Calcineurin Autoinhibitory Peptide

The Ca(2+)-dependent activation of calcineurin phosphatase activity is regulated by an autoinhibitory element (residues 457-482) located 43 residues COOH-terminal of the calmodulin-binding domain (residues 390-414). Removal of residues 457-482 does not result in full Ca(2+)/calmodulin-independent activity. Full activity in the absence of Ca(2+) requires the removal of residues 420-457. In the present study the presence of additional autoinhibitory elements within residues 420-457 was tested using two calcineurin A subunit COOH-terminal region constructs containing residues 420-511 (AI(420-511)) or 328-511 (AI(328-511)). Using recombinant, Ca(2+)/calmodulin-independent calcineurin, AI(420-511) and AI(328-511) were three- to fourfold more potent inhibitors of calcineurin phosphatase activity than the synthetic calcineurin autoinhibitory peptide(457-482). Calmodulin reversed the inhibition of calcineurin phosphatase activity by AI(328-511) but not AI(420-511). Kinetic studies indicated that AI(420-511) exhibited mixed-type inhibition and that the enzyme/substrate/inhibitor complex is partially active. These results indicate that (i) additional autoinhibitory elements are present within residues 420-457, (ii) calmodulin-binding to the autoinhibitory domain neutralizes the inhibitory function of the 420-457 autoinhibitory segment, (iii) the full-length autoinhibitory domain is a mixed-type inhibitor of calcineurin phosphatase activity, and (iv) the enzyme/substrate/inhibitor complex is partially catalytically active.

In glutamate-mediated excitatory neuronal cell death, immunosuppressants (FK506, Cys-A) are powerful agents that protect neurons from apoptosis. Immunosuppressants inhibit two types of enzyme, calcium/calmodulin-dependent protein phosphatase (calcineurin: CaN), and peptidyl-prolyl cis-trans-isomerase (PPIase) activity such as the FKBP family. In this study, we used a protein transduction approach to determine the functional role of CaN and to produce a potential therapeutic agent for glutamate-mediated neuronal cell death. We created a novel cell-permeable CaN autoinhibitory peptide using the 11 arginine protein transduction domain. This peptide was highly efficient at transducing into primary culture neurons, potently inhibited CaN phosphatase activities, and inhibited glutamate-mediated neuronal cell death. These results showed that CaN plays an important role in excitatory neuronal cell death and cell-permeable CaN autoinhibitory peptide could be a new drug to protect neurons from excitatory neuronal death.

Autoinhibitory domain (AID) of calcineurin (CN) was discovered two decades ago. Fewer investigations are reported to find out shortest possible peptide from the AID for CN inhibition. Hence, this study has focused on screening of nearly 150 peptide fragments derived from the AID using in silico method. Therefore, we have employed docking studies, aiming to analyze the best pose of AID-derived peptides on CN active site. We also analyzed binding free energy (ΔG) of docked complex using molecular mechanics/generalized Born surface area (MM/GBSA). MM/GBSA predicts two short peptides P1 and P2 found to be lowest binding free energy. Two peptides exhibit better binding affinity with CN, suggests that the possible candidates for potential CN inhibition. Further, the stability of the docked complex was analyzed using molecular dynamic (MD) simulation. MD study shows that CNA:P2 is the most stable complex than CN A:P1 and CN A:AID. Besides, we have synthesized and purified P1 and P2 peptides over high performance liquid chromatography (HPLC) found to be 90.31% and 98.93% of purity, respectively. In addition, AID peptides were characterized over mass spectral analysis. Peptides were subjected to CN inhibitory assay using malachite green method. Where, P1 and P2 exhibit CN inhibition better than AID. In particular, shortest peptide P2 shows highest inhibitory activity than AID. Enzyme assay reveals CN inhibitory activity of P2 peptide is consistent within silico results. In silico and in vitro, results corroborated each other to confirm short peptide P2 can be used as a potential CN inhibitor.