The Protein Tyrosine Phosphatase (PTP) family of phosphatases regulates cell signaling through dephosphorylation of serine, threonine and tyrosine residues of downstream effector proteins. The role of PTPs is very well known in normal cell functions, as well as in disease states such as diabetes and cardiovascular diseases. We previously reported on the role of the phosphotyrosine recognition loop (PTBL) of PTP1B in protein-protein interactions (Londhe et al.2020, Nat. Chem. Biol., 16(2):122-125) and showed that interfering with protein-protein interactions between PTP1B and 14-3-3zusing R18, a pan-14-3-3 inhibitor, destabilizes the reversibly oxidized form of PTP1B (PTP1B-OX) and activates the phosphatase. We rationally designed peptides using phosphotyrosine recognition loop of PTP1B to determine whether selective and effective activation of PTPs was possible. We first performed a structural analysis to compare the changes occurring in PTP1B-OX compared to its reduced form. Using this approach, we observed profound conformational changes in the phosphotyrosine recognition loop (PTBL) of PTP1B and rationally designed peptides comprised of the sequence of amino acids of the newly exposed loop, with a TAT (transactivator of transcription) peptide sequence in amino or carboxy-terminal to facilitate cell permeability. Targeting the PTP1B-OX-14-3-3z interaction using R18 and TAT-PTBL peptide led to disruption in protein-protein interaction and maintained PTP1B in its active form.Interestingly, destabilizing the PTP1B-OX form by treating cells with rationally designed PTP1B activator peptides also decreased EGFR phosphorylation at PTP1B sites. In cells stimulated with exogenousH2O2 to bypass the NOX-mediated PTP1B oxidation, the pretreatment with R18 and TAT-PTBL prevented the interaction between PTP1B-OX and 14-3-3z. Then we observed that our TAT-PTBL peptide was not interfering with NOX-mediated reactive oxygen species (ROS) generation, whereas R18, a pan-14-3-3 inhibitor, decreased ROS production significantly. Finally, exposing A431 epidermoid carcinoma cells to PTP1B activator peptides prevented EGFR phosphorylation and impaired colony formation. Collectively our data suggest that the biological activity of PTP1B activator peptides is likely to be therapeutically useful, and could translate into drug leads.
This research was supported by the National Institutes of Health grant no. HL138605 and the American Heart Association grant no. 17GRNT33700265 to B.B. and National Institutes of Health grant no. GM55989 to N.K.T. B.B. is also grateful for support from the following foundations: the Heart and Stroke Foundation of Canada and SUNY Research Foundation.
