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

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。小泛素样修饰物（SUMO）是该家族的新成员，但由于其新颖独特的功能，近年来引起了人们的广泛关注。类小泛素化循环是一个多步骤的过程，涉及成熟、活化、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（UBC 9）是唯一的E2结合酶，也是sumoylation循环中最具特征的酶。UBC 9将激活的SUMO转移到靶蛋白，并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。遗传毒性应激诱导参与核功能的大量蛋白质以及参与重要信号传导途径的蛋白质（如核因子κ B（NF-：B））的类小泛素化。UBC 9和SUMO在人类癌前病变中响应于低等级、长期遗传毒性应激而高度表达，这意味着SUMO化的上调可能是对遗传毒性应激的适应性过程。此外，UBC 9在几种恶性肿瘤中过表达，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UBC 9功能可抑制细胞生长，并通过Bcl-2依赖机制增加凋亡。UBC 9的SUMO缀合活性的失活突变增强了对DNA损伤剂的敏感性。UBC 9可能是肿瘤发生和肿瘤进展的基础，通过防止细胞凋亡途径的激活和最小化与肿瘤进展的累积DNA损伤相关的急性细胞应激反应。UBC 9在肝细胞癌（HCC）中是否失调尚不清楚。UBC 9蛋白的稳定性如何在免疫后调节也是未知的。最后，UBC 9的表达是否会因酒精而改变也是未知的。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作为甲硫氨酸代谢的第一产物，由甲硫氨酸腺苷甲硫氨酸（MAT）催化。除了作为甲基供体，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通路。在哺乳动物中，两个MAT基因MAT 1A和MAT 2A分别编码MAT的两个催化亚基11和12。由于MAT 1A mRNA水平降低和MAT 1A编码同工酶的翻译后抑制，慢性肝病（包括酒精）患者的肝脏SAMe生物合成受损。慢性肝脏SAMe缺乏发生在MAT 1A敲除（KO）小鼠中，其表现出增加的胆碱缺乏饮食诱导的脂肪肝倾向、更高水平的脂质过氧化、脂肪性肝炎和HCC的自发发展。我们最近发现，早在一个月大的时候，这个模型中的遗传毒性应激就增加了。一致地，我们发现UBC 9表达在MAT 1A KO肝脏中增加，并且重要的是，SAMe的施用在蛋白质水平上降低了UBC 9表达。用SAMe处理HepG 2和HuH-7细胞（两种人肝癌细胞系）也降低了UBC 9蛋白水平。这是一个令人惊讶的发现，因为我们最近报道了SAMe抑制蛋白酶体活性。由于酒精喂养降低了肝脏SAMe水平，我们还检查了UBC 9表达在该模型中是否改变，并发现与MAT 1A KO肝脏一样，肝脏UBC 9蛋白水平响应于酒精喂养而增加。在该提案中，我们正在测试几种新的假设，1）UBC 9蛋白半衰期受到磷酸化的影响，使得在关键位点的磷酸化防止降解，2）SAMe及其代谢物甲硫腺苷（MTA）可以通过降低UBC 9蛋白在这些关键位点的磷酸化来降低其半衰期，3）已知在肝癌细胞中促凋亡的SAMe和MTA，通过降低UBC 9表达引起细胞凋亡，4）在酒精性肝损伤的发展过程中，UBC 9表达和sumoylation增加，它们可能导致许多观察到的异常。在本申请中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。 公共卫生相关性：S-腺苷甲硫氨酸（SAMe）是一种重要的生物分子，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UBC 9，这是一种在癌症发展中诱导的重要蛋白质。鉴于肝癌是全球癌症死亡的主要原因，如果拟议的研究成功完成，它们可能会发现肝癌和酒精性肝病的发展和治疗的新见解，因此它们具有非常高的公共卫生相关性。

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.