An artificial cell-cycle inhibitor isolated from a combinatorial library.
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.
Cohen
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Department of Molecular Biology, Massachusetts General Hospital, 50 Blossom Street, Boston, MA 02114, USA.
Colas
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Brent
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D001667:Binding, Competitive; D042846:CDC2-CDC28 Kinases; D020134:Catalytic Domain; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D003001:Cloning, Molecular; D003433:Crosses, Genetic; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016193:G1 Phase; D006801:Humans; D007700:Kinetics; D019151:Peptide Library; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000235:genetics
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases