【作者】 刘超；

【导师】 张令强；

【作者基本信息】 安徽医科大学 ， 细胞生物学， 2017， 硕士

【摘要】 泛素化和类泛素化修饰是一种蛋白翻译后修饰,在生命进程中起到非常重要的调控作用,Nedd8是一种类泛素蛋白,在氨基酸序列和三维结构上与泛素都非常相似,最早发现Nedd8在小鼠胚胎的脑发育前期呈现高表达,并随着发育进程逐步降低。目前研究发现Nedd8多与肿瘤的恶性程度、预后等呈正相关。在细胞内泛素广泛分布,而Nedd8主要在细胞核内富集,Nedd8能够对泛素化修饰起到双向调节作用,当Nedd8使Cullin类E3酶发生Neddylation修饰时,会改变其酶的构型,使E3更容易和泛素结合酶(E2)相结合,促进E3的泛素化修饰酶活性,但是当Neddylation与泛素化修饰竞争同一修饰位点时,又可以起到抑制底物泛素化修的作用。RBXs是泛素连接酶Cullin-RING类复合体的一个组分,是目前研究最多的Neddylation修饰连接酶,随着进一步研究发现,泛素连接酶MDM2、Smurf1、ITCH和NEDL2也能起到Neddylation修饰连接酶的作用,Smurf1与同源蛋白Smurf2非常相似,两者都属于泛素连接酶HECT类Nedd4-like亚族,Smurf2在结构上比Smurf1多一个WW结构域,两者在细胞内既有功能上的冗余,又有底物的特异性,在肿瘤的发生发展中Smurf1往往起到促癌的作用,而Smurf2在肿瘤发生的前期是起到抑制肿瘤的作用,但是在肿瘤的发展后期却能够促进肿瘤。Smurf1的Neddylation修饰提示我们Smurf2同样可能作为一个Neddylation修饰的E3。这种翻译后修饰对Smurf2有何影响并没有报道。在高表达Nedd8的结直肠癌细胞系HCT116细胞中我们检测到Smurf2可以发生Neddylation修饰,并通过药物MLN4924和去Neddylation修饰酶对Neddylation修饰抑制的作用进一步确认了Smurf2被Neddylation修饰的可靠性。在研究中我们发现Smurf2和Smurf1虽然都能够发生Neddylation修饰,但是Smurf1和Smurf2并不相同,鉴于Smurf2作为Neddylation修饰连接酶的猜想,我们并没有在Smurf2 HECT结构域内发现一个与Smurf1类似的Neddylation修饰酶活中心。由于Nedd4家族普遍存在C2结构域和HECT结构域相互作用,我们在外源实验中发现Smurf1和Smurf2存在直接相互作用,并发现Smurf1能够促进Smurf2发生Neddylation修饰。进一步研究发现Neddylation可以使Smurf2泛素化修饰增强,并通过蛋白酶体途径使Smurf2蛋白水平下调。在此我们发现了泛素连接酶Smurf2一种新的翻译后修饰——Neddylation修饰,通过这种修饰能够调节Smurf2自身的泛素化修饰和酶活性。但是这种修饰和Smurf1的Neddylation并不相同,这提示我们同源蛋白在修饰层面存在的差异性,并且可以通过其他方式相互影响修饰过程。在设计靶向泛素连接Smurf1或Smurf2的药物时,我们应该充分考虑其同源蛋白的异同,评估药物对靶分子以外蛋白的影响。更多还原

【Abstract】 Nedd8 is a ubiquitin-like protein and neddylation is a posttranslational modification play an important role in the life-cycle.Nedd8 is highly homologous to ubiquitin in amino acid sequence and protein structure.The early study of Nedd8 found Nedd8 was highest in the early mouse embryo and was developmentally down-regulated in the mouse embryo of brain.In current study,Nedd8 is positively correlative with tumorigenesis.Unlike ubiquitin which was equally expression in the nucleus and cytosol,Nedd8 is highly enrich in the nuclear and is weaker in the cytosol.Neddylation regulate ubiquitination though dual direction regulation.When the Cullin-RING is in the neddylation stage,the structure of Cullin-RING will change to facilitate recruitment of E2.This indicate that neddylation is essential for Cullin-RING ligases and promotes ubiquitination.However,when the neddylation site compete with Ubiquitination site in certain substrates,neddylation will inhibit the ubiquitin congection.Currently,all reported Nedd8 E3 enzymes can also function as ubiquitin E3 enzymes.The best-studed Nedd8 ligase are RBXs,a component of the complex of Ubiquitin ligase Culin-RING E3.In recent study,the most reported non Cullin-RING Nedd8 E3 enzymes include MDM2,Smurf1,ITCH and NEDL2.Smurf2 is highly homologous to Smurf1,they are one of the member of HECT Nedd4-like ubiquitin ligases.There are some difference in crystal structure between Smurf1 and Smurf2.Smurf1 have two WW domains,and Smurf2 have three WW domains.They have functional redundancy and substrate specificity,because of the difference and similarity between Smurf1 and Smurf2.For example,Smurf1 promotes tumor progression,while Smurf2 inhibits tumor development in early-stage,while promotes tumor progression in later-stage.The neddylation of Smurf1 gives us a clue that Smurf2 may be a Nedd8 E3 ligase.Neddylation is a new modification of Smurf2.We find that Smurf2 could be modified by Nedd8 in HCT116 cell line which highly expresses Nedd8.We use MLN4924 and deneddylation enzyme which could inhabit Neddylation to conform the result.Although both Smurf1 and Smurf2 could be modified by Nedd8,Our study find that Smurf2 is different from Smurf1 in the Nedd8-modification.We can not find active site of Nedd8 ligase in HECT domain.Multiple sequence alignment of the HECT family indicates that Nedd8-ligase active site is non-conserve in the family.All Smurf2’s domains could be modified by Nedd8,when Nedd8 was over expressed in cell.Previous study have demonstrated that c2 domain could interact with HECT domain.We find Smurf1 and Smurf2 directly interact in vitro.The interaction between Smurf1 and Smurf2 may enhance Smurf2 neddylation.Because of Smurf2 neddylation,the protein level is downregulated and the Smurf2 ubiquitination is upregulated.We also find that Nedd8 promote degredation of Smad3 and RNF20 in protein levels.The study show some similarity and also difference between Smurf1 and Smurf2.A new modification of Smurf2 could be enhanced by its homologous protein Smurf1.This will help us to understand homologous protein in modification level.When design a target drug of Smurf1 or Smurf2,we should think about the drug target validation and identification of secondary drug target effects.更多还原