Han Jinbo, Zhang Ling, Shao Xiaoxia, Shi Jiahao, Chi Chengwu
Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai, China.
FEBS J. 2006 Oct;273(19):4459-69. doi: 10.1111/j.1742-4658.2006.05451.x. Epub 2006 Sep 5.
Many physiologically important proproteins, pathogenic bacterial exotoxins and viral envelope glycoproteins are activated by the proprotein convertase furin, which makes furin inhibitor a hot target for basic research and drug design. Although synthetic and bioengineered inhibitors of furin have been well characterized, its endogenous inhibitor has not been directly purified from mammalian tissues to date. In this study, three inhibitors were purified from the porcine liver by using a combination of chromatographic techniques, and identified to be the C-terminal truncated fragments with different sizes of histone H1.2. The gene of porcine histone H1.2 was cloned and sequenced, further confirming the determined sequences. These three C-terminal fragments inhibited furin with Ki values around 2 x 10(-7) m while the full-length histone H1.2 inhibited it with a lesser activity, suggesting that the inhibitory activity relies on the C-terminal lysine-rich domain. Though the inhibition was temporary, these inhibitors were specific, and the reactive site of one C-terminal fragment was identified. A 36 amino acid peptide around the reactive site was synthesized, which could still inhibit furin with a Ki of 5.2 x 10(-7) m.
许多具有重要生理功能的前体蛋白、致病性细菌外毒素和病毒包膜糖蛋白都是由前体蛋白转化酶弗林蛋白酶激活的，这使得弗林蛋白酶抑制剂成为基础研究和药物设计的热门靶点。尽管弗林蛋白酶的合成抑制剂和生物工程抑制剂已得到充分表征，但迄今为止，其内源抑制剂尚未从哺乳动物组织中直接纯化出来。在本研究中，通过多种色谱技术从猪肝中纯化出三种抑制剂，并鉴定为不同大小的组蛋白H1.2的C端截短片段。克隆并测序了猪组蛋白H1.2的基因，进一步证实了所确定的序列。这三个C端片段对弗林蛋白酶的抑制常数（Ki）约为2×10⁻⁷mol/L，而全长组蛋白H1.2的抑制活性较低，这表明抑制活性依赖于C端富含赖氨酸的结构域。尽管这种抑制是暂时的，但这些抑制剂具有特异性，并且确定了其中一个C端片段的反应位点。合成了反应位点周围的一个36个氨基酸的肽段，其对弗林蛋白酶的抑制常数（Ki）仍为5.2×10⁻⁷mol/L。
