本研究所用人前列腺癌细胞株PC3购自美国ATCC公司。绿色荧光蛋白(green fluorescent protein, GFP)融合蛋白表达载体pEGFP-AR(1-918aa), pEGFP-NES^{AR(743-817aa)}, pEGFP-NAR (1-665aa)和pEGFP-NAR-NES^AR均为美国匹兹堡大学泌尿外科研究中心王洲实验室构建并惠赠。NES^AR的赖氨酸突变体(赖氨酸K突变为精氨酸R)pEGFP-NES^{AR K776R}, pEGFP-NES^{AR K807R}和pEGFP-NES^{AR K776R/K807R}的构建, 参见StarMut 基因定点突变试剂盒说明书, 购自北京康润诚业生物科技有限公司。

RPMI1640(roswell park memorial institute 1640)细胞培养液、胎牛血清(fetal bovine serum, FBS)、100 U/mL青霉素和100 mg/L链霉素购自美国GIBCO公司。蛋白合成抑制剂放线菌酮(cycloheximide, CHX)和蛋白酶体抑制剂MG132均购自美国Sigma-Aldrich公司。PolyJet^TM 体外DNA转染试剂盒购自美国SignaGen公司。GFP抗体购自德国Acris, 泛素蛋白(ubiquitin, Ub)抗体(sc8017)、内参照GAPDH抗体(sc-25778)和辣根过氧化物酶标记的山羊抗鼠/兔 IgG (sc-2004/2005)均购于美国Santa Cruz Biotecnology公司。抗GFP标签的单克隆抗体偶联的琼脂糖凝胶珠(agarose conjugated anti-GFP-tag)购自北京博尔迈生物技术有限公司。二喹啉甲酸(bicinchoninic acid, BCA)蛋白定量试剂盒和增强化学发光(enhanced chemiluminescence, ECL)试剂盒购自美国Thermo公司。

人前列腺癌细胞株PC3用含有10%(体积分数)FBS、100 U/ml青霉素和100 mg/L链霉素的RPMI1640培养液培养于37 ℃、5% (体积分数)CO₂饱和湿度的培养箱内, 当细胞生长融合成单层时, 用胰蛋白酶消化并传代培养。在细胞转染的前一天, 在每个10 cm的培养皿中接种适宜数量的细胞, 使得在转染的时候达到80%~85%的密度; 转染时更换成新鲜的完全培养液; 根据PolyJet^TM 体外DNA转染试剂盒说明进行质粒转染, 转染12~18 h后更换为新鲜的完全培养液; 加入50 mg/L CHX和/或指定浓度的MG132处理指定时间后收集细胞。

按指定时间处理细胞后, 弃去培养液, 用预冷磷酸盐缓冲液(phosphate buffered saline, PBS)清洗两次, 加入一定量改良型的放射免疫沉淀蛋白裂解缓冲液(radioimmune precipitation assay buffer, RIPA)提取总蛋白, 裂解液组成:50 mmol/L Tris (pH 7.4), 1% (体积分数)乙基苯基聚乙二醇(Nonidet P-40), 0.25%(质量分数)脱氧胆酸钠(Na-deoxycholate), 150 mmol/L NaCl, 1 mmol/L EDTA (pH 8.0), 1 mmol/L NaF, 2 mmol/L苯甲基磺酰氟(phenylmethylsulfonyl fluoride, PMSF), 1 mmol/L钒酸钠(Na₃VO₄)和蛋白酶抑制剂。采用BCA法测定蛋白浓度; 加入5× 上样缓冲液, 100 ℃煮沸10 min。取等量蛋白进行8%~12%(质量分数) 十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE)分离蛋白质, 300 mA转膜70 min, 5%(质量分数) 脱脂奶粉室温封闭1 h, 一抗4 ℃孵育过夜, 三羟甲基氨基甲烷缓冲盐水(triethanolamine buffered saline solution-Tween, TBST)洗膜3次× 10 min, 二抗室温孵育1 h, TBST洗膜3次× 10 min; 暗室加入ECL发光液后曝光显影。

对于免疫沉淀, 取一定体积的GFP标记的琼脂糖凝珠, 加入500 μ L预冷细胞裂解液清洗两次, 5 000 r/min 4 ℃离心30 s, 吸除上清液; 加入含等量蛋白的细胞裂解液, 补足细胞裂解液致体积达1 500 μ L, 再4 ℃旋转摇动过夜; 5 000 r/min 4 ℃离心1 min, 吸除上清液; 加入500 μ L冷细胞裂解液, 重复清洗琼脂糖凝珠3次, 4 ℃ 15 min/次; 加入上样缓冲液, 沸水浴中10 min使蛋白质变性, 然后进行蛋白质免疫印记检测分析。

雄激素受体的配体结合域中具有出核转运信号肽的核转出蛋白NES^AR(图1A), 在雄激素受体的细胞浆/细胞核穿梭中具有重要作用。在配体雄激素缺乏的情况下, NES^AR占主导并指导AR的出核; 在配体雄激素存在的情况下, NES^AR的作用被抑制, AR表现为核定位^[9]。同时, 我们发现将融合蛋白表达载体pEGFP, pEGFP-NES^{AR(743-817aa)}, pEGFP-NAR (1-665aa)和pEGFP-NAR-NES^AR瞬时转染雄激素非依赖性前列腺癌PC3细胞后, 在荧光显微镜下观察到, 含NES^AR的融合蛋白(GFP-NES^AR和GFP-NAR-NES^AR)呈胞浆定位, 且荧光信号强度比不含NES^AR的融合蛋白(GFP和GFP-NAR)明显减弱(图1B); 进一步的结果显示, 含NES^AR融合蛋白的表达水平比不含NES^AR的融合蛋白低(图1C)。上述发现提示, NES^AR除了作为出核信号发挥功能外, 在AR蛋白质的降解中也具有潜在的功能。为了确定NES^AR对蛋白稳定性的影响, 我们用蛋白合成抑制剂CHX进行处理, 结果显示含NES^AR的融合蛋白GFP-NES^AR和GFP-NAR-NES^AR具有降解不稳定性, 6 h 内其蛋白发生降解; 而不含NES^AR的GFP-NAR和 GFP, 24 h内其蛋白表达相对稳定(图2)。以上结果提示, NES^AR在前列腺癌细胞中作为一个新的降解基团, 在调节蛋白稳定性中发挥了重要的作用。

Figure 1

图1

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Figure 1 NESAR fusion proteins were expressed at low levels in PC3 cells图1 含NESAR的融合蛋白在PC3细胞中低水平表达 A, protein structure of human androgen receptor and NESAR locus; B, cytoplasmic fluorescence intensity with NESAR is fairly weak. PC3 cells were transiently transfected with expression vectors for green fluorescent protein (GFP), GFP-NESAR, GFP-NAR (1-665aa of AR) and GFP-NAR-NESAR fusion proteins, and visualized under a fluorescence microscope 20 h after transfection. Exposure time for all images was 1 s; C, Western blot analysis of protein levels of transiently transfected fusion proteins in PC3 cells in complete RPMI 1640 medium.

图2

Figure 2

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图2 含NESAR的融合蛋白具有降解不稳定性 Fusion proteins containing NESAR are not stable in the presence of cycloheximide. PC3 cells were transfected with GFP and GFP-NESAR(A), GFP-NAR (1-665aa) and GFP-NAR-NESAR(B) respectively. 20 h later, cells were treated with 50 mg/L CHX for indicated time points. Protein expression was examined by Western blot with anti-GFP antibody.Figure 2 NESARdecreases the stability of its fusion proteins

在一定浓度的蛋白酶体抑制剂MG132处理24 h 的情况下, 分析融合蛋白的表达变化, 发现MG132对GFP蛋白表达没有显著影响, 而含NES^AR的融合蛋白GFP-NES^AR在MG132的处理下, 其蛋白表达显著增加, 并呈现剂量依赖性(图3A、B)。进一步检测在蛋白合成被抑制的情况下, MG132是否可以抑制含NES^AR的融合蛋白的降解, 如图3C、D结果显示, 在蛋白合成被抑制的情况下, MG132显著抑制了GFP-NES^AR融合蛋白的降解, 蛋白表达呈剂量依赖性的增加; 而在对照实验中, MG132对GFP蛋白稳定性没有显著影响, 结果提示, NES^AR通过蛋白酶体依赖的蛋白降解途径调节了融合蛋白的降解。

图3

Figure 3

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图3 NESAR的降解依赖于蛋白酶体途径 A& B, proteasome inhibition increases the level of NESAR containing fusion proteins, transfeced PC3 cells were incubated overnight and then treated with vehicle or different concentrations of MG132 for 24 h. C& D, NESAR fusion proteins’ degradation was blocked by proteasome inhibitor in the presence of cycloheximide, transfeced PC3 cells were incubated overnight and then treated with vehicle or 50 mg/L CHX plus different concentrations of MG132 for 24 h. Protein expression was examined by Western blot with anti-GFP antibody.Figure 3 NESAR undergoes proteasome-dependent degradation

在泛素-蛋白酶体途径中, 蛋白酶体通常特异性地降解泛素化的蛋白。前面实验已证实, 蛋白酶体的抑制可以阻止NES^AR融合蛋白的降解, 那么融合蛋白是否先发生了泛素化?我们在前列腺癌细胞系PC3中, 瞬时共转染带有His 标记的泛素(His-Ub)和GFP或GFP-NES^AR, 然后用DMSO或蛋白酶体抑制剂MG132处理18~20 h。GFP免疫沉淀的结果显示, GFP-NES^AR融合蛋白的泛素化水平明显高于GFP对照(图4), 表明NES^AR作为泛素化识别信号, 引起融合蛋白的泛素化, 进而被蛋白酶体识别降解。

图4

Figure 4

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图4 NESAR是多聚泛素化识别靶标 PC3 cells were transiently co-transfected with expression vectors of his tagged ubiquitin with GFP or GFP-NESAR. Transfected PC3 cells were incubated overnight and then treated with vehicle or 10 μ mol/L MG132 for 20 h. Cell lysates were immunoprecipitated (IP) with anti GFP-tag agarose. Protein expression was examined by Western blot (immunoblot, IB) with anti-ubiquitin and anti-GFP antibodies. Ub, ubiquitin; HC, heavy chain, 55 000; LC, light chain, 25 000.Figure 4 NESAR was the target of polyubiquitination

泛素蛋白是一个非常保守的多肽, 在泛素激活酶(E1)、泛素融合酶(E2)、泛素连接酶(E3)等一系列酶促反应催化下与细胞内靶蛋白上的一个或多个赖氨酸残基发生共价连接, 进行泛素化修饰。通过分析NES^AR的蛋白质序列, 我们发现其存在两个赖氨酸残基(K776和K807), 进而构建了NES^AR的赖氨酸突变体(赖氨酸K突变为精氨酸R), 以期确定NES^AR序列中赖氨酸残基的突变是否会影响其泛素化修饰, 结果显示, 赖氨酸突变的NES^AR的泛素化水平显著降低, 且蛋白稳定性提高(图5), 因此, NES^AR蛋白序列中赖氨酸位点K776和K807是介导其发生泛素化的关键氨基酸残基。

图5

Figure 5

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图5 NESAR泛素化位点的鉴定 A, ubiquitination of GFP-NESAR K776R, GFP-NESAR K807R and GFP-NESAR K776R/K807R. PC3 cells were co-transfected with expression vectors of His tagged ubiquitin with GFP or GFP-NESAR, GFP-NESAR K776R, GFP-NESAR K807R, GFP-NESAR K776R/K807R. Transfected PC3 cells were incubated overnight and then treated with vehicle or 10 μ mol/L MG132 for 20 h. Cell lysates were immunoprecipitated (IP) with anti GFP-tag agarose. Protein expression was examined by Western blot (immunoblot, IB) with anti-ubiquitin and anti-GFP antibodies. Ub, ubiquitin; HC, heavy chain, 55 000; LC, light chain, 25 000. B, protein stability of GFP-NESAR K776R, GFP-NESAR K807R and GFP-NESAR K776R/K807R in the presence of CHX. PC3 cells were transfected with GFP-NESAR, GFP-NESAR K776R, GFP-NESAR K807R and GFP-NESAR K776R/K807Rrespectively. 20 h later, cells were treated with 50 mg/L CHX for indicated time points. Protein expression was examined by Western blot with anti-GFP antibody. K, lysine; R, arginine.Figure 5 Identification of ubiquitination sites of NESAR