Myocardial
2',3'-cyclic nucleotide 3'-phosphodiesterase (
CNPase) metabolizes a
nucleoside 2',3'-cyclic
phosphate to a
nucleoside 2'-phosphate. Recently, the roles of
CNPase in the pathophysiological processes of
heart failure have emerged. The mitochondrial acylome subjected to
SIRT3 regulation give us comprehensive understanding of acylation modifications to a vast array of
protein targets, and the list of acetylated
mitochondrial proteins is still growing. However, it remains elusive whether
CNPase is subjected to the regulation of acetylation and deacetylation, and the effects of which on
CNPase enzymatic activity are still unknown. In this study, the mitochondrial distribution of
CNPase was identified by immunofluorescence and cytosol/mitochondria fractioning. The immunofluorescence staining pattern of
CNPase and
Sirt3 overlapped on the same focal plane. Moreover,
Sirt3 associates directly with
CNPase, and the
CNPase enzymatic activity was subjected to
Sirt3 activity. Then biochemical methods using
acetic anhydride was employed to acetylate the
CNPase proteins, the enzymatic activity of
CNPase decreased. Furthermore, co-immunoprecipitation coupled mass spectrometry identifies K196, K379, K128 as the main acetylation sites. Molecular dynamic simulation shows that acetylation modification suppressed the
CNPase enzymatic activity through decreasing the opening probability of the binding pocket and restricting substrate accessibility. Together with these findings, this study reveals a molecular mechanism underlying
Sirt3 regulating
CNPase enzymatic activity, and suggests that targeting
CNPase's post-translational modifications represents a promising therapeutic strategy.