Revealing the protein propionylation activity of the histone acetyltransferase MOF (males absent on the first)

Abstract

Short-chain acylation of lysine residues has recently emerged as a group of reversible posttranslational modifications in mammalian cells. The diversity of acylation further broadens the landscape and complexity of the proteome. Identification of regulatory enzymes and effector proteins for lysine acylation is critical to understand functions of these novel modifications at the molecular level. Here, we report that the MYST family of lysine acetyltransferases (KATs) possesses strong propionyltransferase activity both in vitro and in cellulo Particularly, the propionyltransferase activity of MOF, MOZ, and HBO1 is as strong as their acetyltransferase activity. Overexpression of MOF in human embryonic kidney 293T cells induced significantly increased propionylation in multiple histone and non-histone proteins, which shows that the function of MOF goes far beyond its canonical histone H4 lysine 16 acetylation. We also resolved the X-ray co-crystal structure of MOF bound with propionyl-coenzyme A, which provides a direct structural basis for the propionyltransferase activity of the MYST KATs. Our data together define a novel function for the MYST KATs as lysine propionyltransferases and suggest much broader physiological impacts for this family of enzymes.

Keywords: Males absent on the first (MOF); acetyltransferase; crystal structure; lysine propionylation; post-translational modification (PTM); protein acylation; proteomics.

Figure 1.

Dual enzymatic activity of eukaryotic KAT enzymes. The three major eukaryotic KAT families catalyze acetylation of lysine residues using acetyl-CoA as the acetyl donor. p300/CBP and GCN5/PCAF KATs have been reported to possess lysine propionyltransferase activity. In this study, we found that all of the MYST KATs possess strong KPT activity, providing a holistic view of KATs as lysine propionyltransferase.

Figure 2.

Quantification of acetyl- and propionyl-CoA abundance in 293T cells using LC-MS/MS. A, LC-MS chromatograms of acetyl- and propionyl-CoA. The cellular acyl-CoA concentration was calculated based on the deuterated acyl-CoA molecules that serve as internal standards. Acetyl-CoA and propionyl-CoA were sufficiently separated with the retention time at 9.43 and 11.43 min, respectively. B, summary of triplicate experiments. The cellular abundance of propionyl-CoA is about 12% of the abundance of acetyl-CoA.

Figure 3.

Test of lysine propionyltransferase activity of the MYST KATs on cellular proteome with Western blot analysis. HEK293T cell lysate was incubated with individual KATs in the presence or absence of propionyl-CoA. Treatment of cell lysate with propionyl-CoA and MYST KATs, especially MOF and HBO1, induced strong lysine propionylation on multiple histone and non-histone proteins.

Figure 4.

Study of MOF acetyl- and propionyltransferase activity on recombinant histones and nucleosomes. Both recombinant free histones and reconstituted nucleosomes were used as substrates for MOF acetyl- and propionyltransferase activity study. Histone proteins with acyl-CoA were incubated with or without MOF; the reaction mixtures were then subjected to Western blot analysis with pan-anti-acetyllysine and pan-anti-propionyllysine antibodies. A, MOF acylates four free core histones; B, MOF acylates histone H2A/H2B and H4 on nucleosome.

Figure 5.

Imaging of MOF in vitro acetylome and propionylome using radioactive gel assay. Carbon-14–labeled acetyl-CoA and propionyl-CoA were used for cell lysate acylation by MOF. Proteins were resolved on SDS-PAGE, and the MOF acylome was imaged using a PhosphorImager.

Figure 6.

Detection of the cellular acetyltransferase and propionyltransferase activities of MOF. A, MOF was overexpressed in transfected 293T cells compared with the control cells where the vector plasmid was used for transfection. B, histone lysine acylation level was tested with pan-anti-acyllysine antibodies. MOF acylated histone H2A/H2B and H4. C, MOF overexpression induced increase of H4 Lys-16 propionylation. D, acylation of the cellular proteome was tested with Western blotting. Increase of lysine acetylation and propionylation of multiple proteins were induced by MOF overexpression.

Figure 7.

A comparison of lysine propionylation sites between the control and MOF-overexpressed cells. A, the numbers of Kpr_D5-modified sites in histones and non-histone proteins were compared between the control and MOF-overexpressed cells. B, the numbers of overlapped and non-overlapped Kpr_D5 sites between both the control and MOF-overexpressed cells.

Figure 8.

X-ray crystal structure of MOF·propionyl-CoA complex. A, overall structure of MOF·propionyl-CoA. MOF structure is shown in a schematic model, with the N-terminal, central, and C-terminal domains colored in green, magenta, and blue, respectively. The bound compounds are shown in sticks, with propionyl-CoA colored in gray and acetyl-CoA (from PDB entry 2GIV) in yellow. B, active site of MOF. Catalytic residues Cys-316, Glu-350, and auto-acetylated Lys-274 are shown in a stick model. F_o − F_c omit map of propionyl-CoA contoured at 2.5σ shows the density for the extra methyl group in propionyl-CoA. C, structural comparison of the binding sites for propionyl-CoA (PDB entry 5WCI) and acetyl-CoA (PDB entry 2GIV) in MOF. The two structures are superimposed, with MOF·propionyl-CoA in pink and MOF·acetyl-CoA in green. The MOF residues interacting with the compounds are shown in a stick model. Dashed lines represent hydrogen bonds. The two extra interactions in MOF·propionyl-CoA are circled.

Figure 9.

Sequence alignment of MYST KATs. A, MOF Pro-349 is a conservative amino acid residue through all the five eukaryotic MYST KATs; B, MOF Val-314 is a conservative amino acid residue through all of the five eukaryotic MYST KATs.