Goping Ing Swie, Sawchuk Tracy, Underhill D Alan, Bleackley R Chris
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7.
J Cell Sci. 2006 Mar 1;119(Pt 5):858-65. doi: 10.1242/jcs.02791.
Cytotoxic lymphocytes induce target cell apoptosis via two major pathways: Fas/FasL and granule exocytosis. The latter pathway has largely been defined by the roles of the pore-forming protein perforin and by the serine proteinases granzymes A and B. Upon entry into target cells, the granzymes cleave substrates that ultimately result in cell death. To gain further insight into granzyme B function, we have identified novel substrates. SDS-PAGE analysis of S100 cell lysates identified a 51 kDa protein that was cleaved by granzyme B. Mass spectrometry analysis revealed that this fragment was the microtubule protein, alpha-tubulin, which was confirmed by western blotting. In addition, two-dimensional gel analysis showed that the truncated form of alpha-tubulin had a more basic isoelectric point than the full-length molecule, suggesting that granzyme B removed the acidic C-terminus. Site-directed mutagenesis within this region of alpha-tubulin revealed the granzyme B recognition site, which is conserved in a subset of alpha-tubulin isoforms. Significantly, we showed that alpha-tubulin was cleaved in target cells undergoing apoptosis as induced by cytotoxic T lymphocytes. Therefore, in addition to its role in the activation of mitochondria during apoptosis, these results suggest a role for granzyme B in the dismantling of the cytoskeleton.
Fas/FasL途径和颗粒胞吐途径。后一种途径很大程度上是由成孔蛋白穿孔素以及丝氨酸蛋白酶颗粒酶A和B的作用所定义的。颗粒酶进入靶细胞后，会切割底物，最终导致细胞死亡。为了进一步深入了解颗粒酶B的功能，我们鉴定了新的底物。对S100细胞裂解物进行SDS-PAGE分析，鉴定出一种被颗粒酶B切割的51 kDa蛋白质。质谱分析表明，该片段是微管蛋白α-微管蛋白，这通过蛋白质印迹法得到了证实。此外，二维凝胶分析表明，α-微管蛋白的截短形式比全长分子具有更碱性的等电点，这表明颗粒酶B去除了酸性的C末端。在α-微管蛋白的该区域内进行定点诱变揭示了颗粒酶B识别位点，该位点在一部分α-微管蛋白同工型中是保守的。重要的是，我们表明在细胞毒性T淋巴细胞诱导的正在经历凋亡的靶细胞中，α-微管蛋白被切割。因此，除了其在凋亡过程中激活线粒体的作用外，这些结果表明颗粒酶B在细胞骨架的拆解中也发挥作用。
