Li F, Zhang D, Fujise K
Research Center for Cardiovascular Diseases, Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas-Houston Medical School, 6431 Fannin St., Houston, TX 77030, USA.
J Biol Chem. 2001 Dec 14;276(50):47542-9. doi: 10.1074/jbc.M108954200. Epub 2001 Oct 11.
Apoptosis is meticulously controlled in living organisms. Its dysregulation has been shown to play a key role in a number of human diseases, including neoplastic, cardiovascular, and degenerative disorders. Bcl-2 family member proteins and inhibitors of apoptosis proteins are two major negative regulators of apoptosis. We report here the characterization of novel antiapoptotic protein, fortilin, which we identified through yeast two-hybrid library screening. Sequence analysis of fortilin revealed it to be a 172-amino acid polypeptide highly conserved from mammals to plants. Fortilin is structurally unrelated to either Bcl-2 family member proteins or inhibitors of apoptosis proteins. Northern blot analysis showed the fortilin message to be ubiquitous in normal tissue but especially abundant in the liver, kidney, and small intestine. Western blot analysis using anti-fortilin antibody showed more extensive expression in cancerous cell lines (H1299, MCF-7, and A549) than in cell lines derived from normal tissue (HEK293). Immunocytochemistry using HeLa cells transiently expressing FLAG-tagged fortilin and immunohistochemistry using human breast ductal carcinoma tissue and anti-fortilin antibody both showed that fortilin is predominantly localized in the nucleus. Functionally, the transient overexpression of fortilin in HeLa cells prevented them, in a dose-dependent fashion, from undergoing etoposide-induced apoptosis. Consistently, U2OS cells stably expressing fortilin protected the cells from cell death induced by etoposide over various concentrations and durations of exposure. In addition, fortilin overexpression inhibited caspase-3-like activity as assessed by the cleavage of fluorogenic substrate benzyloxycarbonyl-DEVD-7-amido-4-(trifluoromethyl)coumarin. Furthermore, the antisense depletion of fortilin from breast cancer cell line MCF-7 was associated with massive cell death. These data suggest that fortilin represents a novel antiapoptotic protein involved in cell survival and apoptosis regulation.
细胞凋亡在生物体中受到精确调控。其失调已被证明在许多人类疾病中起关键作用，包括肿瘤、心血管和退行性疾病。Bcl-2家族成员蛋白和凋亡抑制蛋白是细胞凋亡的两个主要负调控因子。我们在此报告了一种新型抗凋亡蛋白福替林（fortilin）的特性，它是我们通过酵母双杂交文库筛选鉴定出来的。福替林的序列分析表明它是一种由172个氨基酸组成的多肽，从哺乳动物到植物都高度保守。福替林在结构上与Bcl-2家族成员蛋白或凋亡抑制蛋白均无关联。Northern印迹分析显示福替林的信使核糖核酸在正常组织中普遍存在，但在肝脏、肾脏和小肠中尤其丰富。使用抗福替林抗体的蛋白质印迹分析表明，与源自正常组织的细胞系（HEK293）相比，其在癌细胞系（H1299、MCF-7和A549）中的表达更为广泛。使用瞬时表达FLAG标签福替林的HeLa细胞进行免疫细胞化学以及使用人乳腺导管癌组织和抗福替林抗体进行免疫组织化学均显示，福替林主要定位于细胞核。在功能上，福替林在HeLa细胞中的瞬时过表达以剂量依赖的方式阻止它们发生依托泊苷诱导的细胞凋亡。同样，稳定表达福替林的U2OS细胞在不同浓度和暴露时间下均能保护细胞免受依托泊苷诱导的细胞死亡。此外，通过荧光底物苄氧羰基-DEVD-7-氨基-4-（三氟甲基）香豆素的切割评估，福替林过表达抑制了半胱天冬酶-3样活性。此外，乳腺癌细胞系MCF-7中福替林的反义缺失与大量细胞死亡有关。这些数据表明福替林是一种参与细胞存活和凋亡调控的新型抗凋亡蛋白。
