Morris Emma C, Dafforn Timothy R, Forsyth Sharon L, Missen Melinda A, Horvath Anita J, Hampson Lynne, Hampson Ian N, Currie Graeme, Carrell Robin W, Coughlin Paul B
Department of Haematology, University College Hospital, Grafton Way, London WC1E 6AU, UK.
Biochem J. 2003 Apr 1;371(Pt 1):165-73. doi: 10.1042/BJ20021567.
Murine serpin 2A is expressed at high levels in haemopoietic progenitors and down-regulated on differentiation. When it is constitutively expressed in the multipotent haemopoietic cell line, FDCP-Mix, it causes a delay in differentiation and increased clonogenic potential. The serpin is also dramatically up-regulated on T-cell activation. It has an unusual reactive site Cys-Cys sequence, a unique C-terminal extension and lacks a typical cleavable N-terminal signal sequence. In spite of these features, the protein is not a member of the ovalbumin-serpin family, but is instead most closely related to human antichymotrypsin. We have shown that the serpin is intracellular with prominent nuclear localization. Transverse urea gradient gels and CD studies show that the protein undergoes the stressed-relaxed conformational change typical of inhibitory serpins. However, we have not detected complex-forming activity with a set of proteases. Thermal denaturation studies also show that the protein has decreased structural stability under reducing conditions, although it lacks disulphide bonds within the core of the molecule. Our results show that serpin 2A is an intracellular protein with the potential to mediate its biological effects via interaction with non-protease intracellular targets. Furthermore, the results presented suggest a model whereby the serpin interactions could be modulated by redox conditions or conformational change induced by cleavage of the reactive-site loop.
小鼠丝氨酸蛋白酶抑制剂2A在造血祖细胞中高水平表达，在分化时下调。当它在多能造血细胞系FDCP-Mix中组成性表达时，会导致分化延迟并增加克隆形成潜力。该丝氨酸蛋白酶抑制剂在T细胞活化时也显著上调。它具有不寻常的反应位点半胱氨酸-半胱氨酸序列、独特的C末端延伸，并且缺乏典型的可裂解N末端信号序列。尽管有这些特征，该蛋白并非卵清蛋白-丝氨酸蛋白酶抑制剂家族的成员，而是与人类抗胰凝乳蛋白酶关系最为密切。我们已经表明，该丝氨酸蛋白酶抑制剂位于细胞内，具有明显的核定位。横向尿素梯度凝胶和圆二色性研究表明，该蛋白经历了抑制性丝氨酸蛋白酶抑制剂典型的应激-松弛构象变化。然而，我们未检测到它与一组蛋白酶形成复合物的活性。热变性研究还表明，尽管该蛋白在分子核心内缺乏二硫键，但在还原条件下其结构稳定性降低。我们的结果表明，丝氨酸蛋白酶抑制剂2A是一种细胞内蛋白，有可能通过与非蛋白酶细胞内靶点相互作用来介导其生物学效应。此外，所呈现的结果提示了一个模型，即丝氨酸蛋白酶抑制剂的相互作用可能受氧化还原条件或反应位点环裂解诱导的构象变化调节。
