Weinkauf Marc, Zimmermann Yvonne, Hartmann Elena, Rosenwald Andreas, Rieken Malte, Pastore Alessandro, Hutter Grit, Hiddemann Wolfgang, Dreyling Martin
CCG Leukemia, Department of Medicine III, University Hospital Grosshadern/LMU, Munich, Germany, in association with Helmholtz Center Munich, Munich, Germany.
Electrophoresis. 2009 Mar;30(6):974-86. doi: 10.1002/elps.200800508.
Although gene expression following bortezomib treatment has been previously explored, direct effects of bortezomib-induced proteasome inhibition on protein level has not been analyzed so far. Using 2-D PAGE in five mantle cell lymphoma cell lines, we screened for cellular protein level alterations following treatment with 25 nM bortezomib for up to 4 h. Using MS, we identified 38 of the 41 most prominent reliably detected protein spots. Twenty-one were affected in all cell lines, whereas the remaining 20 protein spots were exclusively altered in sensitive cell lines. Western blot analysis was performed for 17 of the 38 identified proteins and 70.6% of the observed protein level alterations in 2-D gels was verified. All cell lines exhibited alterations of the cellular protein levels of heat shock-induced protein species (HSPA9, HSP7C, HSPA5, HSPD1), whereas sensitive cell lines also displayed altered cellular protein levels of energy metabolism (ATP5B, AK5, TPI1, ENO-1, ALDOC, GAPDH), RNA and transcriptional regulation (HNRPL, SFRS12) and cell division (NEBL, ACTB, SMC1A, C20orf23) as well as tumor suppressor genes (ENO-1, FH). These proteins clustered in a tight interaction network centered on the major cellular checkpoints TP53. The results were confirmed in primary mantle cell lymphoma, thus confirming the critical role of these candidate proteins of proteasome inhibition.
尽管先前已经探索了硼替佐米治疗后的基因表达情况，但迄今为止，硼替佐米诱导的蛋白酶体抑制对蛋白质水平的直接影响尚未得到分析。我们使用二维聚丙烯酰胺凝胶电泳（2-D PAGE）对五种套细胞淋巴瘤细胞系进行研究，筛选了用25 nM硼替佐米处理长达4小时后细胞蛋白质水平的变化。通过质谱分析（MS），我们鉴定出了41个最显著且可靠检测到的蛋白质斑点中的38个。其中21个在所有细胞系中均受到影响，而其余20个蛋白质斑点仅在敏感细胞系中发生改变。对鉴定出的38种蛋白质中的17种进行了蛋白质印迹分析，二维凝胶中观察到的蛋白质水平变化有70.6%得到了验证。所有细胞系均表现出热休克诱导蛋白种类（HSPA9、HSP7C、HSPA5、HSPD1）的细胞蛋白质水平发生改变，而敏感细胞系还显示出能量代谢（ATP5B、AK5、TPI1、ENO-1、ALDOC、GAPDH）、RNA和转录调控（HNRPL、SFRS12）以及细胞分裂（NEBL、ACTB、SMC1A、C20orf23）相关的细胞蛋白质水平改变，以及肿瘤抑制基因（ENO-1、FH）的变化。这些蛋白质聚集在一个以主要细胞检查点TP53为中心的紧密相互作用网络中。在原发性套细胞淋巴瘤中证实了这些结果，从而确认了这些蛋白酶体抑制候选蛋白的关键作用。
