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Acetylation site specificities of lysine deacetylase inhibitors in human cells

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Abstract

Lysine deacetylases inhibitors (KDACIs) are used in basic research, and many are being investigated in clinical trials for treatment of cancer and other diseases. However, their specificities in cells are incompletely characterized. Here we used quantitative mass spectrometry (MS) to obtain acetylation signatures for 19 different KDACIs, covering all 18 human lysine deacetylases. Most KDACIs increased acetylation of a small, specific subset of the acetylome, including sites on histones and other chromatin-associated proteins. Inhibitor treatment combined with genetic deletion showed that the effects of the pan-sirtuin inhibitor nicotinamide are primarily mediated by SIRT1 inhibition. Furthermore, we confirmed that the effects of tubacin and bufexamac on cytoplasmic proteins result from inhibition of HDAC6. Bufexamac also triggered an HDAC6-independent, hypoxia-like response by stabilizing HIF1-α, providing a possible mechanistic explanation of its adverse, pro-inflammatory effects. Our results offer a systems view of KDACI specificities, providing a framework for studying function of acetylation and deacetylases.

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Figure 1: Quantitative profiling of the KDACI-regulated acetylome.
Figure 2: Specificity of KDACIs and subcellular distribution of KDACI-upregulated acetylated proteins.
Figure 3: Nicotinamide increases acetylation of nuclear proteins.
Figure 4: Tubacin- and bufexamac-mediated increase in protein acetylation is likely mediated by HDAC6.
Figure 5: Bufexamac inhibits KDACs at lower concentrations and causes hypoxia-like responses at higher concentrations.

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Acknowledgements

We thank the members of the department of proteomics at CPR for their helpful discussions. We thank T. Narita for providing help with bioinformatic analyses. We thank R. Lavallee, D. Bekker-Jensen, H.C. Grell and B. Smith for their technical support. We gratefully acknowledge M.W. McBurney for providing SIRT1 knockout MEFs, D. Reinberg for providing SIRT2 knockout MEFs, and R. Mostoslavsky for providing SIRT6 knockout MEFs. This work was supported by the Hallas Møller Investigator grant from the Novo Nordisk Foundation to C.C. S.A.W. and P.B. were supported by individual postdoctoral grants from the Danish Research Council (FSS: 10-085134, FSS: 12-12610). C.C. is supported by the EMBO Young Investigator program. The Center for Protein Research is supported by a generous grant from the Novo Nordisk Foundation (grant no. NNF14CC0001). J.E.B. is supported by a grant from the Doris Duke Charitable Foundation. We thank the PRIDE team for their support with data storage.

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Authors and Affiliations

Authors

Contributions

C.S. performed most of the experiments and collected data. B.T.W. performed initial experiments, and obtained data in MV4-11 cells, S.A.W. helped with bioinformatic analyses, P.B. assisted with immunofluorescence microscopy, Y.M. provided HDAC6 knockout cells, L.J.J. performed average linkage clustering analysis of KDACIs, W.S. performed UV/VIS spectroscopy, J.Q. synthesized JQ12 and performed in vitro KDAC enzymatic assays for this compound, A.R.M., N.J.W. and S.L. provided tenovin-6, J.C. helped with computational analysis of MS data, P.M. provided critical research reagents, M.M. was involved in the planning of the tenovin-6 experiments and provided infrastructure for initial test experiments, J.E.B. designed experiments, provided pandacostat and JQ12, C.C. planned the project, C.C. and C.S. wrote the manuscript; all co-authors provided input for writing the paper.

Corresponding author

Correspondence to Chunaram Choudhary.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–14, Supplementary Table 1 and Supplementary Notes 1 and 2 (PDF 3622 kb)

Supplementary Table 2

All identified acetylation sites (XLSX 6912 kb)

Supplementary Table 3

Comparison of short- and long-term Nicotinamide treatment in HeLa cells (XLSX 498 kb)

Supplementary Table 4

Nicotinamide upregulated acetylation sites (XLSX 552 kb)

Supplementary Table 5

List of acetylation sites quantified in MV4-11 cells treated with nicotinamide (XLSX 397 kb)

Supplementary Table 6

Comparison of Nicotinamide treatment and SIRT1KO in MEF Cells (XLSX 658 kb)

Supplementary Table 7

Comparison of Nicotinamide treatment and SIRT6KO in MEF Cells (XLSX 572 kb)

Supplementary Table 8

Comparison of Sirtinol treatment and SIRT2KO in MEF Cells (XLSX 392 kb)

Supplementary Table 9

Comparison of AGK2 treatment and SIRT2KO in MEF Cells (XLSX 440 kb)

Supplementary Table 10

EX-527 treatment in HeLa cells (XLSX 649 kb)

Supplementary Table 11

Overview of bufexamac and tubacin upregulated acetylome (XLSX 116 kb)

Supplementary Table 12

Comparison of Bufexamac and Tubacin treatment and HDAC6KO in MEF Cells (XLSX 609 kb)

Supplementary Table 13

High and low dose bufexamac proteome (XLSX 1794 kb)

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Schölz, C., Weinert, B., Wagner, S. et al. Acetylation site specificities of lysine deacetylase inhibitors in human cells. Nat Biotechnol 33, 415–423 (2015). https://doi.org/10.1038/nbt.3130

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