Zhivotovsky B, Gahm A, Orrenius S
Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Stockholm, Sweden.
Biochem Biophys Res Commun. 1997 Apr 7;233(1):96-101. doi: 10.1006/bbrc.1997.6411.
To investigate the involvement of different proteases in the execution step of apoptosis and to determine their intracellular location, isolated rat thymocyte nuclei were incubated either in the presence of Ca2+ and Mg2+ or with cytosolic extract from Jurkat T lymphocytes treated with anti-Fas (APO-1, CD-95) antibody. Inhibitors of caspases, VADcmk and DEVDcho, were not effective in hindering Ca2+-induced apoptotic changes in isolated nuclei, but did prevent similar changes in nuclei treated with the cytosolic extract from apoptotic Jurkat cells. In contrast, the inhibitor of the Ca2+-regulated, nuclear scaffold-associated serine protease, AAPFcmk, was able to inhibit lamin B1 breakdown, as well as chromatin cleavage in nuclei incubated in the presence of Ca2+ and Mg2+, but only partially prevented the same changes induced with cytosolic extract. Our findings provide evidence for the involvement of at least two proteases in lamin cleavage. One belongs to the caspase family and to cleave lamins this enzyme must be translocated from the cytoplasm into the nucleus. The second protease has a nuclear location and is activated by Ca2+. Finally, neither of these two lamin-cleaving proteases is responsible for the cleavage of another nuclear target protein, poly(ADP-ribose)polymerase (PARP), during apoptosis.
为了研究不同蛋白酶在细胞凋亡执行步骤中的作用，并确定它们在细胞内的定位，将分离的大鼠胸腺细胞核在存在Ca2+和Mg2+的条件下孵育，或者与用抗Fas（APO-1，CD-95）抗体处理的Jurkat T淋巴细胞的胞质提取物一起孵育。半胱天冬酶抑制剂VADcmk和DEVDcho在阻碍Ca2+诱导的分离细胞核凋亡变化方面无效，但确实能阻止凋亡Jurkat细胞胞质提取物处理的细胞核发生类似变化。相比之下，Ca2+调节的、与核支架相关的丝氨酸蛋白酶抑制剂AAPFcmk能够抑制在存在Ca2+和Mg2+的条件下孵育的细胞核中的核纤层蛋白B1降解以及染色质裂解，但只能部分阻止胞质提取物诱导的相同变化。我们的研究结果为至少两种蛋白酶参与核纤层蛋白裂解提供了证据。一种属于半胱天冬酶家族，为了裂解核纤层蛋白，这种酶必须从细胞质转运到细胞核中。第二种蛋白酶定位于细胞核，由Ca2+激活。最后，这两种裂解核纤层蛋白的蛋白酶在细胞凋亡过程中都不负责另一种核靶蛋白聚（ADP-核糖）聚合酶（PARP）的裂解。
