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PEPTIDE-APTAMERS: NEW TOOLS TO STUDY PROTEIN INTERACTIONS AND INTRACELLULAR PROCESSES
Giannino Del Sal, Laboratorio Nazionale CIB, AREA Science Park, Trieste and Dipartimento di Biochimica Biofisica e Chimica delle Macromolecole, Trieste
Coordinated protein-protein interactions play a pivotal role in many biological processes, including signal transduction, cell cycle regulation and gene expression. Such interactions can be highlighted by a variety of methodologies including phage-display and the yeast two-hybrid system.
In particular, the yeast two-hybrid system allows the identification of new interactors of a known protein under cellular conditions. Several variations of the original method have been developed in the last years, such as the one-hybrid system that allows the identification of protein-DNA interactions and the three-hybrid system to detect interactions which depend on a third protein. In addition the system has been modified to allow the identification of molecules that specifically disrupt protein complexes and the technique has been called reverse two-hybrid.
One recent and interesting application of the yeast-two hybrid method is represented by the identification of small peptide-aptamers from combinatorial libraries based on their ability to interact to a given particular protein. Once identified, these small peptides can be analysed for their ability to interfere with the function of the target protein and with its ability to bind other cellular partners.
The strategy of using small peptides to interfere with protein complexes has been widely validated. A random 20-mer library can be inserted into the active site of the E.coli Thioredoxin scaffold protein, which allows the constrained peptide to be displayed on the surface of the protein. With this approach peptides able to bind to and interfere with cdk2 and E6 oncoprotein functions have been identified.
These peptide aptamers have been further used to change the localization of target proteins in vivo. Alternatively, peptides deduced from the interacting surfaces of two known proteins and expressed in vivo may act as dominant trans-effectors to dissociate a particular complex, as shown with small p53 peptides capable interfere with the MDM2/p53 association thus leading to activation of p53 response in vivo.
We have developed a set of yeast and mammalian expression vectors containing the E. coli Thioredoxin used as a scaffold protein. For the yeast two-hybrid system the Thioredoxin fused to the VSV tag has been inserted downstream the B42 transactivation domain within the pJG4-5 plasmid and a random peptide library inserted into the active site of the thioredoxin is already available to screen for peptide aptamers that bind to a particular "bait". In addition, a set of plasmid vectors for expression in mammalian cells have been also generated, with the Thioredoxin containing a nuclear localization signal (NLS) cloned downstream the CMV and SV40 promoters as well as in retroviral vectors. These vectors have been validated with peptides know to interfere with MDM2/p53 interaction and with Cdk2.
With these tools we aim at characterizing peptide-aptamers able to bind to mutant p53 and interfere with its association with endogenous proteins (e.g. p73 and p63) Furthermore we would like to extend this approach also to select for peptides that can interfere with other proteins whose function is relevant in controlling the apoptotic process.
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