatocyte; Human; Incidence; Inflammation; Injury; Injury to Liver; Isoenzymes; Knock-out; Knockout Mice; Lead; Ligation; Lipid Peroxidation; Liver; 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; chronic liver disease; 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; 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; Health; Hepatic; Hepatitis C; Hepatocarcinogenesis; Hepatocyte; Human; Incidence; Inflammation; Injury; Injury to Liver; Isoenzymes; Knock-out; Knockout Mice; Lead; Ligation; Lipid Peroxidation; Liver; 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; chronic liver disease; 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.

描述（由申请人提供）：泛素样蛋白介导的翻译后修饰调节多种细胞途径。尽管小型泛素样修饰符（SUMO）是该家族的新成员，但由于其新颖而独特的功能，最近它引起了很多关注。 sumoylation循环是一个多步骤过程，涉及成熟，激活，结合和连接；它被包括E1，E2和E3酶在内的多种酶催化。泛素结合酶9（UBC9）是唯一的E2共轭酶，并且是Sumoylation循环中最佳的特征酶。 UBC9将活化的SUMO转移到靶蛋白上，并被认为在调节底物特异性和增强体内Sumoylation的效率方面起着重要作用。遗传毒性应激诱导参与核功能的广泛蛋白质以及参与重要信号通路的蛋白质（例如核因子kappa b（NF-：b））。在低度，长期的遗传毒性应激的响应中，UBC9和SUMO在人类婚前条件下高度表达，这意味着Sumoylation的上调可能是基因毒性应激的适应性过程。此外，UBC9在几种恶性肿瘤中过表达，例如肺腺癌，卵巢癌和黑色素瘤。在裸鼠中移植的MCF-7乳腺癌细胞中拮抗UBC9的功能可抑制细胞的生长，并通过Bcl-2依赖性机制增加凋亡。 UBC9相和共轭活性的灭活突变会增强对DNA损害剂的敏感性。 UBC9通过防止凋亡途径的激活并最大程度地减少与肿瘤进展的DNA损伤相关的急性细胞应激反应，这可能是肿瘤发生和肿瘤进展的基础。 UBC9在肝细胞癌（HCC）中是否受到UBC9的管制是未知的。在后翻译后如何调节UBC9蛋白的稳定性也是未知的。最后，是否对酒精响应的UBC9表达也未知。 S-腺苷硫氨酸（相同）是在所有哺乳动物细胞中生产的原理生物甲基供体，作为蛋氨酸代谢的第一产物，由蛋氨酸腺苷甲硫代氨酸（MAT）催化。除了作为甲基供体外，积累的证据还显示出相同的调节，这是控制生长和凋亡的许多关键细胞途径。在哺乳动物中，两个MAT1A和MAT2A分别为MAT的两个催化亚基编码11和12。包括酒精在内的慢性肝病患者由于MAT1A mRNA水平降低和对MAT1A编码的同工酶的翻译后抑制作用而损害了肝相同的生物合成。慢性肝相同缺乏发生在MAT1A敲除（KO）小鼠中，这些小鼠表现出增加胆碱缺乏饮食诱导脂肪肝的倾向，脂质过氧化的较高水平，脂肪性肝炎的自发发展和HCC。我们最近表明，该模型最早在一个月大时就会增加遗传毒性应激。一致地，我们发现MAT1A KO肝脏中的UBC9表达增加，并且重要的是在蛋白质水平上施用相同的UBC9表达。治疗HEPG2和HUH-7细胞，两种人肝癌细胞系，也降低了UBC9蛋白水平。这是一个令人惊讶的发现，因为我们最近报道了相同的抑制蛋白体活性。由于酒精喂养降低了肝相同水平，因此我们还检查了该模型中的UBC9表达是否改变，并发现像MAT1A KO肝脏一样，肝UBC9蛋白水平也随着酒精喂养而增加。在这项提案中，我们正在测试几个新的假设，1）UBC9蛋白半衰期受磷酸化的影响，以使关键部位的磷酸化（S）可预防降解，2）相同，其代谢物甲基硫代硫代腺苷（MTA）可以通过降低UBC9蛋白质的磷酸化量来降低这些磷酸化的磷酸化，并在这些关键的情况下降低这些磷酸化量，并在这些磷酸化中降低了这些磷酸化量，并在这些磷酸化中降低了这些磷酸盐的磷酸化。肝癌细胞中的凋亡，通过降低UBC9表达，4）UBC9表达并因此在酒精性肝损伤发展过程中增加了凋亡，因此它们可能有助于看到的许多异常。在此应用中提出了三个目的，以检验这些假设，并阐明在肝癌发生和酒精肝损伤的发育中对信号传导途径的影响。