核转出蛋白对前列腺癌雄激素受体稳定性的调节

发布时间：2018-03-14 18:03

本文选题：雄激素受体　切入点：核转出蛋白　出处：《北京大学学报(医学版)》2017年04期 　论文类型：期刊论文

【摘要】：目的:探讨位于雄激素受体(androgen receptor,AR)配体结合域中具有出核转运信号肽的核转出蛋白(nuclear export signal of androgen receptor,NESAR)对前列腺癌雄激素受体蛋白表达和稳定性调节的机制。方法:绿色荧光蛋白(green fluorescent protein,GFP)融合蛋白表达载体p EGFP-AR(1-918aa),p EGFP-NESAR(743-817aa),p EGFP-NAR(1-665aa)和p EGFP-NAR-NESAR,及NESAR的赖氨酸突变体(赖氨酸K突变为精氨酸R)p EGFPNESAR K776R,p EGFP-NESAR K807R和p EGFP-NESAR K776R/K807R,瞬时转染前列腺癌细胞PC3后,采用荧光显微镜、蛋白质免疫印迹和免疫沉淀,检测NESAR对雄激素受体稳定性的调节。结果:在荧光显微镜下,含NESAR的融合蛋白呈胞浆定位,荧光信号强度比不含NESAR的融合蛋白明显减弱,且含NESAR的融合蛋白表达水平显著低于不含NESAR的融合蛋白表达水平。在蛋白合成抑制剂放线菌酮处理下,GFP-NESAR和GFP-NAR-NESAR的半衰期小于6 h,而对照GFP和GFP-NAR的表达相对更稳定,半衰期大于24 h。融合蛋白GFP-NESAR在蛋白酶体抑制剂MG132的处理下,其蛋白表达显著增加,并呈现剂量依赖性,而MG132对GFP蛋白稳定性没有显著影响;在蛋白合成被抑制的情况下,MG132同样显著抑制了GFP-NESAR融合蛋白的降解,且蛋白表达呈剂量依赖性的增加。GFP免疫沉淀的结果显示,GFP-NESAR融合蛋白的泛素化水平明显高于GFP对照。赖氨酸位点K776和K807突变的NESAR的泛素化水平显著降低,且蛋白稳定性提高,赖氨酸位点K776和K807是介导NESAR发生泛素化的关键氨基酸残基。结论:核转出蛋白NESAR具有降解不稳定性,作为多聚泛素化修饰的识别信号,介导了其融合蛋白在前列腺癌细胞中通过泛素-蛋白酶体途径的降解。本研究为AR蛋白水平和/或活性的调控开辟一种新的研究思路,有助于对AR降解分子机制的了解和在前列腺癌产生去势抗性的过程中对AR靶向的密切调控。
[Abstract]:Objective: to investigate the mechanism of expression and stability of androgen receptor receptor receptor protein in prostate cancer by nuclear export signal of androgen receptor NESAR, which is located in the ligand binding domain of androgen receptor receptor. The fusion protein expression vector pEGFP-ARO1-918aAARP / EGFP-NESARN 743-817aar-EGFP-NARPNAR1-665aaa) and the lysine mutants of NESAR (lysine K mutated to arginine EGFPNESAR K776Rtp EGFP-NESAR K807R and p EGFP-NESAR K776RK807R) were transfected into PC3 cells. The regulation of NESAR on the stability of androgen receptor was detected by fluorescence microscope, Western blot and immunoprecipitation. Results: under fluorescence microscope, the fusion protein containing NESAR was cytoplasmic located. The intensity of fluorescence signal was significantly lower than that of fusion protein without NESAR. The expression level of fusion protein containing NESAR was significantly lower than that of fusion protein without NESAR. The half-life of GFP-NESAR and GFP-NAR-NESAR was less than 6 hours after treatment with protein synthesis inhibitor actinomycin, but the expression of GFP and GFP-NAR in control group was more stable. The half-life of the fusion protein GFP-NESAR was more than 24 h. The protein expression of the fusion protein GFP-NESAR was significantly increased in a dose-dependent manner under the treatment of proteasome inhibitor MG132, but MG132 had no significant effect on the stability of GFP protein. When protein synthesis was inhibited, MG132 significantly inhibited the degradation of GFP-NESAR fusion protein. The protein expression was increased in a dose-dependent manner. The results of immunoprecipitation showed that the ubiquitin level of GFP-NESAR fusion protein was significantly higher than that of GFP control. The ubiquitin level of Lysine K776 and K807 mutant NESAR was significantly decreased, and the protein stability was improved. Lysine sites K776 and K807 are the key amino acid residues that mediate the ubiquification of NESAR. Conclusion: the nucleocapsid NESAR has degradation instability and is used as a recognition signal for polyubiquitin modification. It mediates the degradation of its fusion protein by ubiquitin proteasome pathway in prostate cancer cells. This study provides a new approach for the regulation of AR protein level and / or activity. It is helpful to understand the molecular mechanism of AR degradation and to regulate AR targeting in the process of castration resistance of prostate cancer.
【作者单位】：北京大学第一医院泌尿外科北京大学泌尿外科研究所国家泌尿男生殖系肿瘤研究中心;
【基金】：国家自然科学基金(81402083)资助~~
【分类号】：R737.25

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