Methods development for the identification of lysine acetoacetylation.

(A) Biosynthetic pathways for lysine acetoacetylation (Kacac) and proposed detection methods of Kacac. (B) Dot blot assay verifying the specificity of the pan anti-Kbhb antibody in our developed methods. Synthetic H2BK15acac peptide (Ac-PEPAKSAPAPKKGSKacacKAVTKAQKKDG-NH2) was used for the assay.

Acetoacetate dynamically regulates Kacac levels through the generation of acetoacetyl-CoA.

(A) Western blot analysis of histones from HEK293T cells treated with increasing doses of lithium acetoacetate. (B) Detection of histone Kacac in HCT116 cells. (C) Western blot analysis of histones from HEK293T cells treated with increasing doses of sodium β-hydroxybutyrate. (D) Western blot analysis of histone Kacac in response to treatment with lithium acetoacetate or ketogenic amino acids (leucine and lysine) in HEK293T cells. (E) Western blot analysis of histone Kacac in response to overexpression of ketolysis enzymes (SCOT and AACS) in HEK293T cells. (F) Western blot analysis of histone Kacac in response to treatment with acetohydroxamic acid (AHA), a known SCOT inhibitor, in HEK293T cells.

Identification of writers and eraser responsible for regulating histone Kacac.

(A) p300, PCAF and GCN5 exhibited remarkable acetoacetyltransferase activities (left) and acetyltransferase activities (right) on recombinant histone H3 proteins. (B) Validation of GCN5-mediated Kacac on recombinant histone H3 proteins. (C) Proportional changes of acyl-CoAs result in dynamics of substrates on recombinant histone H3 proteins. (D) Diagram illustrating the catalytic pocket of GCN5 bound with acetyl-CoA (orange) and acetoacetyl-CoA (green). PDB: 5TRL was used for the modeling. (E) Overexpression of HDAC3 abolished acetoacetate-induced Kacac in HEK293T cells.

Proteomic screening of histone Kacac sites in HEK293T cells.

(A) Illustration of histone DKbhb (Kacac) sites identified in HEK293T cells. Green diamond indicates Kacac sites detected in our study. * denotes previously unknown histone Kacac sites. For comparison, the overlapped known Kbhb sites (labeled with an orange dot) described in the literature are also listed. (B) MS/MS spectra of two representative DKbhb peptides derived from HEK293T histones.

Systematic profiling of the Kacac proteome.

(A) Distribution of the Kacac protein based on the site number per protein. (B) The consensus sequence logos show enrichment of amino acid residues among the Kacac sites. (C) Venn diagram shows the cellular compartment distribution of Kacac proteins. (D) Representative ontology annotations and all KEGG pathways enriched within the Kacac proteome. (E) Two protein complexes significantly enriched in the Kacac proteome. The color bar depicts the number of Kacac sites identified in each protein.

Profiling of physiological relevance of Kacac mark in HEK293T cells.

(A) Volcano plot analysis of pairwise comparison of RNA-seq results from HEK293T cells with or without 20 mM acetoacetate treatment. (B) GO (Biological process) enrichment analysis of downregulated (blue) and upregulated (orange) differentially expressed genes (DEGs) after lithium acetoacetate treatment in HEK293T cells, ranked on the basis of adjusted p values. (C) Bubble plots showing the top 10 KEGG pathways enriched in the upregulated (upper) and downregulated (bottom) DEGs after lithium acetoacetate treatment in HEK293T cells, ranked on the basis of adjusted p values and counts. Gradient colors represent enriched significance, and size of circles represents numbers of DEGs. (D) Hallmark gene sets identified by GSEA after lithium acetoacetate treatment in HEK293T cells, ranked on the basis of P values. (E) A graphical model of Kacac. In this model, AACS, not SCOT, is a major player for AcAc-CoA and Kacac generation from acetoacetate.