Molecular characterization of propionyllysines in non-histone proteins

Abstract

Lysine propionylation and butyrylation are protein modifications that were recently identified in histones. The molecular components involved in the two protein modification pathways are unknown, hindering further functional studies. Here we report identification of the first three in vivo non-histone protein substrates of lysine propionylation in eukaryotic cells: p53, p300, and CREB-binding protein. We used mass spectrometry to map lysine propionylation sites within these three proteins. We also identified the first two in vivo eukaryotic lysine propionyltransferases, p300 and CREB-binding protein, and the first eukaryotic depropionylase, Sirt1. p300 was able to perform autopropionylation on lysine residues in cells. Our results suggest that lysine propionylation, like lysine acetylation, is a dynamic and regulatory post-translational modification. Based on these observations, it appears that some enzymes are common to the lysine propionylation and lysine acetylation regulatory pathways. Our studies therefore identified first several important players in lysine propionylation pathway.

Fig. 1.

In Vivo propionylation of p53 determined by p300/CBP and Sirt1. A, p300/CBP catalyzes p53 propionylation in vivo. H1299 cells were transfected with the plasmid DNA-expressing FLAG-p53 with or without FLAG-p300 or CBP-HA. The immunoprecipitated proteins by M2 beads were analyzed by Western blotting using anti-propionyllysine pan antibody, anti-p53, and anti-p300 antibodies. CBP in the total cell extracts was detected by anti-HA antibody. B, p300/CBP are specific propionyltransferases in p53 propionylation. The total cell extracts from H1299 cells transfected with the plasmid DNA-expressing p53 and/or various HATs were analyzed by Western blot using anti-propionyllysine and anti-p53 antibodies. C, Sirt1 de-propionylates p53 in vivo. 293T cells were transfected with plasmid DNA-expressing FLAG-p53 with or without Sirt1-V5-His. Indicated transfection cells were treated with HDAC inhibitor mixture for six hours before harvesting cells for immunoprecipitation. Immunoprecipitates were analyzed by Western blotting using anti-propionyllysine and anti-FLAG antibodies. Ectopic expression of Sirt1 was detected by Sirt1 antibody.

Fig. 2.

Mapping lysine propionylation sites in p53 by mass spectrometry. A, purification of p53, p300, and CBP in vivo. H1299 cells were transfected with plasmids DNA encoding FLAG-p53 (lane 1), FLAG-p53 with FLAG-p300 (lane 2), or FLAG-p53 with FLAG-p300 and SIRT1-V5-His (lane 3), or FLAG-p53 with HA-CBP (lane 4), FLAG-p300 (lane 5), and HA-CBP (lane 6), respectively. Twenty-four hours after transfection, cells were lysed and subjected to immunoprecipitation as described in “Materials and Methods”. The purified target proteins were resolved in SDS-PAGE for mapping modification sites of interest. B, in vivo lysine propionylated and butyrylated peptides and sites identified in p53 when it was co-transfected with (+) or without (−) an enzyme. C, MS/MS spectrum of “K^PropGEPHHELPPGSTK^AcR” that identified Lys²⁹² as in vivo lysine propionylation site in p53.

Fig. 3.

p300 and CBP are lysine propionylated in vivo. A, the p300 propionylation was induced in vivo by HDAC inhibitor mixture. 293T cells were transfected to express FLAG-p300. The indicated transfection cells were treated with HDAC inhibitor mixture for six hours before harvesting for immunoprecipitation. The immunoprecipitated proteins were analyzed by Western blotting using anti-propionyllysine, anti-acetyllysin, and anti-FLAG antibodies, respectively. B, the CBP propionylation levels were enhanced in vivo under HDAC inhibitor mixture treatment. 293T cells were transfected to express HA-CBP. The indicated transfection cells were treated with HDAC inhibitor mixture for six hours before cell harvesting for immunoprecipitation. The immuno-isolated proteins were analyzed by Western blot using anti-propionyllysine, anti-acetyllysine, and anti-FLAG antibodies, respectively. C, Sirt1 depropionylates p300 in vivo. 293T cells were transfected with plasmid DNA expressing FLAG-p300 with or without Sirt1-V5-His. The indicated transfection cells were treated with HDAC inhibitor mixture for six hours before the cells were harvested for immunoprecipitation. The isolated proteins were subjected to Western blotting analysis using anti-propionyllysine and anti-FLAG antibodies. Ectopic expression of Sirt1 was detected by Sirt1 antibody.

Fig. 4.

Mapping lysine propionylation sites in p300 by mass spectrometry. In Vivo lysine propionylated and butyrylated peptides and sites identified in p300 (A) and in CBP (B). C, MS/MS spectrum of “NNKAcKPropTSKAcNKAcSSLSR” that identified Lys¹⁵⁵⁵ as in vivo lysine propionylation site in p300.