Cleavage of nonmuscle myosin heavy chain-A during apoptosis in human Jurkat T cells.
Cleavage of nonmuscle myosin heavy chain-A during apoptosis in human Jurkat T cells.
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人 Jurkat T 细胞凋亡过程中非肌肉肌球蛋白重链 A 的裂解。
DOI：
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发表时间：
2005
期刊：
影响因子：
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通讯作者：
S. Imajoh‐ohmi
中科院分区：
文献类型：
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作者：
Masahiko Kato;H. Fukuda;T. Nonaka;S. Imajoh‐ohmi
我们先前已经报道了钙蛋白酶抑制素，钙蛋白酶的内源性抑制蛋白，在人Jurkat T细胞的凋亡过程中被半胱天冬酶-3样蛋白酶切割[Kato，M.等人（2000）J.Biochem.127，297-305]。在这项研究中，我们发现，非肌肉肌球蛋白重链-A（NMHC-A）在Jurkat细胞凋亡过程中被切割，通过使用切割位点定向抗体的钙蛋白酶抑制蛋白。针对钙蛋白酶抑制蛋白的氨基末端片段产生切割位点定向抗体，并且该抗体检测体外切割的钙蛋白酶抑制蛋白片段。尽管没有检测到切割的钙蛋白酶抑制蛋白，但该抗体在凋亡细胞中检测到了95-kDa的多肽（p95）。根据肽质谱指纹图谱和氨基端测序的结果，该p95被鉴定为NMHC-A的羧基端片段。此外，确定了NMHC-A上的两个切割位点，Asp-1153和Asp-1948，并通过针对每个切割位点的切割位点定向抗体检测了NMHC-A的三个切割片段，一个在Asp-1153切割，另两个在Asp-1948切割。共聚焦免疫荧光显微镜分析结果表明，在细胞凋亡过程中，Asp-1948的裂解比Asp-1153的裂解快。此外，Asp-1153切割片段弥漫性分布在凋亡细胞的细胞质中，而Asp-1948切割片段检测为浓缩点。总之，我们的研究结果可以总结如下：（i）NMHC-A在凋亡过程中在两个位点被切割，（ii）这两个切割位点之间的切割时间不同，（iii）在凋亡细胞中切割片段的分布不同。
We have previously reported that calpastatin, an endogenous inhibitory protein of calpain, is cleaved by a caspase-3-like protease during apoptosis in human Jurkat T cells [Kato, M. et al. (2000) J. Biochem. 127, 297-305]. In this study, we found that nonmuscle myosin heavy chain-A (NMHC-A) is cleaved during apoptosis in Jurkat cells by using a cleavage-site-directed antibody for calpastatin. The cleavage-site-directed antibody was raised against the amino-terminal fragment of calpastatin, and this antibody detected the in vitro cleaved calpastatin fragment. Although cleaved calpastatin was not detected, a 95-kDa polypeptide (p95) was detected in apoptotic cells by this antibody. This p95 was identified as the carboxyl-terminal fragment of NMHC-A based on the results of peptide mass spectrometry fingerprinting and amino-terminal sequencing. Furthermore, two cleavage sites on NMHC-A, Asp-1153 and Asp-1948, were determined, and three cleaved fragments of NMHC-A, one cleaved at Asp-1153 and the other two cleaved at Asp-1948, were detected by cleavage-site-directed antibodies against each cleavage site. The results of confocal immunofluorescence microscopic analysis show that the cleavage at Asp-1948 occurs faster than that at Asp-1153 during apoptosis. In addition, the Asp-1153 cleaved fragment was distributed diffusely in the cytoplasm of apoptotic cells, whereas the Asp-1948 cleaved fragments were detected as condensed dots. In conclusion, our findings can be summarized as follows: (i) NMHC-A is cleaved at two sites during apoptosis, (ii) the timing of cleavage is different between these two cleavage sites, and (iii) the distribution of cleaved fragments is different in apoptotic cells.