An Antiproliferative Genetic Screening Identifies a Peptide Aptamer That Targets Calcineurin and Up-regulates Its Activity

Benoît De Chassey,Anne-Laure Mathieu,Didier Nègre,Pierre Colas,Brian B Rudkin,François-Loïc Cosset,Ivan Mikaelian,Marc Bickle,Delphine Olivier

doi:10.1074/mcp.m600102-mcp200

Abstract

Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein displaying a doubly constrained variable peptide loop. They bind specifically target proteins and interfere with their function. We have built a peptide aptamer library in a lentiviral expression system to isolate aptamers that inhibit cell proliferation in vitro. Using one of the isolated aptamers (R5G42) as a bait protein, we have performed yeast two-hybrid screening of cDNA libraries and identified calcineurin A as a target protein candidate. R5G42 bound calcineurin A in vitro and stimulated its phosphatase activity. When expressed transiently in human cells, R5G42 induced the dephosphorylation of BAD. We have identified an antiproliferative peptide aptamer that binds calcineurin and stimulates its activity. The use of this ligand may help elucidate the still elusive structural mechanisms of activation and inhibition of calcineurin. Our work illustrates the power of phenotypic screening of combinatorial protein libraries to interrogate the proteome and chart molecular regulatory networks.

Highlights

Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein displaying a doubly constrained variable peptide loop
We observed that the best combination was the HA-tagged, human thioredoxin displaying random peptide loops of 10 amino acids, and we constructed pBK1, a high complexity peptide aptamer library (Fig. 1a)
Modulation of Calcineurin Activity—We explored the ability of R5G42 to modulate the enzymatic activity of its target protein

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—We maintained all mammalian cells in a 5% CO2 atmosphere at 37 °C in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% (v/v) fetal calf serum and 100 ␮g/ml penicillin-streptomycin. The resulting plasmid, pVRV6, directs the bicistronic expression of an HA-tagged human thioredoxin (with a modified active site) and of EGFP carrying a farnesylation sequence to anchor the marker protein to plasma membranes. Two-hybrid Screening of R5G42-interacting Proteins—We digested pVRV6-R5G42 with EcoRI and XhoI and ligated the fragment into EcoRI/XhoI-cut pGILDA (Clontech) to create pGILDA-R5G42, a plasmid directing the galactose-inducible expression of a LexAR5G42 fusion protein. After an overnight incubation of the transfection mixture, we washed the cells once with culture medium, and we added fresh medium with or without 0.5 ␮M FK506 (Calbiochem). We collected the cells 24 h later, washed them twice in PBS, and lysed them for 20 min in ice-cold lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, protease inhibitor mixture Complete EDTA-free (Roche Applied Science)). We revealed the blots using the ECL system (PerkinElmer Life Sciences)

RESULTS

Sequence of variable region

Promyelocytic leukemia zinc finger protein

DISCUSSION