Investigating the impact of PACAP on thermal stability of G-actin by differential scanning calorimetry

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a bioactive peptide known for its diverse effects on the nervous system. While numerous studies have demonstrated the neuroprotective properties of PACAP, its role in tissue regeneration and potential as a therapeutic agent remain to be fully understood. Specifically, the understanding of PACAP’s impact on cytoskeletal dynamics, particularly the organization and disorganization of actin filament networks, is limited due to the scarcity of in vitro studies in this area. Additionally, the interaction between PACAP and actin has been minimally explored, and the influence of PACAP on the thermal stability of actin is completely unknown. To address these gaps, the current study aimed to investigate the impact of different forms and fragments of PACAP on the thermal denaturation and renaturation of Ca²⁺-G-actin using a differential scanning calorimetry (DSC) approach. Our primary objective was to determine whether PACAP modulates the thermal stability of Ca²⁺-G-actin and establish a temperature-dependent pattern of any structural alterations that may occur as a result of PACAP interaction. Two PACAP forms exist in vivo: the 38 amino-acid length PACAP38 and the PACAP27, the latter truncated at the C-terminal. In the PACAP38 + Ca²⁺-G-actin mixture, the DSC scans exhibited a mild decrease in actin denaturation temperature compared to the control, plus an exotherm appeared in the high-temperature range with a significantly increased calorimetric enthalpy. The truncated PACAP27 produced a slight increase in actin denaturation temperature with the same exotherm without significant alteration in enthalpy. In PACAP6-38 mixture (i.e., an artificial fragment of PACAP38 + Ca²⁺-G-actin), there was no change in the denaturation temperature of actin and no plus exotherm, but significant ΔH_cal was observed. With the modified PACAP6-27 (another artificial fragment), the exotherm was absent, but the actin denaturation temperature and enthalpy increased compared to the control. Through this research, we sought to elucidate the underlying mechanisms of PACAP’s effects on actin dynamics and provide valuable insights into the potential therapeutic applications of PACAP in the context of cytoskeletal organization and neuronal regeneration. The findings of this study may contribute to the development of novel strategies targeting actin-related processes for neuroprotection and neural tissue repair.