【作者】 陈国栋；

【导师】 毛新良；

【作者基本信息】 苏州大学 ， 药理学， 2013， 博士

【摘要】 第一部分筛选c-Maf关键泛素化位点目的：利用定点突变技术构建c-Maf的赖氨酸位点突变体，结合各突变体对蛋白酶体抑制剂硼替佐咪的不同反应，筛选关键突变位点，确定各赖氨酸位点对c-Maf泛素化的影响。方法：1.通过定点突变PCR技术将c-Maf蛋白质的赖氨酸突变为精氨酸（K R）,得到c-Maf单一赖氨酸突变位点(例如K29R)和M14（全突变体）以及多突变体；同时构建c-Maf含单一赖氨酸的突变体（例如K29），然后将各突变体分别转染到HEK293T细胞中，36hrs后加入蛋白酶体抑制剂硼替佐咪（BZ），进一步处理12hrs，然后用Western blotting方法检测各突变体对c-Maf稳定性的影响，并进一步筛选出可以介导c-Maf泛素化的关键赖氨酸位点。2.将筛选的关键突变体及野生型和全突变体转染到HEK293T细胞中，6hrs后加入蛋白酶体抑制剂MG132处理4hrs，用免疫沉淀法检测各突变体对c-Maf泛素化水平的影响。3.将关键突变体及野生型和全突变体构建到pEGFP-C3中，36hrs后乙醇固定并用DAPI染核，用激光共聚焦检测c-Maf的胞内分布。4.将关键突变体及野生型和全突变体同CCND2-Luc共转染到HEK293T细胞中，36hrs后检测其对荧光素酶活性的影响。结果：1. c-Maf单点突变无法抑制c-Maf的泛素化降解，同时单一赖氨酸的存在也不能单独介导c-Maf的泛素化降解。多个赖氨酸的存在，例如同时保留第85位和350位赖氨酸位点K(85,350)可以介导c-Maf的泛素化降解。2. IP结果显示K(85,350)的c-Maf突变体有明显的多聚泛素化条带，但这两个位点同时突变的多位点突变体也存在多聚泛素化条带，证明这两个位点参与c-Maf泛素化，但也不是唯一的。3.共聚焦结果显示K(85,350)（同时保留85位与350位赖氨酸）、K(85,350)R（同时突变85位与350位赖氨酸）突变体同野生型c-Maf一样主要分布在核内，而完全突变体在核内和胞质中均有分布，提示c-Maf部分赖氨酸位点突变不影响其胞内定位，而完全突变可能由于泛素化修饰的改变导致其细胞内分布的变化。4.荧光素酶活性结果显示，K(85,350)、K(85,350)R多位点突变体以及全突变体相比野生型c-Maf能明显增强CCND2启动子的转录活性，这可能与c-Maf蛋白的降解受到抑制有关。小结：c-Maf的泛素化是由多个赖氨酸位点介导的，例如c-Maf蛋白中K85和K350对于其泛素化很重要，但它们并不是唯一的。K(85,350)和K(85,350)R多突变体不影响蛋白在细胞中的定位，但由于其降解受到一定程度的抑制，可明显增强其对CCND2的转录活性。第二部分c-Maf的溶酶体降解途径目的：全突变c-Maf（M14）突变体的泛素化降解被抑制，但在实验中仍发现其发生降解，由于蛋白质主要存蛋白酶体和溶酶体两种降解途径，我们推测c-Maf可能也通过溶酶体降解。本部分将利用不同的溶酶体抑制剂和蛋白酶体抑制剂抑制蛋白质降解，以证明c-Maf在泛素化降解的同时也存在着溶酶体降解。方法:1.转染WT与M14c-Maf质粒到HEK293T细胞中，用放线菌酮（CHX,100ng/mL）处理细胞不同时间（0、4、8、12hrs）后，收取细胞进行Western Blotting分析，检测蛋白的变化情况。2.转染WT与M14c-Maf质粒到HEK293T细胞中，加入放线菌酮（CHX100ng/mL）抑制蛋白合成，同时加入蛋白酶体抑制剂硼替佐咪（1μM），以及溶酶体抑制剂氯喹（100μM）和氯化铵（1mM）进行处理，12hrs后检测蛋白的变化。结果：1.在0~8hrs，WT-c-Maf降解明显，M14全突变降解不明显，而在8~12hrs间M14开始发生了明显的降解，证明M14仍存在另一种降解方式。2.野生型c-Maf在加入蛋白酶体抑制剂和溶酶体抑制剂后都可以抑制其降解，同时加入抑制效果更明显，而全突变体M14只有在加入溶酶体抑制剂后才能抑制其降解，同时加入溶酶体和蛋白酶体抑制剂后抑制效果与单加溶酶体抑制剂效果相同。小结：c-Maf可以通过蛋白酶体和溶酶体两种方式降解。第三部分c-Maf的E3泛素连接酶的筛选和确证目的:上述研究表明c-Maf可以发生多聚泛素化，但其蛋白泛素化体系还不清楚。本部分利用实验室构建的E3筛选系统，结合LC/MS/MS及免疫共沉淀技术筛选c-Maf的泛素连接酶E3，利用Western Blotting和荧光共定位对筛选的E3进行初步验证。方法：1.将实验室保存的E3表达文库中的E3s质粒转染到NIH3T3/c-Maf+CCND2-luc细胞(稳定表达c-Maf和CCND2-luc的NIH3T3细胞)中，48hrs后检测荧光素酶活性变化，实验重复三次，初步筛选出可能的泛素连接酶。2.将筛选出的E3s与c-Maf质粒共转染到NIH3T3/c-Maf+CCND2-luc细胞中，Western Blotting检测c-Maf蛋白的变化情况，进一步筛选c-Maf的泛素连接酶。3.转染不同量的cMul质粒到NIH3T3/c-Maf+CCND2-luc细胞中, WesternBlotting检测cMul与c-Maf的相关性。4.为了进一步确定c-Maf的泛素化酶体系，我们采用亲和-免疫沉淀偶联蛋白质谱学分析策略从蛋白质组的角度来证实c-Maf的泛素化连接酶。具体方法：共转染HA-c-Maf与Ub质粒到HEK293T细胞中，用MG132处理细胞2hrs后，以Anti-HA agarose微珠进行IP，获得anti-HA的免疫复合物，经纯化，还原一系列步骤后，得到的蛋白质用胰蛋白酶消化以得到多肽，得到的多肽混合物经C18过柱纯化后，应用于LC/MS/MS分析。各样品先经液相色谱分离，各分离组分进一步经串联质谱分析（LC/MS/MS），得到的原始数据经SEQUEST和GPMAlgorithm方法分析各多肽顺序。最后用Scaffold软件识别各蛋白质顺序，与蛋白质质谱学数据库对比分析，从而得到细胞中与c-Maf共沉淀的相互作用蛋白质。这些蛋白质进一步经DAVID3.0和KEGG数据库进行Clustering路径分析，得到c-Maf泛素化相关蛋白质。5.泛素化酶cMul与c-Maf的相互作用分析：共转染HA-c-Maf、Ub及构建的Flag-cMul质粒到HEK293T细胞中，36hrs后加入MG132处理4hrs,分别用Anti-Ub agarose beads和Anti-Flag agarose beads进行co-IP，利用Western Blotting检测c-Maf和cMul的共沉淀情况。6.共转染构建的pEGFP-c-Maf与Flag-cMul到HEK293T细胞中，用Flag抗体做免疫荧光，结合EGFP-c-Maf本身表达的绿色荧光蛋白，于激光共聚焦显微镜分析蛋白的是否存在共定位情况。结果：1.根据荧光素酶活性的变化情况，初步筛选出16个E3基因。2.进一步从16个E3中，筛选出泛素连接酶cMul。3.串联质谱分析（LC/MS/MS）结果显示c-Maf与cMul存在相互作用，且发现了其他几种泛素化相关的蛋白。4.共沉淀与荧光共定位结果进一步证实了c-Maf与cMul有相互作用，而且cMul促进了c-Maf的泛素化降解。小结：筛选出c-Maf的E3泛素连接酶cMul，并在体外证实了该酶有促进c-Maf泛素化降解的作用。结论本研究通过定点突变构建了一系列c-Maf的赖氨酸突变体，证明了c-Maf的泛素化不依赖于单个赖氨酸位点，并找到两个重要的赖氨酸位点K85和K350可以介导c-Maf的泛素化，但是同时发现仅突变这两个赖氨酸位点不能阻断c-Maf的泛素化，说明c-Maf的泛素化由多位点介导。泛素化对调节c-Maf的胞内定位及其生物学功能具有重要重要。此外，我们首次发现c-Maf的降解也同时存在着溶酶体降解途径。通过E3的筛选系统结合LC-MS/MS和免疫共沉淀技术筛选并证实了c-Maf的E3泛素连接酶是cMul。本研究为进一步深入了解c-Maf的泛素化过程奠定基础。更多还原

【Abstract】 Part1The lysine sites of c-Maf for ubiquitination[Objective]To identify the specific lysine residues responsible for c-maf ubiquitinaiton andstability.[Methods]1. The wild-type (WT)-c-Maf and a series of c-Maf mutants with lysine (K) arginine (R) were made by site-directed mutagenesis. These mutants included singlewild type lysine residue or single lysine mutant ones, as well as c-Maf all lysinemutations or multiple lysine mutations. The mutants were transfected into the HEK293Tcell line. Thirty-six hrs later, the cells were treated with MG132for4hrs before celllysates were extracted. The Western Blotting was used to measure the changes of thec-Maf protein.2. The c-Maf and the key mutants were transfected into the HEK293T cell line.Thirty-six hours later, the cells were treated with MG132for4hrs before cell lysateswere extracted. Proteins were then subject to immunoprecitation (IP) and WesternBlotting analysis.3. The c-Maf and the key mutated genes were constructed into the pcDNA-EGFPvector. These plasmids were then transfected into HEK293T cells. Twenty-four hourslater, cells were subject to confocal microscopy analysis to visualize their subcellularlocalization.4. The CCND2-Luc and WT-c-Maf or the mutants were cotransfected into theHEK293T cell line. Thirty-six hrs later, the Luciferase activity was detected. [Results]1. The results showed that a single lysine residue was not sufficient for c-Mafubiquitination, and a single lysine mutant was not able to prevent c-Maf ubiquitination.Further studies found that c-Maf ubiquitination was mediated by multiple lysineresidues, at least two lysine residues were required, such as K(85,350). However,K(85,350)R could not completely block c-Maf ubiquitination.2. The ubiquitination level of c-Maf modulated the cellular discribution. Confocalmicroscopy analysis indicated that wild type c-Maf protein was evenly expressed in thenuclei, but the lysine-free mutant c-Maf protein formed the aggregates and wasdistributed in both the nuclei and cytoplasmic compartments.3. Ubiquitination modulated the biological function of c-Maf. In the analysis of3mutants including K(85,350), K(85,350)R and M14by using WT as the control. TheWT, K(85,350), and K(85,350)R showed the similar effects in transactivating cyclin D2promoter, but the M14mutant showed a higher mudolating activity toward cyclin D2protmoter.[Conclusions]By a series of screening on the lysine residues in c-Maf protein, a single lysinemutant (K to R) was not sufficient for c-Maf ubiquitnation. Multiple lysine residueswere required to mediate c-Maf ubiquitination and turnover. Among these key resides,co-existence of K85and K850is important for c-Maf ubiquitination. The ubiquitinationlevel of c-Maf modulated the cellular discribution and its biological function.Part2c-Maf degradation via lysosomes[Objective]The M14with the lysine residue mutations (14K to14R) also can be degraded. Wewondered whether c-Maf stability was also mediated by other avenues, such as thelysosome pathway. Therefore, we examined wheather c-Maf can be degraded in thelysosmes by using the lysosome inhibitors.[Methods]1. The WT and M14c-Maf were transfected into the HEK293T cells. After48hrs, the cells were treated with the CHX for a certain perioid before being subject to lysisand applied for Western Blotting to measure the protein abundance of c-Maf.2. The WT and M14c-Maf were transfected into the HEK293T cells. After48hrs,the CHX alone or combination with the proteasome inhibitor (Bortezomib) and differentlysosome inhibitors (chloroquine, amino chloride) were added. And then, cells werecollected for protein extracts to measure c-Maf protein levels at0,0.5,1,2,4,6,8, and12hrs after CHX treatment.[Results]1. The WT c-Maf was degraded markedly in0-8hrs after treatment with CHX,while the M14was weakly degraded although M14was also degraded in8-12hrs aftertreatment with CHX, suggesting M14degradation was in a slower rate than its WTcounterpart.2. Both the proteasome inhibitors and lysosme inhibitors could slow down thedegradation of WT c-Maf. And the proteasome inhibitors in combination with lysosomeinhibitors were better than the single one. But only the lysosme inhibitors could slowdown the degradation of M14.[Conclusions]The c-Maf can be degraded via both the proteasome and lysosome pathways.Part3Identificaiton of the ubiquitin ligase E3of c-Maf[Objective]The above studies have clearly stated that c-Maf can be modified by multipleubiquitination, but its specific E3ligase was not identified yet. The prupose of thissection is aimed to identify this E3based on a luciferase screening and a co-IPcoupled-LC/MS/MS strategy.[Methods]1. Individual E3plasmids were transfected into NIH3T3cells which stably expressc-Maf and CCND2promoter-driven luciferase (CCND2-Luci) by lipofectamine2000.After48hrs, luciferase activity from each treatment was detected. 2. To further identify E3ligase for c-Maf, the possible E3s were cotransfected withc-Maf and ubiquitin into NIH3T3, followed by a measurement on c-Maf protein.3. LC/MS/MS analysis of proteins involved in c-Maf ubiquitination. The c-Mafand ubiquitin plasmids were cotransfected into HEK293T cells. Thirty-six hours later,cells were treated with MG132for4hrs. Then the proteins interacting with c-Maf werepulled down by anti-HA agarose beads. Finally, those proteins were identified byLC/MS/MS. The proteins related with the ubiquitination of c-Maf were obtained byDAVID clustering and KEGG pahway analysis.4. The HA-c-Maf, ubiquitin and Flag-E3were cotransfected into the HEK29T cells.After48hrs, proteins were co-IPed by anti-HA or anti-Flag agarose beads. Thewestern-blotting method was used to detect interacting protiens.5. The pEGFP-c-Maf and Flag-E3were co-transfected into HEK29T cells. Theimmunofluorescence was performed by anti-Flag antibody to localize the E3in the cells.The localization of c-Maf and E3were analysed by a laser confocal microscopy.[Results]1. By preliminary screening of the E3library,16possible E3s were narroweddown.2. cMul was singled out from the16possible candidates. It significantly promotedthe degradation of c-Maf.3. The results of LC/MS/MS showed that cMul interacted with c-Maf in HEK293Tcells and also found some other proteins related with ubiquitination of c-Maf.4. cMul was co-localized with c-Maf as observed by confocalfluorescentmicroscopy.[Conclusions]cMul, a probable E3of c-Maf, was identified via E3screening and LC/MS/MSstudy. cMul induces c-Maf degradation in a concentration-dependent manner. Physically,cMul is colocalized with c-Maf.ConclusionsIn the present study, we found c-Maf ubiquitination is mediated with multiple lysine residues, of which K85and K350were proved to play a key role in theubiquitination process of c-Maf protein. We also identified the E3ligase of c-Maf—cMul. These findings will help for in-depth understanding c-Maf biological activity andits role in the pathophysiology of multiple myeloma.更多还原