Activation of histidine decarboxylase through post-translational cleavage by caspase-9 in a mouse mastocytoma P-815
Activation of histidine decarboxylase through post-translational cleavage by caspase-9 in a mouse mastocytoma P-815
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DOI：
10.1074/jbc.m609943200
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发表时间：
2007-05-04
影响因子：
4.8
通讯作者：
Tanaka, Satoshi
中科院分区：
文献类型：
--
作者：
Furuta, Kazuyuki;Nakayama, Kazuhisa;Tanaka, Satoshi
(L)-组氨酸脱羧酶 (HDC) 是哺乳动物组胺合成的限速酶。尽管越来越多的证据表明 HDC 进行翻译后加工，但仍不清楚涉及哪些类型的蛋白酶。我们使用慢病毒表达系统研究了小鼠肥大细胞瘤 P-815 中 HDC 的加工过程。 HDC 以 74 kDa 前体形式表达，用丁酸盐处理后裂解产生 55 kDa 和 60 kDa 形式。丙氨酸扫描突变显示两个串联天冬氨酸残基（Asp(517) -Asp(518)、Asp(550)-Asp(551)）对于加工至关重要。用丁酸盐处理导致表达野生型HDC的细胞的酶活性增加，但不增加表达加工无能突变体的细胞的酶活性。丁酸盐合成组胺的增加伴随着 55-和 60-kDa 形式 HDC 的形成。此外，还发现体外翻译的 74 kDa 形式的 HDC 会被纯化的人 caspase-9 进行有限的切割，而丙氨酸取代的突变体则不会。在 Zn2+ 螯合剂 TPEN 存在下，P-815 细胞中 HDC 的加工和酶促激活得到增强。尽管丁酸盐和 TPEN 处理显着增强了 caspase-3 和 -9 的蛋白酶活性，但没有观察到凋亡细胞死亡。 HDC 的酶激活和加工均被泛 caspase 抑制剂完全抑制，caspase-9 特异性抑制剂部分但显着抑制，但 caspase-3 抑制剂则不然。这些结果表明，在 P-815 细胞中，组胺合成通过 HDC 的翻译后裂解而增强，这是由 caspase-9 介导的。
(L)-Histidine decarboxylase (HDC) is the rate-limiting enzyme for histamine synthesis in mammals. Although accumulating evidence has indicated the post-translational processing of HDC, it remains unknown what kinds of proteases are involved. We investigated the processing of HDC in a mouse mastocytoma, P-815, using a lentiviral expression system. HDC was expressed as a 74-kDa precursor form, which is cleaved to yield the 55- and 60-kDa forms upon treatment with butyrate. Alanine-scanning mutations revealed that two tandem aspartate residues (Asp(517) -Asp(518), Asp(550)- Asp(551)) are critical for the processing. Treatment with butyrate caused an increase in the enzyme activity of the cells expressing the wild type HDC, but not in the cells expressing the processing-incompetent mutant. An increase in histamine synthesis by butyrate was accompanied by formation of the 55- and 60-kDa form of HDC. In addition, the in vitro translated 74-kDa form of HDC was found to undergo a limited cleavage by purified human caspase-9, whereas the alanine-substituted mutants were not. Processing and enzymatic activation of HDC in P-815 cells was enhanced in the presence of a Zn2+ chelator, TPEN. Although treatment with butyrate and TPEN drastically augmented the protease activity of caspase-3, and -9, no apoptotic cell death was observed. Both enzymatic activation and processing of HDC were completely suppressed by a pan-caspase inhibitor, partially but significantly by a specific inhibitor for caspase-9, but not by a caspase-3 inhibitor. These results suggest that, in P-815 cells, histamine synthesis is augmented through the post-translational cleavage of HDC, which is mediated by caspase-9.