ROLE OF S-ADENOSYLMETHIONINE ON UBC9 AND SUMOYLATION IN LIVER CANCER AND ALCOHOLI

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。小泛素样修饰剂（small ubiquitin-like modifier， SUMO）是该家族的新成员，近年来因其新颖而独特的功能而备受关注。sumoylation是一个多步骤的过程，包括成熟、激活、接合和连接；它被多种酶催化，包括E1， E2和E3酶。泛素偶联酶9 （ubitin - conjuconjuenzyme 9, UBC9）是唯一的E2偶联酶，也是summoylation循环中最具特征的酶。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提高体内SUMO化效率方面发挥重要作用。基因毒性应激诱导大量参与细胞核功能的蛋白质以及参与重要信号通路的蛋白质的酰化，如核因子κ B （NF-:B）。UBC9和SUMO在人类恶性前病变中高度表达，以应对低级别、长期的基因毒性应激，这意味着SUMO的上调可能是对基因毒性应激的适应性过程。此外，UBC9在一些恶性肿瘤中过表达，如肺腺癌、卵巢癌和黑色素瘤。裸鼠移植MCF-7乳腺癌细胞拮抗UBC9功能可通过Bcl-2依赖机制抑制细胞生长并增加凋亡。灭活UBC9的SUMO偶联活性突变增强了对DNA损伤剂的敏感性。UBC9可能是肿瘤发生和肿瘤进展的基础，它可以阻止凋亡通路的激活，并最小化与肿瘤进展中累积的DNA损伤相关的急性细胞应激反应。UBC9在肝细胞癌（HCC）中是否失调尚不清楚。UBC9蛋白的稳定性是如何在翻译后调控的也是未知的。最后，UBC9的表达是否因酒精而改变也是未知的。s -腺苷蛋氨酸（SAMe）是所有哺乳动物细胞中主要的生物甲基供体，是蛋氨酸代谢的第一个产物，由蛋氨酸腺苷蛋氨酸（MAT）催化。除了作为甲基供体外，越来越多的证据表明SAMe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。慢性肝病（包括酒精）患者由于MAT1A mRNA水平下降和MAT1A编码同工酶的翻译后抑制，肝脏SAMe生物合成受损。慢性肝脏SAMe缺乏症发生在MAT1A敲除（KO）小鼠中，这些小鼠表现出胆碱缺乏饮食诱导的脂肪肝倾向增加，脂质过氧化水平升高，自发发展为脂肪性肝炎和HCC。我们最近表明，在这个模型中，早在一个月大的时候就有增加的基因毒性应激。一致地，我们发现UBC9在MAT1A KO肝脏中的表达增加，重要的是SAMe在蛋白水平上降低了UBC9的表达。SAMe治疗两种人肝癌细胞系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。由于酒精喂养降低了肝脏SAMe水平，我们也检查了UBC9的表达是否在该模型中改变，并发现与MAT1A KO肝脏一样，肝脏UBC9蛋白水平在酒精喂养的反应中升高。在这个提议中，我们正在测试几个新的假设，1)UBC9蛋白的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及其代谢物甲基硫腺苷（MTA）可以通过降低这些关键位点的磷酸化来降低UBC9蛋白的半衰期，3)SAMe和MTA，已知在肝癌细胞中具有促凋亡作用，通过降低UBC9的表达导致凋亡。4)在酒精性肝损伤的发展过程中，UBC9的表达和sumoylation增加，它们可能导致许多所见的异常。本研究提出了三个目的来验证这些假设，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。

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.