核转出蛋白对前列腺癌雄激素受体稳定性的调节

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pEGFP-AR(1-918aa)，pEGFP-NESAR(743-817aa)，pEGFP-NAR (1-665aa)和pEGFP-NAR-NESAR，及NESAR的赖氨酸突变体（赖氨酸K突变为精氨酸R）pEGFP-NESAR K776R，pEGFP-NESAR K807R和pEGFP-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。

Nuclear export signal of androgen receptor regulated of androgen receptor stability in prostate cancer

Objective: To investigate the mechanisms of nuclear export signal of androgen receptor (NESAR) in the regulation of androgen receptor (AR) protein expression and stability in prostate cancer.Methods: The green fluorescent protein fusion protein expression vectors pEGFP-AR(1-918aa), pEGFP-NESAR (743-817aa), pEGFP-NAR (1-665aa) and pEGFP-NAR-NESAR, and lysine mutants of NESAR pEGFP-NESAR K776R, pEGFP-NESAR K807R and pEGFP-NESAR K776R/K807R, were transiently transfec-ted into prostate cancer cell line PC3.Fluorescence microscopy, Western blot and immunoprecipitation were used to detect NESAR regulation of androgen receptor stability.Results: Under the fluorescence microscope, NESAR-containing fusion proteins were cytoplasmic localization, and their fluorescence intensities were much weaker than those without NESAR.The expression levels of NESAR-containing fusion proteins were significantly lower than those without NESAR.The half-lives of GFP-NESAR and GFP-NAR-NESAR were less than 6 h, while the expression of GFP and GFP-NAR was relatively stable and the half-life was more than 24 h in the presence of cycloheximide.The expression levels of GFP-NESAR were significantly increased by proteasome inhibitor MG132 treatment in a dose-dependent manner;in contrast, MG132 did not show any significant effect on the protein levels of GFP.When new protein synthesis was blocked, MG132 could also prevent the degradation of GFP-NESAR in the transfected cells in the presence of cycloheximide, while it had no significant effect on GFP protein stability in the parallel experiment.GFP immunoprecipitation showed that the ubiquitination level of GFP-NESAR fusion protein was significantly higher than that of the GFP control.The mutations of lysine sites K776 and K807 in NESAR significantly reduced the level of ubiquitination, and showed increased protein stability, indicating that they were the key amino acid residues of NESAR ubiquitination.Conclusion: NESAR was unstable and decreased the stability of its fusion proteins.NESAR was the target of polyubiquitination and mediated the degradation of its fusion proteins through the ubiquitin-proteasome pathway in prostate cancer cells.Our research provides a new way to regulate the level and/or activity of AR proteins, thus helping us understand the molecular mechanisms of AR degradation and strict control of AR in the progression to castration-resistance.