【作者】 杜涛峰；

【导师】 韩文瑜；

【作者基本信息】 吉林大学 ， 预防兽医学， 2013， 博士

【摘要】 转录因子的翻译后修饰，包括磷酸化，乙酰化，甲基化和泛素化，通过调节稳定性，亚细胞定位，寡聚化或DNA结合能力影响其转录活性。FOXP3赖氨酸残基的乙酰化是一个重要的翻译后修饰，调节Treg细胞内FOXP3的表达水平和功能。目前，已鉴定两种组蛋白乙酰化转移酶（HAT），包括Tip60（Tat-interactiveprotein，也称为KAT5）和p300，可以促进FOXP3的乙酰化。乙酰化的FOXP3对多聚泛素化和蛋白酶体降解更耐受，而且具有更好的染色质结合活性。Garcinol，一种分离于印度藤黄果皮的聚异戊二烯二苯甲酮，是组蛋白乙酰化转移酶p300的天然抑制剂。Garcinol可以抑制p300的自身乙酰化和p300介导的p53（又称为TP53）的乙酰化。最近的研究鉴定了一组复杂的组蛋白乙酰化转移酶之间的相互作用导致了FOXP3功能的增强或抑制。本文采用质粒转染，Western blotting和免疫沉淀实验，用Garcinol作为一种工具，分析了p300调节FOXP3乙酰化的机制。实验结果表明，Garcinol抑制了p300和FOXP3复合物的形成，并促进p300溶酶体降解途径，从而降低了FOXP3乙酰化的水平和最终的降解，蛋白酶体抑制剂MG132并不能抑制Garcinol诱导的降解。在FOXP3乙酰化方面，p300对FOXP3的乙酰化影响较为显著，可以通过影响4个赖氨酸残基影响FOXP3总体乙酰化水平和稳定性。分离于小鼠脾脏的CD4^-CD25^highTreg细胞和CD4^-CD25-CD45RB^highT细胞（效应T细胞，Teff），通过Treg细胞体外抑制实验，研究Garcinol对Treg细胞的功能的影响。实验结果表明，Treg细胞呈剂量依赖性抑制Teff细胞的增殖，并且Garcinol可以抑制Treg细胞的抑制活性。本文通过MTT细胞毒性实验和用neu转化的乳腺癌细胞（H2N113）建立的MMTV-neu转基因小鼠，分别在体外和体内研究了Garcinol和抗肿瘤单克隆抗体7.16.4的协同作用。细胞毒性实验结果表明，7.16.4呈剂量依赖性的抑制H2N113细胞的增殖。Garcinol也可以抑制H2N113细胞的增殖，但不能增强7.16.4的体外抑制活性。小鼠肿瘤模型实验结果表明，高浓度的7.16.4（9mg/kg）本身可以显著抑制肿瘤的生长，低浓度的7.16.4（1.8mg/kg）和Garcinol（43.6mg/kg）本身对肿瘤生长的抑制作用很小，但Garcinol和低浓度的7.16.4的联合应用增强了对肿瘤的抑制作用。这些数据说明Garcinol增强了靶向治疗7.16.4抗体在体内的抗肿瘤活性。本研究证明，可以通过小分子破坏p300的稳定性限制Treg细胞的功能，为癌症的靶向治疗提供了一种新的途径。更多还原

【Abstract】 Post-translational modifications, such as phosphorylation, acetylation, methylationand ubiquitination have been shown to occur on many transcriptional factors andaffect transcriptional activity by regulating protein stability, subcellular localization,oligomerization, or DNA binding. FOXP3lysine ε-acetylation is an important post-translational modification that regulates FOXP3expression levels and functions inTreg cells. At least two histone acetyltransferases （HAT）, including Tat-interactiveprotein （Tip60, also known as KAT5） and p300, promote FOXP3acetylation.Acetylated forms of FOXP3appear to be more resistant to poly-ubiquitination andproteasomal degradation, and move from the nucleoplasm to the chromatin.Garcinol, a polyisoprenylated benzophenone derivative from the fruit rind ofGarcinia indica, has been claimed to represent a natural inhibitor of p300.Garcinolinhibits p300auto-acetylation as well as p300-mediated p53（also known as TP53）acetylation.Our recent studies have defined a complex set of histone acetyltransferaseinteractions which can lead to enhanced or repressed changes in FOXP3function. Wehave explored the use of a natural p300inhibitor, Garcinol, as a tool to understandmechanisms by which p300regulates FOXP3acetylation. In the presence of Garcinol,p300appears to become disassociated from the FOXP3complex and undergoeslysosome-dependent degradation. As a consequence of p300’s physical absence,FOXP3becomes less acetylated and eventually degraded, a process that cannot berescued by the proteasome inhibitor MG132. p300plays a complex role in FOXP3acetylation, as it could also acetylate a subset of four Lys residues that repressivelyregulate total FOXP3acetylation.In vitro Treg suppression assay,Mouse CD4^-CD25^highTreg and CD4^-CD25-CD45RB^highT cells（representing effector T cell, Teff） were isolated fromC57BL/6mice. Teffand Treg cells were mixed at different ratios. We next examinedwhether Garcinol affected Treg suppressive function. Treg cells inhibited theproliferation of Teffcells in a dose-dependent manner.Garcinol acts as a degradationdevice to reduce the suppressive activity of Treg cells.In MTT assay demonstrated that7.16.4could inhibit H2N113in adose-dependent manner.We did note that Garcinol slightly inhibited the proliferationof H2N113.However, Garcinol was not able to enhance the activity of7.16.4in this invitro assay. Studying mouse tumor models,we show that the high dose7.16.4（9mg/kg）treatment alone significantly reduced the tumor growth. The growth of tumorappeared to be only modestly reduced by the low dose7.16.4（1.8mg/kg）treatment.While Garcinol （43.6mg/kg） had little discernible effects on the growth of tumors, thecombination of Garcinol and low dose7.16.4led to enhanced inhibition of tumorgrowth.These data indicate that Garcinol enhance the in vivo anti-tumor activity of atargeted therapeutic7.16.4monoclonal antibody in MMTV-neu transgenics implantedwith neu transformed breast tumor cells. Our studies provide the rationale formolecules that disrupt p300stability to limit Treg functions in targeted therapies forcancers.更多还原