Identification of the Regulatory Elements and Protein Substrates of Lysine Acetoacetylation

Reviewer #1 (Public review):

Summary

Lysine acetoacetylation (Kacac) is a recently discovered histone post-translational modification (PTM) connected to ketone body metabolism. This research outlines a chemo-immunological method for detecting Kacac, eliminating the requirement for creating new antibodies. The study demonstrates that acetoacetate acts as the precursor for Kacac, which is catalyzed by the acyltransferases GCN5, p300, and PCAF, and removed by the deacetylase HDAC3. Acetoacetyl-CoA synthetase (AACS) is identified as a central regulator of Kacac levels in cells. A proteomic analysis revealed 139 Kacac sites across 85 human proteins, showing the modification's extensive influence on various cellular functions. Additional bioinformatics and RNA sequencing data suggest a relationship between Kacac and other PTMs, such as lysine β-hydroxybutyrylation (Kbhb), in regulating biological pathways. The findings underscore Kacac's role in histone and non-histone protein regulation, providing a foundation for future research into the roles of ketone bodies in metabolic regulation and disease processes.

Strengths

(1) The study developed an innovative method by using a novel chemo-immunological approach to the detection of lysine acetoacetylation. This provides a reliable method for the detection of specific Kacac using commercially available antibodies.

(2) The research has done a comprehensive proteome analysis to identify unique Kacac sites on 85 human proteins by using proteomic profiling. This detailed landscape of lysine acetoacetylation provides a possible role in cellular processes.

(3) The functional characterization of enzymes explores the activity of acetoacetyltransferase of key enzymes like GCN5, p300, and PCAF. This provides a deeper understanding of their function in cellular regulation and histone modifications.

(4) The impact of acetyl-CoA and acetoacetyl-CoA on histone acetylation provides the differential regulation of acylations in mammalian cells, which contributes to the understanding of metabolic-epigenetic crosstalk.

(5) The study examined acetoacetylation levels and patterns, which involve experiments using treatment with acetohydroxamic acid or lovastatin in combination with lithium acetoacetate, providing insights into the regulation of SCOT and HMGCR activities.

Weakness

(1) There is a limitation to functional validation, related to the work on the biological relevance of identified acetoacetylation sites. Hence, the study requires certain functional validation experiments to provide robust conclusions regarding the functional implications of these modifications on cellular processes and protein function. For example, functional implications of the identified acetoacetylation sites on histone proteins would aid the interpretation of the results.

(2) The authors could have studied acetoacetylation patterns between healthy cells and disease models like cancer cells to investigate potential dysregulation of acetoacetylation in pathological conditions, which could provide insights into their PTM function in disease progression and pathogenesis.

(3) The time-course experiments could be performed following acetoacetate treatment to understand temporal dynamics, which can capture the acetoacetylation kinetic change, thereby providing a mechanistic understanding of the PTM changes and their regulatory mechanisms.

(4) Though the discussion section indeed provides critical analysis of the results in the context of existing literature, further providing insights into acetoacetylation's broader implications in histone modification. However, the study could provide a discussion on the impact of the overlap of other post-translational modifications with Kacac sites with their implications on protein functions.

Impact

The authors successfully identified novel acetoacetylation sites on proteins, expanding the understanding of this post-translational modification. The authors conducted experiments to validate the functional significance of acetoacetylation by studying its impact on histone modifications and cellular functions.

Reviewer #2 (Public review):

In the manuscript by Fu et al., the authors developed a chemo-immunological method for the reliable detection of Kacac, a novel post-translational modification, and demonstrated that acetoacetate and AACS serve as key regulators of cellular Kacac levels. Furthermore, the authors identified the enzymatic addition of the Kacac mark by acyltransferases GCN5, p300, and PCAF, as well as its removal by deacetylase HDAC3. These findings indicate that AACS utilizes acetoacetate to generate acetoacetyl-CoA in the cytosol, which is subsequently transferred into the nucleus for histone Kacac modification. A comprehensive proteomic analysis has identified 139 Kacac sites on 85 human proteins. Bioinformatics analysis of Kacac substrates and RNA-seq data reveals the broad impacts of Kacac on diverse cellular processes and various pathophysiological conditions. This study provides valuable additional insights into the investigation of Kacac and would serve as a helpful resource for future physiological or pathological research.

The following concerns should be addressed:

(1) A detailed explanation is needed for selecting H2B (1-26) K25 sites over other acetylation sites when evaluating the feasibility of the chemo-immunological method.

(2) In Figure 2(B), the addition of acetoacetate and NaBH4 resulted in an increase in Kbhb levels. Specifically, please investigate whether acetoacetylation is primarily mediated by acetoacetyl-CoA and whether acetoacetate can be converted into a precursor of β-hydroxybutyryl (bhb-CoA) within cells. Additional experiments should be included to support these conclusions.

(3) In Figure 2(E), the amount of pan-Kbhb decreased upon acetoacetate treatment when SCOT or AACS was added, whereas this decrease was not observed with NaBH4 treatment. What could be the underlying reason for this phenomenon?

(4) The paper demonstrates that p300, PCAF, and GCN5 exhibit significant acetoacetyltransferase activity and discusses the predicted binding modes of HATs (primarily PCAF and GCN5) with acetoacetyl-CoA. To validate the accuracy of these predicted binding models, it is recommended that the authors design experiments such as constructing and expressing protein mutants, to assess changes in enzymatic activity through western blot analysis.

(5) HDAC3 shows strong de-acetoacetylation activity compared to its de-acetylation activity. Specific experiments should be added to verify the molecular docking results. The use of HPLC is recommended, in order to demonstrate that HDAC3 acts as an eraser of acetoacetylation and to support the above conclusions. If feasible, mutating critical amino acids on HDAC3 (e.g., His134, Cys145) and subsequently analyzing the HDAC3 mutants via HPLC and western blot can further substantiate the findings.

(6) The resolution of the figures needs to be addressed in order to ensure clarity and readability.

Reviewer #3 (Public review):

Summary:

This paper presents a timely and significant contribution to the study of lysine acetoacetylation (Kacac). The authors successfully demonstrate a novel and practical chemo-immunological method using the reducing reagent NaBH4 to transform Kacac into lysine β-hydroxybutyrylation (Kbhb).

Strengths:

This innovative approach enables simultaneous investigation of Kacac and Kbhb, showcasing their potential in advancing our understanding of post-translational modifications and their roles in cellular metabolism and disease.

Weaknesses:

The paper's main weaknesses are the lack of SDS-PAGE analysis to confirm HATs purity and loading consistency, and the absence of cellular validation for the in vitro findings through knockdown experiments. These gaps weaken the evidence supporting the conclusions.