【作者】 王瑾；

【导师】 李淑晶；

【作者基本信息】 大连理工大学 ， 生物学， 2020， 硕士

【摘要】 细胞凋亡对于维持机体生长发育至关重要。细胞内凋亡信号通路的紊乱会直接导致各种疾病的发生。例如,在癌症中经常会出现凋亡失调的现象。髓样细胞白血病蛋白1(Myeloid cell leukemia 1,MCL1)属于BCL-2家族蛋白,其在细胞中发挥的主要功能为抗凋亡。研究发现在多种人类恶性肿瘤(如乳腺癌、非小细胞肺癌(NSCLC)、淋巴瘤、急性髓系白血病(AML)和多发性骨髓瘤等)的生存中,MCL1都发挥着重要作用。同时MCL1的稳定性直接影响其功能的发挥,降低其稳定性会增强放、化疗的治疗效果,提高患者生存率。SUMO修饰是一种关键的翻译后修饰,可以调节底物功能,包括蛋白质活性、稳定性、转录活性、亚细胞分布和蛋白质相互作用等。通过对MCL1的蛋白序列分析,我们发现了两个潜在的SUMO修饰位点。因此,本研究探讨了MCL1的SUMO化修饰作用以及该修饰对MCL1功能的具体调控机制。主要工作如下:1.通过在COS7细胞系中进行免疫共沉淀实验,我们发现SUMO蛋白可以修饰MCL1。此外,突变掉SUMO1的结合位点,MCL1的SUMO修饰带消失,而加入E2结合酶Ubc9后其修饰可以被增强。同时,我们发现内源MCL1也具有SUMO修饰带。因此,我们确定MCL1能够进行SUMO修饰。另外,我们通过定点突变确定了MCL1的SUMO修饰位点为234和238处的赖氨酸。2.在免疫荧光实验中,我们发现野生型MCL1与SUMO修饰位点突变体MCL1-2K的亚细胞定位没有显著差异,暗示SUMO修饰不影响MCL1的亚细胞定位。通过蛋白质稳定性实验发现,突变体MCL1-2K的蛋白稳定性降低,暗示SUMO修饰可以稳定MCL1。3.酵母双杂交实验再次证实了,MCL1与TRIM11蛋白(一种新发现的MCL1泛素E3连接酶)可以直接结合。通过免疫共沉淀实验发现,MCL1的SUMO化可以抑制TRIM11介导的MCL1的泛素化进而增加其稳定性。4.细胞增殖、细胞克隆形成和细胞凋亡实验结果显示,野生型MCL1显著增强肿瘤细胞的增殖能力和抗凋亡能力,而突变体MCL1-2K的促增殖和抗凋亡作用显著减弱。综上所述,本论文发现MCL1可以被SUMO修饰,探讨了SUMO修饰调控MCL1稳定性和功能的具体分子机制。更多还原

【Abstract】 Apoptosis is important to maintain the growth and development of the body.In cancers,apoptosis evasion through dysregulation of pro?apoptotic and anti?apoptotic intracellular signals is a recurring event.Myeloid cell leukemia 1(MCL1)is an anti-apoptotic protein of the BCL-2 family,which is overexpressed in many cancers.MCL1 is essential for the survival of human malignancies,including breast cancer,non - SCLC,acute myeloid leukaemia,MYC-driven lymphomas and multiple myeloma.Studies have shown that reducing the stability of MCL1 can improve the therapeutic effects of cancer.SUMOylation is a crucial post-translational modification that can regulates substrate functions including protein activity,stability,transcriptional activity,subcellular distribution,and control of protein interactions.Sequence analysis of MCL1 reveals two potential SUMO conjugation sites.Therefore,we hypothesize that MCL1 is a target of SUMO.The main works of the paper are as follows:1.We observed that MCL1 could be modified by SUMO at K234 and K238 sites using immunoprecipitation assay in COS7 cells.In addition,the SUMOylation level of MCL1 was enhanced by Ubc9.And we found that endougenous MCL1 could also be modified by SUMO.So,we believed that MCL1 was a target of SUMO.2.We found the SUMOylation did not affect subcellular distribution between wide-type MCL1 or its mutant 2K by immunofluorescence staining.We also observed that the half-life of wild-type MCL1 was longer than the mutant 2K.It demonstrated that the SUMOylation could stabilize the protein level of MCL1.3.The direct association of TRIM11(a novel MCL1 ubiquitin E3 ligase)and MCL1 was identified using the mammalian two-hybrid system.In addition,we found that the SUMOylation of MCL1 could improve its stability by inhibiting the MCL1ubiquitin-proteasome pathway mediated by TRIM11 using immunoprecipitation assay.4.Cell growth assay,colony formation assay and apoptosis assay demonstrated that the SUMOylation of MCL1 promoted the proliferation of cancer cells by inhibiting apoptosis.Collectively,our data identified MCL1 a novel target of SUMO,and showed the specific molecular mechanism of regulating the stability and function of MCL1 by SUMO.更多还原