Directed evolution of p53 variants with altered DNA-binding specificities by in vitro compartmentalization.

The p53 tumour suppressor governs cell fate by differential transactivation of a spectrum of target genes. To further understand how p53 discriminates between target promoters, we have for the first time used in vitro compartmentalization (IVC) to evolve variants with greater affinity for the distal p53 response element in the promoter of the p21 gene involved in cell-cycle arrest, and for the low affinity BS1 response element of the pro-apoptotic PUMA gene. These variants have mutations in the L1 loop of the p53 DNA binding domain and in the N-terminal proline-rich domain. The in vitro binding phenotype of these variants extends to both increased transactivation of promoters containing the response elements in reporter gene studies and increased up-regulation of endogenous p21 as compared to wild-type p53. One variant was co-selected for increased binding to both response elements yet displayed increased apoptotic function. This result supports the notion that prediction of phenotypic outcome based on transcriptional activation of individual genes is confounded by the networked complexity of the p53 response.

Rational design and biophysical characterization of thioredoxin-based aptamers: insights into peptide grafting.

Peptide aptamers are simple structures, often made up of a single-variable peptide loop constrained within a constant scaffold protein. Aptamers were rationally designed by inserting peptides into a solvent-exposed loop on thioredoxin (Trx). They were designed to interact with the proteins elongation initiation factor 4E (eIF4E) and mouse double minute 2 (MDM2) and were then validated by competitive fluorescence anisotropy experiments. The constructed aptamers interacted with eIF4E and MDM2 with apparent K(d) values of 1.25+/-0.06 microM and 0.09+/-0.01 microM, respectively, as determined by isothermal titration calorimetry (ITC). The MDM2 aptamer (SuperTIP) interacted approximately 2-fold more tightly with MDM2 than the free linear peptide (12.1 peptide), while the eIF4E aptamer elongation initiation factor 4GI-SG interacted approximately 5-fold less strongly than the free linear peptide (elongation initiation factor 4GI). These differences in binding with respect to each aptamer's free peptide reveal that there are more factors involved than just constraining a peptide in a scaffold that lead to tighter binding. ITC studies of aptamer interactions reveal an enthalpic component more favorable than that for the free linear peptides, as well as a larger unfavorable entropic component. These results indicated that stapling of the free peptide in the scaffold increases the favorable enthalpy of the interaction with the target protein. Thermostability studies also revealed that peptide insertion significantly destabilized the Trx scaffold by approximately 27 degrees C. It is this destabilization that leads to an increase in the flexibility of the Trx scaffold, which presumably is lost upon the aptamer's interaction with the target protein and is the cause of the increase in unfavorable entropy in the ITC studies. The precise origin of the enthalpic effect was further studied using molecular dynamics for the MDM2-SuperTIP system, which revealed that there were also favorable electrostatic interactions between the Trx scaffold and the MDM2 protein itself, as well as with the inserted peptide. This work reveals that any increase in the binding affinity of an aptamer over a free peptide is dependent on the increase in the favorable enthalpy of binding, which is ideally caused by stapling of the peptide or by additional interactions between the aptamer protein and its target. These need to be sufficient to compensate for the destabilization of the scaffold by peptide insertion. These observations will be useful in future aptamer designs.

Oligomerization of the human ARF tumor suppressor and its response to oxidative stress.

The tumor suppressor ARF plays an important role as an inhibitor of the Mdm2-mediated degradation of p53. Here we demonstrate that human ARF (p14ARF) can form homo-oligomers. The stability of the oligomers is favored by oxidizing agents in a reversible fashion and involves all three cysteine residues in p14ARF. Furthermore, the effect of p14ARF in clonogenic assays is moderately but reproducibly increased by the mutation of its cysteine residues. We also observed that altering the amino terminus of p14ARF resulted in the appearance of remarkably stable oligomers. This indicates that the amino terminus of p14ARF interferes with the ability of the protein to form multimeric complexes. These observations suggest that p14ARF activity may be linked to its oligomerization status and sensitive to the redox status of the cell.