Sinensky M, Fantle K, Dalton M
Eleanor Roosevelt Institute, Denver, Colorado 80206.
Cancer Res. 1994 Jun 15;54(12):3229-32.
A polyclonal antibody [anti-prelamin A antibody (alpha-PA)] has been obtained against the peptide LLGNSSPRTQSPQN which is proteolytically removed during the farnesylation-dependent processing of prelamin A to mature lamin A. We tested the ability of this antibody to detect inhibition of farnesylation-dependent protein processing of prelamin A. The alpha-PA antibody was shown to immunoprecipitate prelamin A from lovastatin-treated HeLa cells but not mature lamin A from untreated cells. Further studies were performed after antigen-affinity chromatographic purification of the antibody. Western blotting of lovastatin-treated HeLa cell extract demonstrated that the purified alpha-PA antibody recognizes prelamin A. Furthermore, this signal could be competed away by incubation with the peptide. Indirect immunofluorescence helped detect nuclear accumulation of the antigen in response to treatment of HeLa cells with lovastatin or in Chinese hamster ovary K1 cells transiently transfected with a prelamin A mutant blocked in farnesylation. This antibody should be useful for screening compounds that may block any of the three common steps in the farnesylation-dependent processing of proteins (farnesylation, endoproteolysis, and carboxymethylation) since it appears that prelamin A undergoes all of these reactions prior to removal of the antigenic peptide. Inhibitors of these reactions have been proposed as potential anticancer drugs, since they would be expected to block the biological activity of oncogenic p21ras proteins. Since such screening would be performed most efficiently by enzyme-linked immunosorbent assays, we can detect the accumulation of prelamin A after treatment with lovastatin by performing this procedure as well. Application of alpha-PA in an enzyme-linked immunosorbent assay, which demonstrates the activity of a peptidomimetic farnesyltransferase inhibitor, supports the use of this antibody in large scale screening for inhibitors of farnesylation-dependent protein processing.
已获得一种多克隆抗体[抗前层粘连蛋白A抗体(α-PA)]，其针对的肽段为LLGNSSPRTQSPQN，该肽段在法尼基化依赖性的前层粘连蛋白A加工为成熟层粘连蛋白A的过程中被蛋白水解去除。我们测试了该抗体检测前层粘连蛋白A法尼基化依赖性蛋白加工抑制作用的能力。结果表明，α-PA抗体可从洛伐他汀处理的HeLa细胞中免疫沉淀前层粘连蛋白A，但不能从未处理细胞中免疫沉淀成熟层粘连蛋白A。在对抗体进行抗原亲和层析纯化后进行了进一步研究。对洛伐他汀处理的HeLa细胞提取物进行蛋白质印迹分析表明，纯化的α-PA抗体可识别前层粘连蛋白A。此外，与该肽段孵育可使此信号消失。间接免疫荧光有助于检测在用洛伐他汀处理HeLa细胞后或在用法尼基化受阻的前层粘连蛋白A突变体瞬时转染的中国仓鼠卵巢K1细胞中抗原的核内积累。该抗体对于筛选可能阻断蛋白质法尼基化依赖性加工的三个常见步骤（法尼基化、内切蛋白水解和羧甲基化）中任何一个步骤的化合物应该是有用的，因为似乎前层粘连蛋白A在去除抗原肽之前会经历所有这些反应。这些反应的抑制剂已被提议作为潜在的抗癌药物，因为预期它们会阻断致癌性p21ras蛋白的生物学活性。由于通过酶联免疫吸附测定法进行这种筛选将最为有效，我们也可以通过执行此程序来检测洛伐他汀处理后前层粘连蛋白A的积累。α-PA在酶联免疫吸附测定中的应用证明了一种拟肽法尼基转移酶抑制剂的活性，支持将该抗体用于大规模筛选法尼基化依赖性蛋白加工的抑制剂。
