Nuclear transport of granzyme B (fragmentin-2) - Dependence on perforin in vivo and cytosolic factors in vitro
Nuclear transport of granzyme B (fragmentin-2) - Dependence on perforin in vivo and cytosolic factors in vitro
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DOI：
10.1074/jbc.271.48.30781
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发表时间：
1996-11-29
影响因子：
4.8
通讯作者：
Trapani, JA
中科院分区：
文献类型：
--
作者：
Jans, DA;Jans, P;Trapani, JA
细胞毒性T细胞和自然杀伤细胞能够通过孔形成蛋白穿孔素和丝氨酸蛋白酶颗粒酶B的协同作用杀死其靶细胞，导致非常独特的细胞凋亡典型的核变化。而穿孔素的行为在膜，颗粒酶B似乎是既能够进入靶细胞的细胞质和积累在分离的细胞核，在这项研究中，我们研究了核转运的荧光素化颗粒酶B在体内完整的细胞中存在的穿孔素和在体外semipermeabilized细胞使用共聚焦激光扫描显微镜。观察到单独的颗粒酶B进入完整细胞的细胞质，但不在细胞核中积累。然而，在亚溶解浓度的穿孔素存在下，它在完整细胞核中强烈积累，在约2.5小时后达到最大水平约1.5倍于细胞质中的水平。体外核转运试验表明，颗粒酶B在细胞核和核仁中的最大积累水平约为细胞质中的2.5和3倍，与体外SV 40大肿瘤抗原（T-Ag）融合蛋白的信号依赖性核蓄积相反，颗粒酶B的核/核仁输入不依赖于ATP，并且不可被不可水解的GTP类似物GTP γ S（鸟苷5 '-O-（3-硫代三磷酸））复制。然而，与T-Ag融合蛋白类似，颗粒酶B在核和核仁中的积累依赖于外源加入的胞质溶胶，颗粒酶B蛋白酶活性的特异性抑制剂对核/核仁积累没有影响，这意味着蛋白水解活性对于核靶向不是必需的，结果表明，颗粒酶B（32 kDa）可能通过被动扩散从细胞质转运到细胞核，并通过与细胞核结合而积累。在未标记颗粒酶B的存在下，主动和被动核转运性质是正常的，这意味着核膜和孔复合物不是颗粒酶B底物。
Cytotoxic T and natural killer cells are able to kill their target cells through synergistic action of the pore-forming protein perforin and the serine protease granzyme B, resulting in very distinctive nuclear changes typical of apoptosis. Whereas perforin acts at the membrane, granzyme B appears to be both capable of entering the cytoplasm of target cells and accumulating in isolated nuclei, In this study we examine nuclear transport of fluoresceinated granzyme B both in vivo in intact cells in the presence of perforin and in vitro in semipermeabilized cells using confocal laser scanning microscopy. Granzyme B alone was observed to enter the cytoplasm of intact cells but did not accumulate in nuclei, In the presence of sublytic concentrations of perforin, however, it accumulated strongly in intact cell nuclei to levels maximally about 1.5 times those in the cytoplasm after about 2.5 h, In vitro nuclear transport assays showed maximal levels of nuclear and nucleolar accumulation of granzyme B of about 2.5- and 3-fold those in the cytoplasm, In contrast to signal-dependent nuclear accumulation of SV40 large tumor antigen (T-Ag) fusion proteins in vitro, nuclear/nucleolar import of granzyme B was independent of ATP and not inhibitable by the non-hydrolyzable GTP analog GTP gamma S (guanosine 5'-O-(3-thiotriphosphate)). Similar to T-Ag fusion proteins, however, granzyme B nuclear and nucleolar accumulation was dependent on exogenously added cytosol, Specific inhibitors of granzyme B protease activity had no effect on nuclear/nucleolar accumulation, implying that proteolytic activity was not essential for nuclear targeting, The results imply that granzyme B (32 kDa) may be transported from the cytoplasm to the nucleus through passive diffusion and accumulate by binding to nuclear/nucleolar factors in a cytosolic factor-mediated process, Active and passive nuclear transport properties were normal in the presence of unlabeled granzyme B, implying that the nuclear envelope and pore complex are not granzyme B substrates.