Characterization and Prediction of Lysine (K)-Acetyl-Transferase Specific Acetylation Sites

Xuegong Zhang,Lei Huang,Tingting Li,Wei-Guo Zhu,Wenlin Li,Ming Lu,Yipeng Du,Likun Wang

doi:10.1074/mcp.m111.011080

Abstract

Lysine acetylation is a well-studied post-translational modification on both histone and nonhistone proteins. More than 2000 acetylated proteins and 4000 lysine acetylation sites have been identified by large scale mass spectrometry or traditional experimental methods. Although over 20 lysine (K)-acetyl-transferases (KATs) have been characterized, which KAT is responsible for a given protein or lysine site acetylation is mostly unknown. In this work, we collected KAT-specific acetylation sites manually and analyzed sequence features surrounding the acetylated lysine of substrates from three main KAT families (CBP/p300, GCN5/PCAF, and the MYST family). We found that each of the three KAT families acetylates lysines with different sequence features. Based on these differences, we developed a computer program, Acetylation Set Enrichment Based method to predict which KAT-families are responsible for acetylation of a given protein or lysine site. Finally, we evaluated the efficiency of our method, and experimentally detected four proteins that were predicted to be acetylated by two KAT families when one representative member of the KAT family is over expressed. We conclude that our approach, combined with more traditional experimental methods, may be useful for identifying KAT families responsible for acetylated substrates proteome-wide.

Highlights

Significant peptides (p value Յ1e-4)Significant peptides (p value Յ1e-3)Significant peptides (p value Յ1e-2)Significant peptides (p value Յ1e-1)proteins that can’t be acetylated after overexpression of p300 or p300/CBP-associated factor (PCAF), there might be several reasons
We developed an Acetylation Set Enrichment Based (ASEB) method to predict acetylated proteins or sites and the KAT families responsible for the acetylation
Using an immunoprecipitation assay combined with Western blotting we evaluated the acetylation of these proteins

Summary

EXPERIMENTAL PROCEDURES

Data Preparation—We collected human proteins acetylated by each of the three families by searching the PubMed literature with key words (supplemental Table S2). Taking advantage of the idea of GSEA, we proposed a new method called ASEB to detect new sites acetylated by a specific KAT family. The smaller the p value, the more significant the chance that the given peptides were acetylated by the KAT family. It did not require the balanced number of positive and negative datasets, as support vector machine did. It directly calculates the similarity between two single peptides. The ASEB method is fit to these cases, because the ES of a given peptide would be significant as soon as it is similar to some but not all peptides in the KAT set. Pan lysine acetylation antibody was used to detect acetylation of immunoprecipitated proteins

RESULTS

Background

DISCUSSION