Ultradeep Lysine Crotonylome Reveals the Crotonylation Enhancement on Both Histones and Nonhistone Proteins by SAHA Treatment

Lysine crotonylation is a newly discovered protein post-translational modification and was reported to share transferases and deacylases with lysine acetylation. The acetyltransferase p300 was reported to also contain crotonyltransferase activity, and class I histone deacetylases were demonstrated to be the major histone decrotonylases. However, the decrotonylases for nonhistone proteins are unclear. Moreover, because of the lack of high-quality pan-antibodies, large-scale analysis of crotonylome still remains a challenge. In this work, we comprehensively studied lysine crotonylome on both histones and nonhistone proteins upon SAHA treatment and dramatically identified 10 163 lysine crotonylation sites in A549 cells. This is the first identification of tens of thousands of lysine crotonylation sites and also the largest lysine crotonylome data set up to now. Moreover, a parallel-reaction-monitoring-based experiment was performed for validation, which presented highly consistent results with the SILAC experiments. By intensive bioinformatic analysis, it was found that lysine crotonylation participates in a wide range of biological functions and processes. More importantly, it was revealed that both the crotonylation and acetylation levels of most core histones sites and a number of nonhistone proteins as well as some known substrates of class IIa and IIb HDACs were up-regulated after SAHA treatment. These results suggest that SAHA may have decrotonylation inhibitory activities on both histones and nonhistone proteins by inhibiting HDACs

SAHA Treatment Reveals the Link between Histone Lysine Acetylation and Proteome in Nonsmall Cell Lung Cancer A549 Cells

Suberoylanilide hydroxamic acid (SAHA) is a well-known pan HDAC inhibitor, and its clinical application (Vorinostat) has been demonstrated to treat nonsmall cell lung cancer (NSCLS). Nevertheless, the impact of SAHA treatment on histone lysine acetylation and proteome in NSCLS cells still need further elucidate. In NSCLS A549 cells, by using stable isotope labeling for cell culture (SILAC)-based quantitative proteomics, biochemistry assay, and bioinformatic analysis, here we for the first time comprehensively identified and quantified histone lysine acetylation in A549 cells toward SAHA treatment. Despite the fact that SAHA treatment significantly increased histone lysine acetylation in specific sites, unexpectedly, some important “histone markers” showed markedly decreased acetylation level. Further quantitative proteome studies showed that among totally quantifiable 2818 nonredundant proteins, 1355 proteins were with increased level and 1463 with decreased level in response to SAHA treatment. Bioinformatic analysis further revealed that those quantifiable proteins were mainly involved in multiple biological functions and metabolic and enzyme-regulated pathways as well as protein complexes. By establishing the link between histone modification and whole proteome in response to SAHA treatment in NSCLS cells, this study therefore may deepen our understanding of HDAC inhibitor-mediated cancer therapeutics

Ultradeep Lysine Crotonylome Reveals the Crotonylation Enhancement on Both Histones and Nonhistone Proteins by SAHA Treatment

Lysine crotonylation is a newly discovered protein post-translational modification and was reported to share transferases and deacylases with lysine acetylation. The acetyltransferase p300 was reported to also contain crotonyltransferase activity, and class I histone deacetylases were demonstrated to be the major histone decrotonylases. However, the decrotonylases for nonhistone proteins are unclear. Moreover, because of the lack of high-quality pan-antibodies, large-scale analysis of crotonylome still remains a challenge. In this work, we comprehensively studied lysine crotonylome on both histones and nonhistone proteins upon SAHA treatment and dramatically identified 10 163 lysine crotonylation sites in A549 cells. This is the first identification of tens of thousands of lysine crotonylation sites and also the largest lysine crotonylome data set up to now. Moreover, a parallel-reaction-monitoring-based experiment was performed for validation, which presented highly consistent results with the SILAC experiments. By intensive bioinformatic analysis, it was found that lysine crotonylation participates in a wide range of biological functions and processes. More importantly, it was revealed that both the crotonylation and acetylation levels of most core histones sites and a number of nonhistone proteins as well as some known substrates of class IIa and IIb HDACs were up-regulated after SAHA treatment. These results suggest that SAHA may have decrotonylation inhibitory activities on both histones and nonhistone proteins by inhibiting HDACs