PMID: 31422747

Interaction of alpha Carboxyl Terminus 1 Peptide With the Connexin 43 Carboxyl Terminus Preserves Left Ventricular Function After Ischemia-Reperfusion Injury.

Abstract

Background alpha Carboxyl terminus 1 (alphaCT1) is a 25-amino acid therapeutic peptide incorporating the zonula occludens-1 (ZO-1)-binding domain of connexin 43 (Cx43) that is currently in phase 3 clinical testing on chronic wounds. In mice, we reported that alphaCT1 reduced arrhythmias after cardiac injury, accompanied by increases in protein kinase Cepsilon phosphorylation of Cx43 at serine 368. Herein, we characterize detailed molecular mode of action of alphaCT1 in mitigating cardiac ischemia-reperfusion injury. Methods and Results To study alphaCT1-mediated increases in phosphorylation of Cx43 at serine 368, we undertook mass spectrometry of protein kinase Cepsilon phosphorylation assay reactants. This indicated potential interaction between negatively charged residues in the alphaCT1 Asp-Asp-Leu-Glu-Iso sequence and lysines (Lys345, Lys346) in an alpha-helical sequence (helix 2) within the Cx43-CT. In silico modeling provided further support for this interaction, indicating that alphaCT1 may interact with both Cx43 and ZO-1. Using surface plasmon resonance, thermal shift, and phosphorylation assays, we characterized a series of alphaCT1 variants, identifying peptides that interacted with either