Kolonin M G, Finley R L
Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201, USA.
Proc Natl Acad Sci U S A. 1998 Nov 24;95(24):14266-71. doi: 10.1073/pnas.95.24.14266.
Two-hybrid technology provides a simple way to isolate small peptide aptamers that specifically recognize and strongly bind to a protein of interest. These aptamers have the potential to dominantly interfere with specific activities of their target proteins and, therefore, could be used as in vivo inhibitors. Here we explore the ability to use peptide aptamers as in vivo inhibitors by expressing aptamers directed against cell cycle regulators in Drosophila. We expressed two peptide aptamers, each of which specifically recognizes one of the two essential cyclin-dependent kinases (Cdks), DmCdk1 and DmCdk2, in Drosophila. Expression of each Cdk aptamer during organogenesis caused adult eye defects typical of those caused by cell cycle inhibition. Co-overexpression of DmCdk1 or DmCdk2 resulted in suppression of the eye phenotypes, indicating that each aptamer interacts with a Cdk target in vivo and suggesting that these peptides disrupt normal eye development by inhibiting Cdk function. Moreover, the specificity of each aptamer for one of the two Cdks as determined in two-hybrid assays was retained in Drosophila. Combined, our results demonstrate that peptide aptamers generated by yeast two-hybrid methods can serve as inhibitory reagents to target specific proteins in vivo.
双杂交技术提供了一种简单的方法来分离能特异性识别并强烈结合目标蛋白的小肽适配体。这些适配体有可能显性干扰其靶蛋白的特定活性，因此可作为体内抑制剂使用。在这里，我们通过在果蝇中表达针对细胞周期调节因子的适配体，探索将肽适配体用作体内抑制剂的能力。我们在果蝇中表达了两种肽适配体，每种适配体都能特异性识别两种必需的细胞周期蛋白依赖性激酶（Cdks）之一，即DmCdk1和DmCdk2。在器官发生过程中表达每种Cdk适配体都会导致典型的因细胞周期抑制而引起的成虫眼缺陷。共过表达DmCdk1或DmCdk2可导致眼表型的抑制，这表明每种适配体在体内与Cdk靶标相互作用，并表明这些肽通过抑制Cdk功能破坏正常的眼发育。此外，在双杂交实验中确定的每种适配体对两种Cdk之一的特异性在果蝇中得以保留。综合来看，我们的结果表明，通过酵母双杂交方法产生的肽适配体可作为体内靶向特定蛋白的抑制试剂。
