Identification of the regulatory elements and protein substrates of lysine acetoacetylation
- Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, United States
- Complex Carbohydrate Research Center, University of Georgia, United States
Figures
Figure 1 with 5 supplements
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.
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Figure 1—source data 1
PDF file containing original dot blot for Figure 1B, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/104123/elife-104123-fig1-data1-v1.pdf
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Figure 1—source data 2
Original files for dot blot analysis displayed in Figure 1B.
- https://cdn.elifesciences.org/articles/104123/elife-104123-fig1-data2-v1.zip
Figure 1—figure supplement 1
Scheme of chemical synthesis of the 2,5-dioxopyrrolidin-1-yl 2-(2-methyl-1,3-dioxolan-2-yl)acetate.
Figure 1—figure supplement 2
Mass spectra (top) and 1H NMR spectrum (bottom) of ethyl 2-(2-methyl-1,3-dioxolan-2-yl)acetate.
ESI-HRMS calc for C8H14NaO4 [M+Na]+: 197.0784 found 197.0779 1H NMR (500 MHz, DMSO-d6) δ 3.95 (q, J = 7.1 Hz, 2H), 3.77 (s, 4H), 1.29 (s, 3H), 1.08 (t, J = 7.1 Hz, 3H).
Figure 1—figure supplement 3
Mass spectra (top) and 1H NMR spectrum (bottom) of 2-(2-methyl-1,3-dioxolan-2-yl)acetic acid.
ESI-HRMS calc for C6H10NaO4 [M+Na]+: 169.0471 found 169.0466 1H NMR (500 MHz, DMSO-d6) δ 3.84 (s, 4H), 3.15 (s, 2H), 1.37 (s, 3H).
Figure 1—figure supplement 4
Mass spectra (top) and 1H NMR spectrum (bottom) of 2,5-dioxopyrrolidin-1-yl 2-(2-methyl-1,3-dioxolan-2-yl)acetate.
ESI-HRMS calc for C10H13NNaO6 [M+Na]+: 266.0635 found 266.0628 1H NMR (500 MHz, DMSO-d6) δ 3.86–3.79 (m, 4H), 2.90 (s, 2H), 2.71 (s, 4H), 1.34 (s, 3H).
Figure 1—figure supplement 5
Structure (top) and ESI mass spectra (bottom) of the synthetic K15acac-H2B(1–26) peptide with the deconvoluted spectra below it.
ESI-HRMS calc for C122H210N36O37 [M]+: 2771.5657 found 2771.5587.
Figure 2 with 1 supplement
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.
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Figure 2—source data 1
PDF file containing original western blots for Figure 2, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/104123/elife-104123-fig2-data1-v1.pdf
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Figure 2—source data 2
Original files for western blot analysis displayed in Figure 2.
- https://cdn.elifesciences.org/articles/104123/elife-104123-fig2-data2-v1.zip
Figure 2—figure supplement 1
Dynamic regulation of histone Kacac in vivo.
(A) Time-dependent histone Kacac in HEK293T cells treated with acetoacetate for varying durations. (B) Validation of SCOT and AACS overexpression in HEK293T cells. (C) Validation of AACS overexpression in HepG2 cells. (D) Western blot analysis of histone Kacac in response to AACS and HMG-CoA reductase (HMGCR) overexpression in HepG2 cells. (E) Western blot analysis of histone Kacac in response to treatment with lovastatin, a known HMGCR inhibitor, in HepG2 cells.
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Figure 2—figure supplement 1—source data 1
PDF file containing original western blots for Figure 2—figure supplement 1, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/104123/elife-104123-fig2-figsupp1-data1-v1.pdf
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Figure 2—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/104123/elife-104123-fig2-figsupp1-data2-v1.zip
Figure 3 with 6 supplements
Identification of writers and erasers 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.
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Figure 3—source data 1
PDF file containing original western blots for Figure 3, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/104123/elife-104123-fig3-data1-v1.pdf
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Figure 3—source data 2
Original files for western blot analysis displayed in Figure 3.
- https://cdn.elifesciences.org/articles/104123/elife-104123-fig3-data2-v1.zip
Figure 3—figure supplement 1
Identification of writers responsible for regulating Kacac in vitro.
(A) Testing the HAT identity and purity by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). (B) Testing the HAT activities for transferring the acetyl motif to recombinant histone H4 proteins. (C, D) Screening of HAT activities involved in transferring the acetoacetyl motif to recombinant histone H4 proteins. (E) Validation of p300-mediated Kacac on recombinant histone H3 proteins. (F) Validation of p300, GCN5, and PCAF activities for transferring the acetoacetyl motif to histone extracts from HEK293T cells. (G) Western blot analysis of histone Kacac in response to p300 overexpression in HEK293T cells.
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Figure 3—figure supplement 1—source data 1
PDF file containing original gel or western blots for Figure 3—figure supplement 1, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/104123/elife-104123-fig3-figsupp1-data1-v1.pdf
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Figure 3—figure supplement 1—source data 2
Original files for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) or western blot analysis displayed in Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/104123/elife-104123-fig3-figsupp1-data2-v1.zip
Figure 3—figure supplement 2
p300, GCN5, and PCAF act as acetoacetyltransferases in vitro.
(A) Validation of p300, GCN5, and PCAF activities as acetoacetyltransferases on synthetic histone H3(1–20) peptide. Proportional changes of acyl-CoAs result in dynamics of PCAF-mediated substrates (B) and p300-mediated substrates (C, D) on recombinant histone H3 proteins.
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Figure 3—figure supplement 2—source data 1
PDF file containing original western blots for Figure 3—figure supplement 2, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/104123/elife-104123-fig3-figsupp2-data1-v1.pdf
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Figure 3—figure supplement 2—source data 2
Original files for western blot analysis displayed in Figure 3—figure supplement 2.
- https://cdn.elifesciences.org/articles/104123/elife-104123-fig3-figsupp2-data2-v1.zip
Figure 3—figure supplement 3
Predicted binding modes of GCN5 with acyl-CoAs.
2D diagram indicating the interaction details of GCN5 bound with acetyl-CoA (A) and acetoacetyl-CoA (B). PDB: 5TRL was used for the modeling.
Figure 3—figure supplement 4
Predicted binding modes of PCAF with acyl-CoAs.
(A) 3D diagram illustrating the catalytic pocket of PCAF bound with acetyl-CoA (orange) and acetoacetyl-CoA (green). 2D diagram indicating the interaction details of PCAF bound with acetyl-CoA (B) and acetoacetyl-CoA (C). PDB: 4NSQ was used for the modeling.
Figure 3—figure supplement 5
Predicted binding modes of p300 with acyl-CoAs.
(A) 3D diagram illustrating the catalytic pocket of p300 bound with acetyl-CoA (orange) and acetoacetyl-CoA (green). 2D diagram indicating the interaction details of p300 bound with acetyl-CoA (B) and acetoacetyl-CoA (C). PDB: 5LKU was used for the modeling.
Figure 3—figure supplement 6
Predicted binding modes of HDAC3 with its substrate mimics.
(A) 3D diagram illustrating the catalytic pocket of HDAC3 bound with acetyl-lysine mimic (orange) and acetoacetyl-lysine mimic (green). 2D diagram indicating the interaction details of HDAC3 bound with acetyl-lysine mimic (B) and acetoacetyl-lysine mimic (C). PDB: 4A69 was used for the modeling.
Figure 4
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.
Figure 5 with 1 supplement
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 Kyoto Encyclopedia of Genes and Genomes (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.
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Figure 5—source data 1
Complete list of identified Kacac (DKbhb) sites.
- https://cdn.elifesciences.org/articles/104123/elife-104123-fig5-data1-v1.xlsx
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Figure 5—source data 2
Kacac sites (A) and neighbor sites (B) that are critical for biological functions.
- https://cdn.elifesciences.org/articles/104123/elife-104123-fig5-data2-v1.xlsx
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Figure 5—source data 3
Protein complex analysis of Kacac proteins.
- https://cdn.elifesciences.org/articles/104123/elife-104123-fig5-data3-v1.xlsx
Figure 5—figure supplement 1
Functional annotation of Kacac marks in HEK293T cells.
(A) Protein class enrichment of Kacac proteins, ranked on the basis of raw p values. (B) Heatmap of differentially expressed genes in ‘Control’ and ‘AcAc’ groups in RNA sequencing (RNA-seq) data. ‘Control’ indicates untreated HEK293T cells; ‘AcAc’ indicates lithium acetoacetate treated HEK293T cells.
Figure 6
Profiling of physiological relevance of Kacac mark in HEK293T cells.
(A) Volcano plot analysis of pairwise comparison of RNA sequencing (RNA-seq) results from HEK293T cells with or without 20 mM acetoacetate treatment. (B) Gene ontology (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 Kyoto Encyclopedia of Genes and Genomes (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 gene set enrichment analysis (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.
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Figure 6—source data 1
Differentially expressed genes (DEGs) upon acetoacetate treatment in RNA sequencing (RNA-seq).
- https://cdn.elifesciences.org/articles/104123/elife-104123-fig6-data1-v1.xlsx
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Figure 6—source data 2
Gene ontology (GO) terms identified by using differentially expressed genes (DEGs).
- https://cdn.elifesciences.org/articles/104123/elife-104123-fig6-data2-v1.xlsx
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Figure 6—source data 3
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways identified by using differentially expressed genes (DEGs).
- https://cdn.elifesciences.org/articles/104123/elife-104123-fig6-data3-v1.xlsx
Chemical structure 1
Ethyl 2-(2-methyl-1,3-dioxolan-2-yl)acetate.
Chemical structure 2
2-(2-Methyl-1,3-dioxolan-2-yl)acetic acid.
Chemical structure 3
2,5-Dioxopyrrolidin-1-yl 2-(2-methyl-1,3-dioxolan-2-yl)acetate.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Human) | HEK293T | Gift | N/A | Further STR profiling showed a close match (~80%) to HEK293-derived cell lines (e.g., 293TT), consistent with the known genetic similarity and instability among HEK293 derivatives; no mycoplasma contamination was found. |
| Cell line (Human) | HCT116 | Gift | N/A | Further STR profiling identified the cells as HCT116 (90% match); no mycoplasma contamination was found. |
| Cell line (Human) | HepG2 | ATCC | Cat#: HB-8065, RRID:CVCL_0027 | Further STR profiling identified the cells as HepG2 (100% match); no mycoplasma contamination was found. |
| Transfected construct (Human) | pCMVβ-p300-myc | Addgene | Cat#: 30489 | |
| Transfected construct (Human) | flag-HDAC3 | Addgene | Cat#: 13819 | |
| Transfected construct (Human) | AACS | OriGene Technologies | Cat#: RC206247 | |
| Transfected construct (Human) | SCOT | OriGene Technologies | Cat#: RC203764 | |
| Transfected construct (Human) | HMGCR | Addgene | Cat#: 86085 | |
| Antibody | Pan anti-Kbhb (Rabbit polyclonal) | PTM BioLabs | Cat#: PTM-1201, RRID:AB_2927634 | WB (1:1000) |
| Antibody | Pan anti-Kac (Rabbit polyclonal) | PTM BioLabs | Cat#: PTM-105, RRID:AB_2877698 | WB (1:2000) |
| Antibody | Anti-Flag (Mouse monoclonal) | Thermo Fisher Scientific | Cat#: MA1-91878, RRID:AB_1957945 | WB (1:1000) |
| Antibody | Anti-β-actin (Mouse monoclonal) | Santa Cruz Biotechnology | Cat#: sc-47778, RRID:AB_626632 | WB (1:1000) |
| Antibody | Anti-H3 (Mouse monoclonal) | Santa Cruz Biotechnology | Cat#: sc-517576, RRID:AB_2848194 | WB (1:3000) |
| Antibody | Anti-rabbit IgG HRP-linked antibody | Cell Signaling Technology | Cat#: 7074S, RRID:AB_2099233 | WB (1:3000) |
| Antibody | Anti-mouse IgG HRP-linked antibody | Cytek Biosciences | Cat#: 72-8042, RRID:AB_3750732 | WB (1:3000) |
| Peptide, recombinant protein | Histone H3.1 Human | New England Biolabs | Cat#: M2503 | |
| Peptide, recombinant protein | Histone H4 Human | New England Biolabs | Cat#: M2504 | |
| Peptide, recombinant protein | H3(1–20)/H4(1–20) | This paper | N/A | Described in in vitro HAT activity screening |
| Peptide, recombinant protein | K15acac-H2B(1–26) | This paper | N/A | Described in dot blot assay |
| Commercial assay or kit | ECL Western Blotting Substrate | Thermo Fisher Scientific | Cat#: 32209 | |
| Commercial assay or kit | M-PER Mammalian Protein Extraction Reagent | Thermo Fisher Scientific | Cat#: 78501 | |
| Commercial assay or kit | EpiQuik Total Histone Extraction Kit | Epigentek | Cat#: OP-0006-100 | |
| Commercial assay or kit | RNeasy Plus Mini Kit | QIAGEN | Cat#: 74134 | |
| Commercial assay or kit | Lipofectamine 3000 Transfection Reagent | Thermo Fisher Scientific | Cat#: L3000008 |
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Identification of the regulatory elements and protein substrates of lysine acetoacetylation
eLife 14:RP104123.
https://doi.org/10.7554/eLife.104123.4
