线性泛素链组装复合体LUBAC与泛素连接酶Smurfs相互作用与调控机制的研究

发布时间：2018-06-05 16:21

本文选题：线性泛素链组装复合体LUBAC + 线性泛素链去泛素化酶OTULIN　；参考：《中国人民解放军军事医学科学院》2016年博士论文

【摘要】：蛋白质泛素化修饰过程在调节各种细胞生物学功能的过程中发挥了非常重要的作用,如细胞周期进程、DNA损伤修复、信号转导和各种蛋白质膜定位等。泛素化修饰可分为多聚泛素化修饰和单泛素化修饰。多聚泛素化修饰系统可以通过对底物连接不同类型的多泛素化链调节蛋白质的功能。多聚泛素化修饰中已知7种泛素链连接方式均为泛素内赖氨酸连接方式。近几年发现了第8种类型的泛素链连接形式即线性泛素链,其泛素链的连接方式是由泛素甲硫氨酸的氨基基团与另一泛素甘氨酸的羧基基团相连形成泛素链标记。2006年日本大阪大学Kazuhiro Iwai教授实验室首次发现了线性泛素链组装复合体(Linear ubiquitin chain assembly complex,LUBAC)的存在,它是线性泛素化修饰的E3。LUBAC复合体是由HOIL-1L,HOIP,Sharpin组成。目前已知的线性泛素链组装复合体的底物主要有NEMO、RIP2、RIP1等,它们在TNF-α激活的信号通路过程中发挥着非常重要的作用。LUBAC可参与多个信号通路的激活。LUBAC可以特异性激活NF-B信号通路,在ERK磷酸化激活过程也发挥着重要的作用。去泛素化酶属于Ub信号的关键调节因子,目前已知去线性泛素化特异性DUB主要包含OTULIN(OTU domain-containing deubiquitinase with linear linkage specificity)可以对线性泛素链的多聚泛素化信号起到调节作用。去线性泛素化酶OTULIN在调控Wnt信号通路、神经发育以及血管生成等多种生物学功能中发挥着重要作用。泛素化系统一般通过适时和有选择性的在其底物上加上泛素链的方式调控细胞的功能,所以识别线性泛素链组装复合体的底物和功能非常必要,它将有助于阐明线性泛素链参与调控的各种细胞生物学功能的具体机制。寻找新的参与体内线性泛素化连接和识别的蛋白复合体,成为线性泛素化研究领域的热点。解析LUBAC介导的线性泛素化是否参与其他信号通路以及识别LUBAC新底物同样十分重要。Smurfs(Smad ubiquitination regulatory factor 1/2)是HECT E3 Nedd4家族的主要成员,目前报道参与多种细胞生物学的功能。Smurfs最初确定来源于调节BMP/TGFβ家族信号通路,但最近的研究表明,其调节范围广泛,包括Wnt信号通路调控。TGFβ家族信号通路调节广泛的生物学过程包括细胞的迁移、生长、分化和发育。本课题组在研究Smurfs调控机制的过程中,利用酵母双杂交技术,通过Smurf2-HECT结构域对脾脏文库筛选得到HOIP(HOIL-1L Interacting Protein),HOIP作为线性泛素链组装复合体LUBAC的核心分子,我们猜想Smurfs与HOIP是否存在真实的相互作用,同时Smurfs与LUBAC线性复合体有什么关联,Smurfs在线性泛素化的调控过程中是否发挥了重要的作用,LUBAC线性泛素化修饰是否参与新的信号通路调控发挥重要的功能,Smurfs是否在NF-κB信号通路的过程中发挥着重要的作用,这些问题将在本课题中展开深入的研究。本课题通过实验研究发现:Smurf2与Smurf1相似,都可以与HOIP互作。Smurfs作为泛素连接酶E3其活性位点突变体不影响其与HOIP的相互关系。Smurfs与LUBAC复合体的其他组分HOIL-1L,Sharpin不能直接相互作用,但是加入HOIP后Smurfs可以与HOIL-1L和Sharpin相互作用。体内CO-IP实验发现以Smurf1为例,HOIP、HOIL-1L、Sharpin均可以与Smurfs相互作用。HOIP是一个具有多结构域分子量为120KD的蛋白,通过研究我们发现HOIP的C端的LDD结构域可以与Smurf1-HECT-N-Lobe结合。LDD被称为线性泛素决定区,在所有RBR类E3中,LDD结构域是特异存于HOIP,特异性决定线性泛素链组装复合体LUBAC的功能。该结合区为泛素由E2转移到泛素硫酯键中间体最后停留的位置,该区域中ZF结构域可以与Ub结合。Smurfs作为一个泛素连接酶与线性泛素链组装复合体LUBAC之间是否存在调控关系Smurfs是否参与线性泛素化修饰?我们发现单表达LUBAC复合体的各个组分,Smurfs表达水平不变,共表达LUBAC复合体的3个成员HOIP、HOIL-1L、Sharpin后,Smurfs蛋白表达水平下调,LUBAC对于Smurfs E3活性突变体Smurf1 C699A和Smurf2C716A没有影响。敲低LUBAC复合体的各个组分HOIP、HOIL-1L、Sharpin、Smurfs的表达水平上调。检测Smurfs的泛素化水平,发现单一过表达HOIP,Smurfs的泛素化水平没有变化,过表达LUBAC复合体后,Smurfs的泛素化水平明显上调。敲低LUBAC复合体的各个组分HIOP/HOIL-1L/Sharpin后,Smurfs泛素化水平下调,体外泛素化实验同样得到相同的结论。HOIP C885是LUBACE3活性中心,C885A突变体抑制Smurfs体外泛素化水平的上调。OTULIN作为线性去泛素化酶,可以抑制Smurfs泛素化水平的上调。Smurfs泛素水平上调,被认为是LUBAC促进Smurfs募集线性泛素链使Smurfs发生线性泛素化修饰。LUBAC可以增强Smurfs E3活性的机制在于HOIP与Smurfs的相互作用抑制了Smurf1分子间二聚体形成,促进活性单体的比例,进而我们还发现LUBAC可以抑制TGFβ/BMP信号通路活性。敲低HOIP后,CAGA12-luc活性上调,TGFβ信号通路相关的靶基因Smad7,SNON,CTGF,PAI-1表达上调。同时发现,BMP激活的BRE-luc的活性被LUBAC抑制。LUBAC可以抑制TGFβ/BMP信号通路的活性的机制是什么?LUBAC复合体可以增强Smurfs的泛素化水平,那么对于Smurfs的重要底物R-Smad,具有怎么样的重要作用呢,我们研究发现LUBAC线性泛素链组装复合体可以加速Smurfs对Smads的降解,这一过程是依赖于Smurfs E3活性的。TGFβ促进细胞迁移、侵袭和转移,那么LUBAC复合体与OTULIN对细胞迁移有什么作用呢?通过Transwell实验,我们发现敲低HOIP以后可以增强细胞的迁移能力,相对地,敲低OTULIN后,细胞迁移能力降低,同时利用RT-PCR实验发现,有关细胞迁移、侵袭和转移相关的TGFβ信号通路相关的靶基因PTHr P、MMP2、MMP9在HOIP敲低和OTULIN敲低的细胞中也发生相应的上调和下调。综上所述,本课题主要以HECT类泛素连接酶Smurfs通过与线性泛素链组装复合体LUBAC核心分子HOIP的相互作用为联系的基础,发现线性泛素链组装复合体LUBAC可以通过线性泛素化修饰Smurfs,增强Smurfs E3活性,加速Smurfs的底物降解,抑制TGFβ/BMP信号通路的活性,相对地,我们也发现去线性泛素化酶OTULIN和LUBAC的C885A的E3活性突变体可以废除Smurfs的泛素化增强效应。LUBAC负调控TGFβ/BMP信号通路的活性,TGFβ促进细胞迁移、转移,LUBAC抑制了该效应。这是首次发现线性泛素链组装复合体和线性泛素化修饰参与TGFβ/BMP信号通路的调控。我们也发现LUBAC可以促进Smurfs募集线性泛素化从而增强Smurfs E3的活性,这也是对Smurfs调控机制又一补充。Nedd8(neural precursor cell-expressed developmentally downregulated 8)分子是一类结构上与泛素相似的分子,参与蛋白质翻译后修饰,这一过程被称为Neddylation,Neddylation的发生机制与泛素化相似,需要E1(UBA3-APPBP1)、E2(Ubc12)、E3介导的一系列酶促反应。Neddylation修饰在Cullin-Roc类泛素连接酶的活性调控中具有至关重要的作用。与泛素化研究相比,目前在真核细胞内还只有很少的Neddylation修饰底物被发现,Neddylation的生理功能也有待深入研究。目前对于Ub与底物的结合研究的比较清楚,已发现接近20种结构域(即UBD)可以与泛素结合,但是对于类泛素蛋白Nedd8非共价结合蛋白的研究,目前鲜有报道,所以研究Nedd8与蛋白相互作用的特异性规律,可以更好的了解Neddylation的生理功能。Smurf1是HECT泛素连接酶Nedd4家族的成员,在骨形成、胚胎发育等过程中发挥重要作用。Smurf2与Smurf1高度相似,两者负调控TGFβ和BMP信号通路。本课题以Smurf1为诱饵采用酵母双杂交的方法,筛选到类泛素蛋白Nedd8。我们通过CO-IP、GST-Pull down等实验证明Smurf1与Smurf2均可以与Nedd8在体内外发生相互作用,利用截短体相互作用研究,我们发现Smurf1的WW结构域,HECT的N-lobe Small结构域,HECT的C-lobe结构域可以结合Nedd8。Smurf2与Smurf1相似,HECT结构域中的N-lobe Small domain和C-lobe介导结合,但是WW结构域未参与与Nedd8的结合。分析Nedd4家族9个成员与Nedd8的相互作用发现NEDL1和NEDL2与Smurfs同样可以结合Nedd8。随后,我们利用Nedd8的晶体结构与Smurf2-HECT晶体结构进行结构对接,模拟相互作用,以期发现结合模序。通过序列比对,序列L(X7)R(X5)F(X)ALQ保守。突变体相互作用实验证明,该模序介导结合。通过突变体功能实验表明废除Nedd8与Smurf的非共价相互作用削弱了Smurf的Neddylation修饰,稳定了Smurf蛋白水平,同时改变了Smurf与Ub形成硫酯键中间体的能力。Nedd8与Smurf相互作用对于Smurf的E3活性和底物的泛素化水平起到了关键作用。同时对于Smurfs所调控的BMP和TGFβ信号通路以及细胞迁移发挥了重要的作用。同时,我们也研究发现Nedd8可共价修饰Smurf。Smurf1可以作为一个Nedd8泛素连接酶激活Smurf1发生自身Neddylation修饰。Smurf1的N-lobe中的C426是其Neddylation E3催化活性中心,区别于泛素修饰E3活性中心C699。Neddylaiton对HECT E3调控过程中发挥重要作用。
[Abstract]:The process of ubiquitination of proteins plays a very important role in regulating various cell biological functions, such as cell cycle process, DNA damage repair, signal transduction and various protein membrane localization. Ubiquitination modification can be divided into polyubiquitin modification and mono ubiquitination modification. The substrate connects different types of polyubiquitin chains to regulate the function of proteins. The 7 ubiquitin chain connections in polyubiquitin modification are all ubiquitin lysine connections. In recent years, eighth types of ubiquitin chain connections, namely, linear ubiquitin chains, are found in the ubiquitin chain by the amino groups of the ubiquitin methionine. The existence of a linear ubiquitin chain assembly complex (Linear ubiquitin chain assembly complex, LUBAC) was first found in Professor Kazuhiro Iwai of Osaka University, Japan, for the first time linked to the carboxyl group of another ubiquitin glycine for.2006. The E3.LUBAC complex of the linear ubiquitination trimming is the HOIL-1L, HOIP, and group. The substrates of the linear ubiquitin chain assembly complexes known at present are mainly NEMO, RIP2, RIP1 and so on. They play a very important role in the signaling pathway of TNF- alpha activation..LUBAC can participate in the activation of.LUBAC pathway in multiple signaling pathways, and the activation process of ERK phosphorylation also plays an important role in the activation process of ERK phosphorylation. Use. De ubiquitination enzyme is a key regulator of Ub signal. Currently, the known de linear ubiquitination specific DUB contains OTULIN (OTU domain-containing deubiquitinase with linear linkage specificity), which can regulate the polyubiquitination signal of linear ubiquitin chain. Pathways, neurodevelopment, and angiogenesis play an important role. Ubiquitination systems generally regulate cell functions by adding a ubiquitin chain on their substrates by timely and selective substrates, so it is necessary to identify the substrates and functions of the linear ubiquitin chain assembly complex, which will help to clarify the linearity. The ubiquitin chain participates in the specific mechanisms of various cellular biological functions regulated by the ubiquitin chain. Finding new protein complexes involved in linear ubiquitination and recognition has become a hot spot in the field of linear ubiquitination. It is also very important to analyze whether LUBAC mediated linear ubiquitination is involved in other signaling pathways and identification of LUBAC NEW substrates. (Smad ubiquitination regulatory factor 1/2) is a major member of the HECT E3 Nedd4 family. Currently, the function.Smurfs involved in multiple cell biology is originally determined from the regulation of the BMP/TGF beta family signal pathway, but recent studies have shown that the regulatory range is wide, including Wnt signaling pathway regulating the regulation of the.TGF beta family signal pathway. Extensive biological processes include cell migration, growth, differentiation and development. In the process of studying the regulatory mechanism of Smurfs, we use yeast two hybrid technology to screen HOIP (HOIL-1L Interacting Protein) through the Smurf2-HECT domain to the spleen library, and HOIP as the core molecule of the linear ubiquitin chain assembly complex LUBAC. We conjecture whether there is a real interaction between Smurfs and HOIP, and what is the correlation between Smurfs and the LUBAC linear complex, and whether Smurfs plays an important role in the regulation of linear ubiquitination, and whether LUBAC linear ubiquitination modification plays an important role in the regulation of new signaling pathways, and whether Smurfs is in NF- kappa B signaling pathway. The problem will play an important role in the process. Through the experimental study, we find that Smurf2 is similar to Smurf1 and can interact with HOIP as.Smurfs as a ubiquitin ligase E3 its active site mutant does not affect its relationship with HOIP, the other component HOIL- of.Smurfs and LUBAC complex. 1L, Sharpin can not interact directly with each other, but after adding HOIP, Smurfs can interact with HOIL-1L and Sharpin. In vivo CO-IP experiment found that HOIP, HOIL-1L, Sharpin can interact with Smurfs as an egg white with a multi domain molecular weight. .LDD, which can be combined with Smurf1-HECT-N-Lobe, is called a linear ubiquitin decision area. In all RBR class E3, the LDD domain is specific to HOIP and specifically determines the function of the linear ubiquitin chain assembly complex LUBAC. The binding area is the final stop of ubiquitin from E2 to the ubiquitin thiol bond intermediate, and the ZF domain in this region can be with Ub knots. Is there a regulatory relationship between.Smurfs and LUBAC as a ubiquitin ligase and linear ubiquitin chain assembly complex? Is Smurfs involved in linear ubiquitination? We found that each component of the single expression of LUBAC complex, the Smurfs expression level is unchanged, and the expression of 3 members of the LUBAC complex, HOIP, HOIL-1L, Sharpin, Smurfs protein expression The level of LUBAC was not affected by the Smurfs E3 active mutant Smurf1 C699A and Smurf2C716A. The expression level of HOIP, HOIL-1L, Sharpin, Smurfs of the low LUBAC complex was up-regulated. The level of ubiquitination is obviously up-regulated. After knocking down each component of the LUBAC complex for HIOP/HOIL-1L/Sharpin, the level of ubiquitination of Smurfs is down. The same conclusion is found that.HOIP C885 is the active center of LUBACE3 in vitro. The C885A mutant inhibits the up-regulation of.OTULIN as a linear ubiquitination enzyme, which can inhibit the ubiquitination of Smurfs in vitro as a linear ubiquitination enzyme. The up regulation of Smurfs ubiquitination level up regulation of.Smurfs ubiquitin level, it is considered that LUBAC promotes Smurfs to raise linear ubiquitin chain to make Smurfs linear ubiquitination.LUBAC can enhance Smurfs E3 activity. The interaction between HOIP and Smurfs inhibits the formation of two polymer between Smurf1 molecules and promotes the proportion of active monomers. We also found that LUBAC can inhibit the activity of TGF beta /BMP signaling pathway. After knocking down HOIP, CAGA12-luc activity up-regulated. The target genes of the TGF beta signaling pathway, Smad7, SNON, CTGF and PAI-1 are up regulated. It can enhance the ubiquitination level of Smurfs, so how important is the important role of the Smurfs substrate R-Smad? We have found that the LUBAC linear ubiquitin chain assembly complex can accelerate the degradation of Smurfs to Smads. This process is dependent on the.TGF beta of Smurfs E3 activity to promote cell migration, invasion and metastasis, then LUBAC complex What is the effect of chimerism and OTULIN on cell migration? Through Transwell experiments, we found that after knocking down HOIP, the migration ability of cells could be enhanced. Relatively, after knocking down OTULIN, the cell migration ability decreased, and the RT-PCR experiment found the target gene PTHr P related to cell migration, invasion and metastasis associated TGF beta signaling pathway. MMP2, MMP9 also up-regulated and down-regulated at HOIP knockout and OTULIN knockout cells. To sum up, the topic is mainly based on the interaction of HECT ubiquitin ligase Smurfs with the interaction of the linear ubiquitin chain assembly complex LUBAC core molecule HOIP, and it is found that the linear ubiquitin chain assembly complex LUBAC can be linear through linear flooding. Modified Smurfs, enhanced the activity of Smurfs E3, accelerated the degradation of Smurfs substrate and inhibited the activity of TGF beta /BMP signaling pathway. Relatively, we also found that the C885A E3 active mutant of the linear ubiquitinase OTULIN and LUBAC can abolish the activity of Smurfs ubiquitination enhancement effect.LUBAC negatively regulated beta signaling pathway activity. Migration, transfer, and LUBAC inhibit this effect. This is the first time that the linear ubiquitin chain assembly complex and linear ubiquitination modification are involved in the regulation of the TGF beta /BMP signaling pathway. We also found that LUBAC can promote linear ubiquitination of Smurfs to enhance the activity of Smurfs E3, which is also a supplement to the Smurfs regulation mechanism (neural PRECUR). SOR cell-expressed developmentally downregulated 8) is a class of molecules similar to ubiquitin and participates in post-translational modification of protein. This process is called Neddylation, the mechanism of Neddylation is similar to ubiquitination, and a series of enzyme catalyzed reaction.Neddylation modified by E1 (UBA3-APPBP1), E2 (Ubc12) and E3 are modified. N-Roc ubiquitin ligase plays an important role in the regulation of the activity of ubiquitin ligase. Compared with ubiquitination, only a few Neddylation modified substrates have been found in eukaryotic cells, and the physiological function of Neddylation remains to be studied. At present, the study of the binding of Ub to substrates is clear and close to 20 structures have been found. The domain (UBD) can be combined with ubiquitin, but there are few reports on the non covalent binding protein of Nedd8 like ubiquitin protein. Therefore, the study of the specific rules of the interaction between Nedd8 and protein can better understand the physiological function of Neddylation,.Smurf1 is a member of the HECT ubiquitin linked enzyme Nedd4 family, in bone formation, embryo development and so on. The important role of.Smurf2 is highly similar to that of Smurf1, which negatively regulates the TGF beta and BMP signaling pathways. This subject uses Smurf1 as the bait using yeast two hybrid method to screen ubiquitin like protein Nedd8.. We have proved that Smurf1 and Smurf2 can interact with Nedd8 in vivo and in vivo by CO-IP, GST-Pull down and other experiments. The study of intertruncation interaction shows that the WW domain of Smurf1, the N-lobe Small domain of HECT, the C-lobe domain of HECT can be similar to Nedd8.Smurf2 and Smurf1, and the N-lobe Small in the HECT domain is combined with them. Interaction found that NEDL1 and NEDL2 can be combined with Smurfs to combine with Nedd8., and then we use the crystal structure of Nedd8 to simulate the structure of the Smurf2-HECT crystal structure and simulate the interaction, in order to find the binding order. By sequence alignment, the sequence L (X7) R (X5) F (X) is preserved. The experiment of mutants interaction proves that the model sequence is mediated by binding. The non covalent interaction between Nedd8 and Smurf weakened the Neddylation modification of Smurf, stabilized the Smurf protein level, and changed the ability of.Nedd8 and Smurf interaction between Smurf and Ub to form the sulfur ester bond intermediate, which played a key role in the E3 activity of Smurf and the ubiquitination of substrates. The BMP and TGF beta signaling pathways regulated by Smurfs and cell migration play an important role. At the same time, we also found that Nedd8 covalently modified Smurf.Smurf1 can be used as a Nedd8 ubiquitin ligase to activate Smurf1 to occur Neddylation modified.Smurf1 N-lobe C426 is its Neddylation catalytic active center. The ubiquitin modified E3 active center C699.Neddylaiton plays an important role in the regulation of HECT E3.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q75

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