Pak Stephen C, Kumar Vasantha, Tsu Christopher, Luke Cliff J, Askew Yuko S, Askew David J, Mills David R, Brömme Dieter, Silverman Gary A
Department of Pediatrics, Harvard Medical School, Children's Hospital, Boston, Massachusetts 02115.
J Biol Chem. 2004 Apr 9;279(15):15448-59. doi: 10.1074/jbc.M400261200. Epub 2004 Jan 22.
High molecular weight serpins are members of a large superfamily of structurally conserved proteins that inactivate target proteinases by a suicide substrate-like mechanism. In vertebrates, different clades of serpins distribute predominantly to either the intracellular or extracellular space. Although much is known about the function, structure, and inhibitory mechanism of circulating serpins such as alpha(1)-antitrypsin (SERPINA1) and antithrombin III (SERPINC1), relatively little is known about the function of the vertebrate intracellular (clade B) serpins. To gain a better understanding of the biology of the intracellular serpins, we initiated a comparative genomics study using Caenorhabditis elegans as a model system. A screen of the C. elegans genomic and cDNA databases revealed nine serpin genes, tandemly arrayed on chromosome V. Although the C. elegans serpins represent a unique clade (L), they share significant functional homology with members of the clade B group of intracellular serpins, since they lack typical N-terminal signal peptides and reside intracellularly. To determine whether nematode serpins function as proteinase inhibitors, one family member, srp-2, was chosen for further characterization. Biochemical analysis of recombinant SRP-2 protein revealed SRP-2 to be a dual cross-class inhibitor of the apoptosis-related serine proteinase, granzyme B, and the lysosomal cysteine proteinases, cathepsins K, L, S, and V. Analysis of temporal and spatial expression indicated that SRP-2 was present during early embryonic development and highly expressed in the intestine and hypoderm of larval and adult worms. Transgenic animals engineered to overexpress SRP-2 were slow growing and/or arrested at the first, second, or third larval stages. These data suggest that perturbations of serpin-proteinase balance are critical for correct postembryonic development in C. elegans.
高分子量丝氨酸蛋白酶抑制剂（serpins）是一个结构保守的蛋白质大家族的成员，它们通过类似自杀底物的机制使靶蛋白酶失活。在脊椎动物中，不同分支的丝氨酸蛋白酶抑制剂主要分布在细胞内或细胞外空间。尽管人们对循环丝氨酸蛋白酶抑制剂如α1-抗胰蛋白酶（SERPINA1）和抗凝血酶III（SERPINC1）的功能、结构和抑制机制了解很多，但对脊椎动物细胞内（B分支）丝氨酸蛋白酶抑制剂的功能了解相对较少。为了更好地理解细胞内丝氨酸蛋白酶抑制剂的生物学特性，我们启动了一项以秀丽隐杆线虫为模型系统的比较基因组学研究。对秀丽隐杆线虫基因组和cDNA数据库的筛选揭示了9个丝氨酸蛋白酶抑制剂基因，它们串联排列在第五条染色体上。尽管秀丽隐杆线虫的丝氨酸蛋白酶抑制剂代表一个独特的分支（L），但它们与细胞内丝氨酸蛋白酶抑制剂B分支的成员具有显著的功能同源性，因为它们缺乏典型的N端信号肽且位于细胞内。为了确定线虫丝氨酸蛋白酶抑制剂是否作为蛋白酶抑制剂发挥作用，选择了一个家族成员srp-2进行进一步表征。对重组SRP-2蛋白的生化分析表明，SRP-2是凋亡相关丝氨酸蛋白酶颗粒酶B以及溶酶体半胱氨酸蛋白酶组织蛋白酶K、L、S和V的双重跨类抑制剂。对时空表达的分析表明，SRP-2在胚胎发育早期存在，并在幼虫和成虫的肠道和皮下高度表达。经基因工程改造过表达SRP-2的转基因动物生长缓慢和/或在第一、第二或第三幼虫阶段停滞。这些数据表明，丝氨酸蛋白酶抑制剂-蛋白酶平衡的扰动对于秀丽隐杆线虫胚胎后期的正确发育至关重要。
