SIRT1 Top 40 Hits: Use of One-Bead, One-Compound Acetyl-Peptide Libraries and Quantum Dots to Probe Deacetylase Specificity

2005/12/13 by Adam L. Garske, John M. Denu · 21 citations
Medicine · Immunology and Microbiology · Biochemistry, Genetics and Molecular Biology · #Sirtuins and Resveratrol in Medicine #Antimicrobial Peptides and Activities #RNA Interference and Gene Delivery

paper · doi:10.1021/bi052015l

Abstract

A novel, high-throughput method for determining deacetylase substrate specificity was developed using a one-bead, one-compound (OBOC) acetyl-peptide library with a quantum dot tagging strategy and automated bead-sorting. A 5-mer OBOC peptide library of 104,907 unique sequences was constructed around a central epsilon-amino acetylated lysine. The library was screened using the human NAD+-dependent deacetylase SIRT1 for the most efficiently deacetylated peptide sequences. Beads preferentially deacetylated by SIRT1 were biotinylated and labeled with streptavidin-coated quantum dots. After fluorescent bead-sorting, the top 39 brightest beads were sequenced by mass spectrometry. In-solution deacetylase assays on randomly chosen hit and nonhit sequences revealed that hits correlated with increased catalytic activity by as much as 20-fold. We found that SIRT1 can discriminate peptide substrates in a context-dependent fashion.

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