SIRT6 Lysine‐Demyristoylates ATF2 to Ameliorate Vascular Injury via PRKCD/VE‐Cadherin Pathway Regulating Vascular Endothelial Barrier

Abstract Cardiovascular diseases (CVDs) progression is significantly modulated by epigenetic mechanisms, particularly through Sirtuin 6 (SIRT6), a key NAD⁺‐dependent deacetylase in the sirtuin family. Though essential for cardiovascular homeostasis, the effects of SIRT6‐mediated lysine myristoylation on CVDs progression remain largely unexplored due to detection limitations. This study developes an innovative lysine‐myristoylated peptide enrichment technique, identifying mutant SIRT6 (H133Y) with high myristoyl affinity but deficient demyristoylase activity. This advancement enables identification of 15 previously unrecognized human lysine‐myristoylated proteins. Further study demonstrates that SIRT6 demyristoylates activating transcription factor 2 (ATF2) at K296 and regulates its nucleoplasmic translocation. Through 4D label‐free mass spectrometry and molecular approaches, it is revealed that decreased nuclear localization of ATF2 results in reduced Protein kinase C delta type (PRKCD) expression, establishing a SIRT6/Myr‐ATF2/PRKCD/VE‐Cadherin pathway that enhances endothelial barrier integrity under high myristate conditions. These findings are validated in vitro (gene overexpression/knockdown cells) and in vivo (SIRT6 knockout/double‐transgenic mice). The study provides both a novel method for identifying lysine‐myristoylated proteins and critical insights into SIRT6 demyristoylation biology. Modulating the SIRT6 pathway might yield therapies to strengthen endothelial integrity and mitigate vascular dysfunction in CVDs, offering promising clinical translation avenues.