An acetylome peptide microarray reveals specificities and deacetylation substrates for all human sirtuin isoforms

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Sirtuin enzymes are crucial for regulating metabolism and aging via protein deacetylation.
• Understanding sirtuin isoform-specific functions is limited due to few identified protein targets.
• Over 6,800 mammalian acetylation sites are known, but most sirtuins lack assigned substrates.

Purpose of the Study:

• To develop a high-throughput method for characterizing sirtuin deacetylation activity.
• To identify novel substrates for all seven human sirtuin isoforms.
• To investigate isoform-specific substrate preferences of sirtuins.

Main Methods:

• Development of a peptide microarray displaying 6,802 human acetylation sites.
• Parallel characterization of deacetylation activity for all seven human sirtuins using the microarray.
• Validation of identified substrates through biochemical assays.

Main Results:

• The microarray system enabled parallel deacetylation assays for all human sirtuins.
• Isoform-specific substrate preferences were revealed, identifying candidates for all sirtuins, including Sirt4.
• Malate dehydrogenase confirmed as a Sirt3 substrate; Peroxiredoxin 1 and HMG B1 identified as Sirt5 and Sirt1 substrates, respectively.

Conclusions:

• The developed peptide microarray is a powerful tool for studying sirtuin deacetylation.
• The study provides a valuable resource for identifying novel in vivo sirtuin substrates.
• This work significantly enhances the understanding of sirtuin functions in metabolism and aging.