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

描述(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通路。尽管小泛素样修饰物(SUMO)是该家族的新成员，但由于其新颖而独特的功能，近年来引起了人们的极大关注。苏莫化循环是一个多步骤的过程，包括成熟、活化、连接和连接，它由多个酶催化，包括E1、E2和E3酶。泛素结合酶9(UBC9)是唯一的E2结合酶，也是苏木酰化循环中最具特性的酶。UBC9将激活的相扑转移到靶蛋白上，被认为在调节底物特异性和提高体内相扑效率方面发挥着重要作用。基因毒性应激可诱导多种与核功能有关的蛋白质以及参与重要信号转导途径的蛋白质如核因子-kappaB(NFkappaB)等的总甲基化。UBC9和SUMO在人类癌前状态下高度表达，以响应低级别、长期的遗传毒性应激，这意味着SUMO的上调可能是对遗传毒性应激的一种适应过程。此外，UBC9在几种恶性肿瘤中过表达，如肺腺癌、卵巢癌和黑色素瘤。在裸鼠体内移植的MCF-7乳腺癌细胞中，拮抗UBC9功能抑制了细胞生长，并通过依赖于Bcl2的机制增加了细胞的凋亡率。UBC9‘S相扑结合活性失活突变增强了对DNA损伤剂的敏感性。UBC9可能是肿瘤发生和发展的基础，通过阻止凋亡通路的激活和最小化与肿瘤进展过程中积累的DNA损伤相关的急性细胞应激反应。UBC9在肝细胞癌(HCC)中是否被解除调控尚不清楚。UBC9蛋白的稳定性是如何在翻译后调节的也是未知的。最后，UBC9的表达是否会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，甲基供体还调节着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。包括酒精在内的慢性肝病患者由于MAT1A mRNA水平降低和MAT1A编码的同工酶翻译后抑制而损害了肝脏的相同生物合成。Mat1a基因敲除(KO)小鼠存在慢性肝脏同样缺陷，表现为胆碱缺乏饮食诱发脂肪肝的倾向增加，脂质过氧化水平升高，脂肪性肝炎和肝细胞癌的自发发展。我们最近发现，在这个模型中，早在一个月大的时候就有增加的遗传毒性应激。我们一致地发现，UBC9在MAT1A KO肝脏中的表达增加，重要的是，在蛋白质水平上同样降低了UBC9的表达。同样的作用于人肝癌细胞系HepG2和HH-7，也降低了UBC9的蛋白水平。这是一个令人惊讶的发现，因为我们最近报道，同样会抑制蛋白酶体的活性。由于酒精喂养降低了肝脏中UBC9的水平，我们还检查了该模型中UBC9的表达是否发生了变化，并发现与MAT1AKO肝脏一样，肝脏的UBC9蛋白水平在酒精喂养后增加。在这个提议中，我们验证了几个新的假说，1)UBC9蛋白质的半衰期受到磷酸化的影响，从而防止关键位点的磷酸化(S)，2)Same及其代谢产物甲硫腺苷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，已知在肝癌细胞中促进凋亡，通过降低UBC9的表达引起细胞凋亡，4)UBC9的表达增加，因此苏木糖化在酒精性肝损伤的发展过程中，它们可能导致了许多异常。在这项应用中提出了三个目标来检验这些假说，并阐明它们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。 公共卫生意义：S-腺苷蛋氨酸(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。同样由蛋氨酸腺苷转移酶(MAT)蛋白合成。同样的水平在酒精性肝损伤和癌症期间也会下降。这个项目旨在了解Same是如何控制UBC9的，UBC9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，它们可能会发现对肝癌和酒精性肝病的发展和治疗的非常新的见解，因此它们与公共卫生具有很高的相关性。

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.