Cohen B A, Colas P, Brent R
Department of Molecular Biology, Massachusetts General Hospital, 50 Blossom Street, Boston, MA 02114, USA.
Proc Natl Acad Sci U S A. 1998 Nov 24;95(24):14272-7. doi: 10.1073/pnas.95.24.14272.
Understanding the genetic networks that operate inside cells will require the dissection of interactions among network members. Here we describe a peptide aptamer isolated from a combinatorial library that distinguishes among such interactions. This aptamer binds to cyclin-dependent kinase 2 (Cdk2) and inhibits its kinase activity. In contrast to naturally occurring inhibitors, such as p21(Cip1), which inhibit the activity of Cdk2 on all its substrates, inhibition by pep8 has distinct substrate specificity. We show that the aptamer binds to Cdk2 at or near its active site and that its mode of inhibition is competitive. Expression of pep8 in human cells retards their progression through the G1 phase of the cell cycle. Our results suggest that the aptamer inhibits cell-cycle progression by blocking the activity of Cdk2 on substrates needed for the G1-to-S transition. This work demonstrates the feasibility of selection of artificial proteins to perform functions not developed during evolution. The ability to select proteins that block interactions between a gene product and some partners but not others should make sophisticated genetic manipulations possible in human cells and other currently intractable systems.
要理解细胞内运行的基因网络，需要剖析网络成员之间的相互作用。在此，我们描述了一种从组合文库中分离出的肽适配体，它能区分此类相互作用。这种适配体与细胞周期蛋白依赖性激酶2（Cdk2）结合并抑制其激酶活性。与天然存在的抑制剂（如p21（Cip1））不同，p21（Cip1）抑制Cdk2对其所有底物的活性，而pep8的抑制作用具有独特的底物特异性。我们表明，该适配体在Cdk2的活性位点或其附近与之结合，其抑制模式是竞争性的。pep8在人类细胞中的表达会延缓它们通过细胞周期G1期的进程。我们的结果表明，该适配体通过阻断Cdk2对G1期到S期转换所需底物的活性来抑制细胞周期进程。这项工作证明了选择人工蛋白质来执行进化过程中未产生的功能的可行性。选择能够阻断基因产物与某些而非其他伙伴之间相互作用的蛋白质的能力，应该会使在人类细胞和其他目前难以处理的系统中进行复杂的基因操作成为可能。
