Dickinson J L, Norris B J, Jensen P H, Antalis T M
Queensland Cancer Fund Experimental Oncology Unit, The Queensland Institute of Medical Research, Brisbane, 4029, Australia.
Cell Death Differ. 1998 Feb;5(2):163-71. doi: 10.1038/sj.cdd.4400324.
The serine proteinase inhibitor (serpin), plasminogen activator inhibitor type 2 (PAI-2), has been reported to inhibit tumor necrosis factor-alpha (TNF) induced apoptosis. In order to begin to understand the molecular basis for this protection, we have investigated the importance of a structural domain within the PAI-2 molecule, the C-D interhelical region, in mediating the protective effect. The C-D interhelical region is a 33 amino acid insertion which is unique among serpins and has been implicated in transglutaminase catalyzed cross-linking of PAI-2 to cell membranes. We have constructed a mutant of PAI-2 wherein 23 amino acids are deleted from the C-D interhelical region generating a structure predicted to be homologous to the closely related, but non-inhibitory serpin, chicken ovalbumin. The PAI-2Delta65/87 deletion mutant retained inhibitory activity against its known serine proteinase target, urokinase-type plasminogen activator (uPA); however expression of this mutant in HeLa cells failed to protect from TNF-induced apoptosis. Analyses of the cellular distribution of PAI-2 showed that intracellular PAI-2, and not secreted or cell-surface PAI-2, was likely responsible for the observed protection from TNF-induced apoptosis. No evidence was found for specific cross-linking of PAI-2 to the plasma membrane in either control or TNF/cycloheximide treated cells. The data demonstrate that the PAI-2 C-D interhelical domain is functionally important in PAI-2 protection from TNF induced apoptosis and suggest a novel function for the C-D interhelical domain in the protective mechanism.
丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂），纤溶酶原激活物抑制剂2型（PAI-2），已被报道可抑制肿瘤坏死因子-α（TNF）诱导的细胞凋亡。为了开始理解这种保护作用的分子基础，我们研究了PAI-2分子内一个结构域，即C-D螺旋间区域，在介导保护作用中的重要性。C-D螺旋间区域是一个33个氨基酸的插入片段，在丝氨酸蛋白酶抑制剂中是独特的，并且与转谷氨酰胺酶催化的PAI-2与细胞膜的交联有关。我们构建了一个PAI-2突变体，其中从C-D螺旋间区域删除了23个氨基酸，产生了一种预测与密切相关但无抑制作用的丝氨酸蛋白酶抑制剂鸡卵清蛋白同源的结构。PAI-2Delta65/87缺失突变体对其已知的丝氨酸蛋白酶靶点尿激酶型纤溶酶原激活物（uPA）仍保留抑制活性；然而，该突变体在HeLa细胞中的表达未能保护细胞免受TNF诱导的凋亡。对PAI-2细胞分布的分析表明，细胞内的PAI-2而非分泌型或细胞表面的PAI-2可能是观察到的免受TNF诱导凋亡保护作用的原因。在对照细胞或TNF/环己酰亚胺处理的细胞中均未发现PAI-2与质膜特异性交联的证据。数据表明，PAI-2的C-D螺旋间结构域在PAI-2保护细胞免受TNF诱导凋亡中具有重要功能，并提示C-D螺旋间结构域在保护机制中具有新功能。
