【作者】 钟加滕；

【导师】 孙连坤；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2014， 博士

【摘要】 MTSS1(肿瘤转移抑制基因-1)首先作为一种肿瘤转移抑制基因被鉴定出来，其在转移性膀胱癌细胞系中不表达。在对MTSS1功能的进一步研究中， MTSS1也可以作为一种支架蛋白，与多个伴侣分子相互作用来调控肌动蛋白动力学。近来的研究显示，MTSS1蛋白与肿瘤的发生发展密切相关，特别是前列腺癌和乳腺癌。前列腺癌中MTSS1的表达下调可以促进肿瘤的发生、发展和转移。而乳腺癌中MTSS1的低表达与乳腺癌的预后呈现出明显的相关性。但是，前列腺癌和乳腺癌细胞内MTSS1蛋白水平下调的机制还不是很清楚。泛素蛋白酶体系统是真核细胞中主要的蛋白质降解通路，通过细胞周期、DNA复制和修复、凋亡以及一些重要的信号通路相关蛋白的降解，下调细胞内蛋白的表达水平，间接的调控细胞的多种生物学过程。泛素蛋白酶体系统通过E1泛素活化酶，E2泛素结合酶和E3泛素连接酶将泛素分子共价修饰至底物上，蛋白酶体通过识别和降解泛素标记的蛋白对底物进行降解。E3泛素连接酶决定了泛素蛋白酶体系统对于底物蛋白质降解的特异性。β-TRCP是E3连接酶的一种，其可以识别蛋白质的特异性氨基酸序列，通过与Cullin1、Skp1及Rbx1形成SCFβ-TRCPE3泛素连接酶复合物对底物进行降解。底物被降解之前，首先被细胞内的激酶磷酸化，然后SCFβ-TRCP复合物将其携带的泛素分子转移至底物，底物被泛素化后被蛋白酶体所降解。我们前期对于MTSS1蛋白结构的观察发现，MTSS1蛋白存在可以被E3泛素连接酶SCFβ-TRCP1识别的氨基酸序列DSG(X)2+nS，提示MTSS1蛋白可能被泛素蛋白酶体途径识别和降解。基于E3泛素连接酶SCFβ-TRCP1降解底物的特征，我们设计了本实验。首先通过检测MTSS1与E3泛素连接酶SCFβ-TRCP复合物之间的结合，观察抑制E3泛素连接酶SCFβ-TRCP1复合物对底物水平的调节以及寻找磷酸化MTSS1蛋白的激酶及其磷酸化位点，验证了E3泛素连接酶SCFβ-TRCP是否介导了MTSS1蛋白的降解。其次，分别在前列腺癌和乳腺癌中构建了高表达MTSS1野生型和突变了激酶磷酸化位点的细胞系，通过细胞增殖和转移实验，探讨了SCFβ-TRCP1对MTSS1细胞功能的影响。方法：（1）免疫共沉淀和免疫印迹的方法探讨MTSS1蛋白与SCFβ-TRCP1E3泛素连接酶复合物之间的结合。（2）利用shRNA片段连入慢病毒载体，包被病毒，感染目的细胞方法建立稳定基因敲除的细胞系，观察抑制SCFβ-TRCP1E3泛素连接酶复合物中基因的表达对MTSS1蛋白表达水平的影响。（3）利用半衰期实验检测细胞内不同细胞系MTSS1蛋白降解速度。（4） RT-PCR检测抑制细胞内β-TRCP对细胞内MTSS1和β-TRCP mRNA水平的影响。（5）瞬时转染后利用免疫印迹方法检测CKIδ介导的外源性MTSS1的降解。利用点突变的方法突变被CKIδ磷酸化的位点，观察突变位点对于β-TRCP和MTSS1结合以及MTSS1降解的影响。（6）利用逆转录病毒建立高表达MTSS1野生型以及突变型细胞系。划痕实验观察高表达野生型或者突变型MTSS1基因对细胞迁移速度的影响。（7） Bromodeoxyuridine (BrdU)实验检测细胞增殖的改变。（8） Transwell实验检测细胞迁移能力。结果：（1）免疫共沉淀结果显示，MTSS1蛋白可以与SCFβ-TRCP1E3泛素连接酶复合物成分β-TRCP1、Cullin1、Skp1及Rbx1相结合。（2）利用shRNA抑制β-TRCP和Cullin1可以增加细胞内MTSS1蛋白表达水平。蛋白质半衰期实验显示，抑制细胞内β-TRCP水平可以明显的降低MTSS1蛋白的降解速度。（3）蛋白质降解实验显示，激酶CKIδ参与了SCFβ-TRCP1E3泛素连接酶复合物对于底物蛋白质的降解。（4）突变MTSS1322位点的丝氨酸可以抑制β-TRCP介导的MTSS1蛋白的泛素化。突变型MTSS1（S322A）可以抑制MTSS1蛋白质与β-TRCP之间的结合以及CKIδ介导的MTSS1蛋白的降解。（5）构建高表达MTSS1野生型以及突变型细胞系，细胞计数以及BrdU实验结果显示，高表达MTSS1野生型细胞系细胞增殖速度明显的降低，处于S期细胞数目明显减少，而MTSS1突变型细胞系细胞增殖速度进一步被抑制。（6）划痕和Transwell实验结果显示，高表达MTSS1可以明显的抑制细胞的迁移，而突变了β-TRCP识别区域后，细胞的迁移能力进一步降低。结论：E3泛素连接酶SCFβ-TRCP1通过CKIδ对MTSS1蛋白322位点的丝氨酸的磷酸化修饰介导了MTSS1蛋白泛素化降解过程。SCFβ-TRCP1E3泛素连接酶复合物对MTSS1蛋白的降解调控参与了MTSS1蛋白在前列腺癌和乳腺癌细胞生长抑制和转移中的作用。更多还原

【Abstract】 MTSS1(metastasis suppressor-1) was first identified as a metastasis suppressorissing in metastatic bladder carcinoma cell lines. In the further study of MTSS1unction, MTSS1also acts as a scaffold protein that interacts with multiple partners toegulate actin dynamics. Recent research shows that MTSS1protein is closely relatedo the development of tumor, especially prostate cancer and breast cancer. The downegulation of MTSS1expression in prostate cancer contributes to the occurrence,evelopment and metastasis of tumor. MTSS1low expression is related to therognosis of breast cancer. However, the mechanism of down regulation of MTSS1inrostate and breast cancer cells is not clear.Ubiquitin proteasome system is the main protein degradation pathway in theukaryotic cells, through degradation of the cell cycle, DNA replication and repair,poptosis, and the degradation of some important signal pathways related proteins,own regulation intracellular protein expression level, indirect regulates a variety ofiological processes of cells. Ubiquitin proteasome system through E1biquitin-activating enzyme, E2ubiquitin-conjugating enzyme, and E3ubiquitinigase enzyme covalently attachs of multiple ubiquitin molecules to a substrate androteasome identifying and degradation of ubiquitin marker proteins for degradationf the substrate. The substrate specificity of ubiquitin proteasome system isetermined by the E3enzymes.β-TRCP is a kind of E3ligase, it can identify the specificity amino acid sequencef the protein, by combination with Cullin1, Skp1and Rbx1, SCFβ-TRCPcompoundsor degradation of the substrate. Before degradation, the substrate is first phosphorylated by intracellular kinase, then SCFβ-TRCPtransfer ubiquitin molecules tothe substrate and after the ubiquitin the substrate is degradated by proteasome.Dependent the observation of MTSS1protein structure, we found that theMTSS1protein has the amino acid sequence DSG(X)2+nS, which can be identified byE3ubiquitin ligase SCFβ-TRCP1, and its suggest MTSS1protein may be identificationand degradation by ubiquitin proteasome pathway.Based on the degradation characteristics of E3ubiquitin ligase SCFβ-TRCP1substrate, we designed this experiment. First, we detect the combination of theMTSS1and E3ubiquitin ligase SCFβ-TRCP1complexes，observe influence of substratelevel by inhibition of E3ubiquitin ligase complex and search phosphorylation MTSS1protein kinase and its phosphorylation site, to verify MTSS1protein degradation byE3ubiquitin ligase SCFβ-TRCP1complexes. Second, we respectively constructedoverexpression of MTSS1wild type and mutant kinase phosphorylation sites of celllines in prostate and breast cancer, to explored the affection of SCFβ-TRCP1on MTSS1cell function through cell proliferation and metastasis the experiment.Methods:（1） The combination between MTSS1protein and SCFβ-TRCP1E3ubiquitin ligasecompounds was detected by co-immunoprecipitation and immunoblotting.（2） We used the method of link shRNA fragments to slow virus vector, packagedvirus, infected cell to establish a stable knockout cell line, to observe theinhibition of the expression of genes within SCFβ-TRCP1E3ubiquitin ligasecompounds for the effect of MTSS1protein expression level.（3） We used the half-life experimental to detect the degradation rate of MTSS1protein in different cell lines.（4） The inhibiton of β-TRCP for the effect of MTSS1and β-TRCP mRNA level incell detected by RT-PCR. （5） We applied immunoblotting to detect exogenous MTSS1degradationmediated by CKIδ after transient transfection. We used the method of pointmutations to mutate the site phosphorylated by CKIδ, to observe the effect ofmutation site for the combination of β-TRCP and MTSS and the degradationof MTSS1.（6） We used retroviruses to establish the wide-type cell line of MTSS1highexpression and mutant cell line. And applied nick experimental to observe theeffect of wild-type or mutant MTSS1gene for cell migration velocity.（7） The change of cell proliferation was detected by bromodeoxyuridineexperimental.（8） The cell migration ability was detected by transwell experimental.Results:（1） The results of co-immunoprecipitation showed that MTSS1protein cancombine with Cullin1、β-TRCP1、Skp1and Rbx1Which are the compositionsof SCFβ-TRCP1E3ubiquitin ligase compounds.（2） The inhibition of Cullin1and β-TRCP by shRNA increased the expression ofMTSS1protein level. The half-life experimental showed that the inhibition ofβ-TRCP level obviously decreased the degradation rate of MTSS1protein.（3） Protein degradation experimental showed that kinase CKIδ involved in thedegradation of substrates protein by SCFβ-TRCP1E3ubiquitin ligase compounds.Mutate the serine of MTSS1322site inhibited the ubiquitylation of MTSS1protein mediated by β-TRCP.（4） Mutant-type MTSS1(S322A) inhibited the combination of β-TRCP and MTSSand the degradation of MTSS1mediated by CKIδ.（5） The wide-type cell line of MTSS1high expression and mutant cell line wereestablished, the results of cell counting and BrdU showed that cellproliferation rate of wide-type cell line of MTSS1high expression be notablydecreased, the cell number in S phase be obviously reduced, however the cell proliferation rate of MTSS1mutant-type cell line further be suppressed.（6） The results of nick experimental and transwell experimental showed that thehigh expression of MTSS1can obviously inhibite cell migration, and aftermutate the identification domain of β-TRCP, the cell migration ability furtherbe decreased.Conclusions:E3ubiquitin ligase SCFβ-TRCP1mediated the MTSS1ubiquitin proteindegradation process through phosphorylate322serine of MTSS1by CKIδ. Thedegradation of regulation MTSS1protein by SCFβ-TRCP1E3ubiquitin ligase involvedin cell growth and metastasis in prostate and breast cancer.更多还原