Histidine and tyrosine residues are targets for SIRT6 ADP-ribosylation activity

SIRT6, a highly conserved member of the sirtuin family, plays a critical role in diverse cellular processes, including gene regulation, DNA damage response and maintaining nuclear lamina integrity. These functions are essential in contexts such as differentiation, metabolic regulation, cancer development and ageing. Given the multifaceted influence of SIRT6 on cellular activities, there is an increasing interest in elucidating the regulatory mechanisms governing its enzymatic functions. SIRT6 exhibits two NAD+-dependent activities: deacetylation and ADP-ribosylation, with current research predominantly focusing on the former. However, the latter—its (ADP-ribosyl)transferase activity—remains underexplored, particularly concerning the specific amino acid targets it modifies and the (ADP-ribosyl)hydrolases that can reverse these modifications. In this study, we have utilized biochemical assays and proteomic techniques to investigate these aspects, revealing that SIRT6 transfers ADP-ribosyl moieties onto histidine and tyrosine residues. In addition, we reveal that the (ADP-ribosyl)hydrolase ARH3 has significant activity in erasing SIRT6-derived ADP-ribosylation in cells.