Lactylation at lysine 145 fosters KAT8-TIP60 complex formation to promote p53 acetylation at lysine 120 and its pro-apoptotic function

Domain: biology
Published: 2026-03-26
Editorial outcome: major_revision

Abstract
p53 is crucial for cellular functions and disease mechanisms, yet effective clinical strategies targeting it remain challenging. Lactylation has emerged as a key factor in understanding disease pathology and offering therapeutic options. Herein, we identify lactylated KAT8 at lysine 145 (K145) as a modulator of p53 activity. GCN5 and SIRT6 function as the acyltransferase and delactylase for KAT8, respectively. K145 lactylation fosters the formation of KAT8-TIP60 complex, which couples with p53 to facilitate its acetylation at lysine 120 (K120). The KAT8-TIP60 complex promotes K120-acetylated p53 binding to the BAX and PUMA promoters, activating their transcription. Furthermore, we link KAT8 lactylation to doxorubicin-induced cardiotoxicity (DIC), showing that doxorubicin increases K145 lactylation, amplifying p53's pro-apoptotic function and triggering cardiomyocyte apoptosis. Glimepiride, a therapeutic agent for type 2 diabetes, could target KAT8, disrupt its interaction with GCN5, inhibit KAT8 K145 lactylation, and mitigate DIC. These findings provide insight into how KAT8 K145 lactylation modulates p53 activity and contributes to DIC.

Introduction
Post-translational modifications (PTMs) are essential regulators of cellular functions and disease mechanisms1,2. Among them, lactylation, a recently identified PTM, has emerged as a significant form of epigenetic regulation3. Research has highlighted the impact of histone lactylation on transcriptional control, metabolic pathways, and cancer progression4-6. The discovery of lactylation in non-histone proteins has further expanded our understanding of its influence on cellular processes7-9. As research progresses, lactylation is increasingly recognized for its potential to identify disease pathologies, offer prognostic insights, and providing therapeutic options.

Lysine acetyltransferase 8 (KAT8) is a crucial acetyltransferase involved in a variety of cellular processes, such as homeostasis, cell proliferation, apoptosis, differentiation, DNA repair, and immune responses10,11. Given its broad functional relevance, KAT8 has attracted considerable attention, particularly in the context of PTMs that regulate its activity12-15. For instance, Li and colleagues demonstrated that USP10 deubiquitinates KAT8 at lysine 410, preventing its proteasomal degradation12. Additionally, Qiu’s research revealed acetylation of KAT8 at lysines 168 and 175 by GCN5 modulates its interaction with other proteins15. These findings highlight the multifaceted role of KAT8 in cellular processes and underscore the need to explore additional regulatory mechanisms. With the growing recognition of lactylation as a key PTM, we enquired whether KAT8 undergoes lactylation and how this modification influences its function.

Since its discovery in 1979, p53 has been recognized for its essential role in maintaining cellular integrity and regulating various pathological conditions16. As a transcription factor, p53 controls genes involved in numerous cellular processes16-18. Its activity is finely regulated by multiple PTMs, with acetylation being a key modification16. Gu and colleagues first identified p53 acetylation, making it the first non-histone protein known to undergo this modification19. Acetylation enhances p53 stability, promotes binding to low-affinity promoters, and strengthens protein-protein interactions, while also boosting its antiviral function20-22. Moreover, acetylation is crucial for p53’s response to DNA damage and activation of target genes23. Given its pivotal role, further exploration of p53 acetylation is essential for understanding disease mechanisms and developing therapeutic options for p53-related disorders.

In this study, we demonstrate that KAT8 undergoes lactylation at K145, regulated by GCN5 and SIRT6. This modification promotes the formation of the KAT8-TIP60 complex, which interacts with p53 to enhance its acetylation at K120. The KAT8-TIP60 complex facilitates the binding of K120-acetylated p53 to the BAX and PUMA promoters, activating their transcription. Furthermore, increased lactylation of KAT8 at K145 functions as a key driver of doxorubicin-induced cardiotoxicity (DIC). Glimepiride, a clinically used therapeutic agent for type 2 diabetes, could target KAT8, disrupt its interaction with GCN5, inhibit KAT8 K145 lactylation, and alleviate DIC. This work uncovers a fresh mechanism by which KAT8 lactylation at K145 regulates p53 activity and contributes to DIC.

Results
KAT8 undergoes lactylation at lysine 145
Previous studies have identified various PTMs of KAT8, including phosphorylation, ubiquitination, and acetylation12,13,15. However, whether KAT8 undergoes lactylation remains unclear. To address this question, we performed immunoprecipitation of HEK293T cell lysates using a KAT8-specific antibody, followed by immunoblot (IB) with pan-anti-lactylated (Pan-Kla) antibodies that selectively recognize either L- or D-lactyl-lysine. The results demonstrated that endogenous KAT8 predominantly undergoes L-lactylation, but not D-lactylation (Fig. 1A). In addition, reciprocal immunoprecipitation with the Pan-Kla antibodies further confirmed the presence of L-lactylation on endogenous KAT8 (Fig. 1B). Next, we generated KAT8 knockout (KO) cells in HEK293T cells using CRISPR-Cas9 technology (Supplementary Fig. 1A). The KAT8 KO effectively eliminated KAT8 lactylation (Supplementary Fig. 1A), while re-expression of exogenous KAT8 in KO cells successfully restored lactylation (Fig. 1C).

Next, mass spectrometry revealed potential lactylation at lysine 145 (K145) (Fig. 1D) and lysine 410 (K410) on KAT8 (Supplementary Fig. 1B). To determine which lysine serves as the major lactylation site, we generated lysine-to-arginine (K-to-R) mutant to mimic lactylation-deficient conditions (K145R and K410R, respectively). When wild-type (WT) KAT8 and the two mutants (K145R and K410R) were re-expressed in KAT8 KO cells, lactylation levels were significantly reduced in the K145R mutant group compared to WT KAT8, whereas the K410R mutant exhibited only modest effects on KAT8 lactylation (Supplementary Fig. 1C). These findings suggest that K145 is the predominant site of lactylation on KAT8. In addition, the K145 residue of KAT8 and its surrounding residues were highly conserved across different species (Fig. 1E). To facilitate the specific detection of KAT8 K145 lactylation, a custom antibody (referred to hereafter as K145Lac) was generated (Supplementary Fig. 1D-G). Using K145Lac, K145-lactylated KAT8 was readily detected in WT KAT8 but not in the K145R or K145E (lactyl-mimetic glutamate) mutant (Fig. 1F, Supplementary Fig. 1H, I).

Given the link between lactate and lactylation3, we hypothesized that modulating intracellular lactate production would affect KAT8 lactylation. When HEK293T cells were cultured under native conditions with 1 g/L glucose, the intracellular lactate concentration was approximately 5 mM (Fig. 1G), which was sufficient to induce KAT8 lactylation (Fig. 1H). Furthermore, WT KAT8 cells were exposed to higher concentrations of glucose, the primary source of intracellular lactate24. Both lactate production and K145 lactylation of KAT8 were induced by glucose (Fig. 1G, H). Conversely, treatment with 2-deoxy-D-glucose (2-DG), a non-metabolizable glucose analog25, reduced both lactate production (Fig. 1I) and KAT8 lactylation (Fig. 1J). Collectively, these results indicate that KAT8 is predominantly lactylated at K145.

GCN5 serves as the acyltransferase responsible for KAT8 K145 lactylation
Previous research has indicated that lactylation can be facilitated by both enzymatic and non-enzymatic processes8,26. To elucidate the mechanism underlying KAT8 K145 lactylation, we first investigated the involvement of GLO1, which plays a key role in non-enzymatic lactylation26. Results found that altering GLO1 expression, either by overexpression or knockdown, did not significantly influence KAT8 lactylation levels (Supplementary Fig. 2A, B). In contrast, screening of multiple acyltransferases showed that GCN5 silencing notably reduced the endogenous KAT8 K145 lactylation (Fig. 2A, B, Supplementary Fig. 2C, D), whereas GCN5 overexpression markedly elevated it (Fig. 2C, Supplementary Fig. 2E-G). This effect of GCN5 on the lactylation of exogenously expressed KAT8 was similarly observed (Supplementary Fig. 2H, I), indicating that GCN5 predominantly facilitates KAT8 lactylation.

As acyltransferases typically catalyze protein lactylation through protein interactions27, we observed an interaction between endogenous GCN5 and KAT8 (Fig. 2D, Supplementary Fig. 2J), with co-immunoprecipitation (co-IP) confirming this association in exogenously expressed proteins (Fig. 2E, Supplementary Fig. 2K). GST pull-down assays purified GST-GCN5 and KAT8 further validated this direct interaction (Fig. 2F). In vitro lactylation assays provided further evidence for GCN5's role in promoting KAT8 K145 lactylation (Fig. 2G), confirming that GCN5 can lactylate KAT8, whereas this capacity was absent in a catalytically inactive GCN5 mutant (Y621A and F622A) (Fig. 2G). Moreover, GCN5 overexpression did not enhance lactylation of KAT8 in K145R or K145E mutants (Fig. 2H, Supplementary Fig. 2L), and GCN5 also failed to lactylate KAT8 K145R mutant in vitro (Supplementary Fig. 2M).

In addition to its catalytic domain, GCN5 contains a bromodomain (BrD) that is critical for substrate recruitment and lysine acylation28. Building on this structural insight (Fig. 2I), we hypothesized that the BrD in GCN5 plays an essential role in binding to KAT8. To evaluate this hypothesis, we deleted the BrD region (amino acids 727-837, referred to as Del), which abolished the interaction between GCN5 and KAT8 (Fig. 2J, K, Supplementary Fig. 2N). This result clearly demonstrates that the BrD is essential for this binding. Further molecular docking analysis revealed that F753, V757, Y765 and N808 on GCN5 are key residues forming the interaction interface with KAT8 at K145 (Fig. 2I). Site-directed mutagenesis further confirmed that the replacement of amino acids tyrosine 765 (Y765) or asparagine 808 (N808) with alanine in GCN5 significantly disrupted the GCN5-KAT8 interaction (Fig. 2L, M, Supplementary Fig. 2O), suggesting that these amino acids in GCN5 are critical for maintaining the binding to KAT8. More importantly, the ability of GCN5 to lactylate KAT8 at K145 was significantly reduced by the deletion of GCN5 BrD and the mutant version of GCN5 (Y765A or N808A) (Fig. 2N, O, Supplementary Fig. 2P), indicating that the lactyltransferase activity of GCN5 is contingent upon its association with KAT8. Collectively, these findings reinforce the conclusion that GCN5 interacts with KAT8 and facilitates its lactylation at K145.

SIRT6 functions as the delactylase for KAT8 K145 lactylation
Given the dynamic nature of lactylation and delactylation processes8, we aimed to identify potential delactylases of KAT8. Since nicotinamide (NAM), an inhibitor of the sirtuin family29, significantly increased both endogenous and exogenous KAT8 lactylation (Supplementary Fig. 3A, B), we focused on sirtuin family members as potential "eraser" of KAT8 lactylation. Overexpression studies identified SIRT6 as the only sirtuin family member that significantly reduced KAT8 K145 lactylation levels (Supplementary Fig. 3C, D). Moreover, increasing SIRT6 expression resulted in a dose-dependent decrease in endogenous KAT8 K145 lactylation (Supplementary Fig. 3E, F), with similar effects observed for exogenous KAT8 (Supplementary Fig. 3G). Conversely, knockdown of SIRT6 notably elevated endogenous KAT8 K145 lactylation (Supplementary Fig. 3H, I). An in vitro assay confirmed that SIRT6 can delactylate KAT8 (Supplementary Fig. 3J). Furthermore, silencing SIRT6 increased KAT8 lactylation in WT cells but had no effect on K145R mutant (Supplementary Fig. 3K, L). Co-IP experiments demonstrated an interaction between endogenous SIRT6 and KAT8, consistent with similar findings for their exogenous forms (Supplementary Fig. 3M-P). A GST pull-down assay further demonstrated a direct interaction between KAT8 and SIRT6, with KAT8 binding specifically to GST-SIRT6 but not to GST alone (Supplementary Fig. 3Q). Collectively, these results indicate that SIRT6 serves as the primary regulator of KAT8 delactylation.

K145-lactylated KAT8 facilitates p53 acetylation
Protein lactylation can impact protein stability, intracellular localization, and binding affinity30,31. To investigate whether lactylation at K145 influences KAT8 stability, we treated WT, K145R, and K145E cells with cycloheximide (CHX) over various time points and assessed KAT8 expression levels. The absence of significant differences in KAT8 expression among these cell types suggested that K145 lactylation did not affect KAT8 stability (Supplementary Fig. 4A, B). Next, we examined whether lactylation at K145 influences the intracellular localization of KAT8. Consistent with previous findings showing KAT8 in both the cytoplasm and nucleus32,33, our results confirmed its presence in these compartments in WT, K145R, and K145E cells, with a comparable cytoplasm-to-nucleus ratio across all types (Supplementary Fig. 4C). Furthermore, altering lactylation levels by silencing GCN5 or overexpressing SIRT6 did not affect KAT8 distribution (Supplementary Fig. 4D, E), indicating minimal impact of K145 lactylation on KAT8 intracellular localization.

Building on previous findings that identified KAT8 as an acyltransferase for various target proteins34-36, we then determined the effect of lactylation at K145 on the acetylation levels of these targets. A comparison between KAT8 WT and K145R cells revealed that reduced lactylation at K145 resulted in decreased acetylation of several important proteins, including FASN, IRF3, p53, and PAX7 (Fig. 3A). Additionally, enhancing KAT8 lactylation through glucose exposure significantly increased p53 acetylation, with a response more pronounced than that of FASN, IRF3, and PAX7 (Fig. 3B). To determine more specifically if KAT8 K145 lactylation regulates p53 acetylation, we knocked out p53 in KAT8 WT, K145R, and K145E cells (Supplementary Fig. 4F) and reintroduced it using a p53-Flag plasmid. Our results showed a significant decrease in acetylation of exogenously expressed p53 in K145R cells compared to WT, while K145E cells showed a modest increase (Fig. 3C, Supplementary Fig. 4G). A similar pattern was observed with endogenous p53 acetylation in relation to KAT8 K145 lactylation (Fig. 3D, Supplementary Fig. 4H). Additionally, p53 preferentially interacted with WT KAT8 compared to the K145R mutant (Supplementary Fig. 4I, J). These findings prompted us to focus on the effects of KAT8 K145 lactylation on p53 acetylation in subsequent experiments.

Given that GCN5 serves as the lactyltransferase responsible for lactylating KAT8 at K145, we aimed to investigate how alterations in GCN5 expression levels affect p53 acetylation. Our results indicated that in KAT8 WT cells, increased GCN5 expression led to a dose-dependent rise in endogenous p53 acetylation (Fig. 3E, Supplementary Fig. 5A). Notably, this increase was not observed in KAT8 K145R mutant cells (Fig. 3F, Supplementary Fig. 5B). Similarly, GCN5 upregulation in KAT8 WT cells enhanced the acetylation of exogenous p53 (Fig. 3G, Supplementary Fig. 5C), while this effect was absent in KAT8 K145R cells (Supplementary Fig. 5D). Furthermore, silencing GCN5 resulted in reduced p53 acetylation specifically in KAT8 WT cells (Fig. 3H and Supplementary Fig. 5E-G), with no changes noted in K145R cells (Supplementary Fig. 5H, I). These findings indicate that the influence of GCN5 on p53 acetylation is contingent upon the lactylation status of KAT8 at K145.

In a parallel investigation, we explored the role of SIRT6, which acts as the delactylase for KAT8 at K145, in modulating p53 acetylation. Our experiments demonstrated that SIRT6 overexpression in KAT8 WT cells led to a dose-dependent decrease in endogenous p53 acetylation (Fig. 3I, Supplementary Fig. 6A), a trend not observed in K145R cells (Supplementary Fig. 6B, C). Additionally, SIRT6 upregulation similarly reduced exogenous p53 acetylation in KAT8 WT cells (Fig. 3J, Supplementary Fig. 6D), while no such reduction was evident in K145R cells (Supplementary Fig. 6E). In contrast, when SIRT6 was silenced in KAT8 WT cells, we observed an increase in both endogenous (Fig. 3K, Supplementary Fig.6F) and exogenous (Fig. 3L, Supplementary Fig. 6G) p53 acetylation, with no effect seen in K145R cells (Supplementary Fig. 6H, I). Collectively, these results suggest that p53 acetylation is intricately regulated by the dynamic processes of lactylation and delactylation of KAT8 at K145.

K145-lactylated KAT8 preferentially interacts with TIP60 to acetylate p53
Next, we aimed to elucidate how KAT8 K145 lactylation promotes p53 acetylation. An analysis of the KAT8 structure revealed that K145 is located distally from the MYST domain, which contains the acetyl-CoA-binding site37 (Fig. 4A, B). This spatial separation suggests that K145 lactylation is unlikely to directly influence the acetyltransferase activity of KAT8. Consistent with its known role as a histone H4 acetyltransferase35, WT KAT8 acetylated histone H4 in vitro (Supplementary Fig. 7A). Notably, the K145R mutant displayed enzymatic activity comparable to WT KAT8 on histone H4 (Supplementary Fig. 7A). This finding indicates that KAT8 lactylation may influence the acetylation status of p53 through mechanisms independent of its acyltransferase function. Further supporting this hypothesis, experiments in KAT8 KO cells reintroduced with a catalytically inactive KAT8 mutant (C316S/E350Q) revealed that glucose-induced lactylation of KAT8 at K145 could lead to increased p53 acetylation (Fig. 4C, Supplementary Fig. 7B). This effect closely mirrored the outcomes seen when WT KAT8 was reintroduced in KAT8 KO cells (Fig. 4D, Supplementary Fig. 7C), suggesting a similar mechanism of action. Consequently, we next aimed to explore alternative mechanisms through which KAT8 K145 lactylation might promote p53 acetylation.

Considering that K145 lactylation of KAT8 occurs on a surface accessible to binding partners (Fig. 4B), we next focused on potential interactions with p53 regulatory proteins that may modulate its acetylation dynamics. Based on this insight, we investigated the interaction between KAT8 and several proteins involved in the acetylation and deacetylation processes of p53, including TIP60, p300, SIRT1 and HDAC119,38-40. In comparing KAT8 WT and K145R cells, we observed that blocking KAT8 lactylation at K145 led to reduced interaction between KAT8 and endogenous TIP60 (Fig. 4E, Supplementary Fig. 7D), the acetyltransferase responsible for p53 acetylation. Additionally, K145 delactylation diminished the binding of KAT8 to exogenously express TIP60, while the K145E mutant maintained its ability to interact with TIP60 (Fig. 4F, Supplementary Fig. 7E). Further GST pull-down assays showed that WT KAT8 purified from HEK293F cells preferentially binds TIP60 (Fig. 4G), and this interaction was independently validated by biolayer interferometry (BLI) (Fig. 4H). The preferential binding observed with WT KAT8 compared to its K145R mutant underscores the significance of K145 lactylation in promoting this interaction.

Notably, increasing KAT8 K145 lactylation levels through GCN5 overexpression or SIRT6 silencing enhanced the interaction between KAT8 and TIP60 (Fig. 4I, J, Supplementary Fig. 7F, G), whereas the KAT8 K145R mutant showed no change in this interaction (Supplementary Fig. 7H, I). Conversely, reducing KAT8 K145 lactylation via GCN5 silencing or SIRT6 overexpression weakened the interaction with TIP60 (Fig. 4K, L, Supplementary Fig. 7J, K), again with no effect on the K145R mutant (Supplementary Fig. 7L, M). To directly test whether K145 lactylation enhances TIP60 binding, we performed an in vitro peptide pull-down assay. Biotin-labeled peptides encompassing the K145 residue of KAT8 were synthesized in unmodified (K), K145-lactylated (Kla), or K145-acetylated (Kac) forms. Strikingly, the Kla peptide, but not the K or Kac controls, pulled down substantially more TIP60 protein (Supplementary Fig. 7N). These findings provide direct evidence that K145 lactylation is sufficient and specific for enhancing the KAT8-TIP60 interaction.

Previous studies have demonstrated that TIP60 can form complexes with other proteins, such as TRRAP41 and p40042. The next question we addressed was whether all TIP60 proteins within the cells were associated with KAT8. Our findings revealed that endogenous p400 and TRRAP did not interact with either WT KAT8 or its K145R and K145E mutants (Supplementary Fig. 8A), although they did interact with TIP60 (Supplementary Fig. S8B). This suggests that the TIP60 protein associated with KAT8 is not part of the classical TIP60 complexes. Further investigation revealed that overexpression of TRRAP or p400 increased the amount of these proteins immunoprecipitated by TIP60 (Supplementary Fig. 8C, D). However, this overexpression disrupted the interaction between KAT8 and TIP60 (Supplementary Fig. 8C, D). In contrast, glucose-induced lactylation of KAT8 at K145 enhanced its binding to TIP60, while simultaneously reducing the interaction between TIP60 and TRRAP/p400 (Supplementary Fig. 8E). Additional analysis using sucrose gradient fractionation revealed TIP60 cosedimented with p400 and TRRAP, but a significant portion of TIP60 also cosedimented with K145-lactylated KAT8 (Supplementary Fig. 8F). Notably, K145-lactylated KAT8 and p400/TRRAP were largely separated into distinct fractions (Supplementary Fig. 8F), suggesting that KAT8/TIP60 complexes and the classical TIP60 complexes are mutually exclusive. When lactylation of KAT8 at K145 was blocked, TIP60 was predominantly found in the high-molecular-weight fraction, where it co-migrated with p400 and TRRAP (Supplementary Fig. 8G). In contrast, its presence in the low-molecular-weight fraction was dramatically reduced (Supplementary Fig. 8G). These findings are consistent with the observation that blocking KAT8 K145 lactylation favors the formation of the classical TIP60 complex (Supplementary Fig. 8H). Taken together, these findings suggest that KAT8 K145 lactylation leads to the formation of a distinct TIP60 complex, separate from the classical TIP60 complexes.

Given the preferential binding of p53 to WT KAT8 compared to the K145R mutant (Supplementary Fig. 4I, J), we explored whether p53 associates with the KAT8-TIP60 complex. Under normal salt conditions, KAT8, TIP60, and p53 co-immunoprecipitated, indicating their association within a shared complex (Fig. 5A). In contrast, under high salt conditions, the interactions between KAT8-p53 and TIP60-p53 could not be detected, while the KAT8-TIP60 interaction persisted (Fig. 5A). This KAT8-TIP60 interaction was completely abolished when KAT8 was replaced by the K145R mutant (Fig. 5B). These results indicate that TIP60 and KAT8 are tightly associated, and that K145 lactylation promotes their association. To further explore their interactions, we examined the impact of gene silencing. First, silencing KAT8 had minimal effects on the overall levels of TIP60 and p53, but as expected reduced the levels of K145-lactylated KAT8, and weakened the interaction between TIP60 and p53 (Fig. 5C, Supplementary Fig. 9A). This interaction was also decreased in cells expressing K145R mutant KAT8 compared to WT KAT8 (Fig. 5D, Supplementary Fig. 9B). Next, p53 knockdown did not alter the KAT8-TIP60 interaction or the overall levels of KAT8, K145-lactylated KAT8, and TIP60 (Fig. 5E, Supplementary Fig. 9C). Furthermore, in cells expressing WT KAT8 (Fig. 5F, Supplementary Fig. 9D), but not the K145R mutant (Supplementary Fig. 9E), silencing TIP60 weakened the interaction between KAT8 and p53, despite unchanged levels of KAT8, K145-lactylated KAT8, and p53. Additionally, p53 retained a similar ability to bind KAT8, regardless of its mutation status (WT, K145R, or K145E) in TIP60 KO cells (Supplementary Fig. 9F). These findings suggest that KAT8, TIP60, and p53 are independent of each other for stability, while TIP60 facilitates the preferential binding of p53 to WT KAT8 over the K145R mutant. Further supporting this, GST pull-down assays showed comparable binding of p53 to WT and K145R KAT8 (Fig. 5G). Interestingly, the addition of TIP60 significantly strengthened the interaction between p53 and WT KAT8, an effect not observed with K145R KAT8 (Fig. 5G). Overall, these findings specifically emphasize TIP60's role in mediating the effects of KAT8 K145 lactylation on p53.

Subsequently, we aimed to further clarify TIP60's role in mediating the effects of KAT8 K145 lactylation on p53. Results found that TIP60 overexpression significantly enhanced p53 acetylation, while its knockdown reduced acetylation levels (Fig. 5H, I). This effect was more pronounced in cells expressing WT KAT8 compared to the K145R mutant (Fig. 5H, I). Consistently, overexpression of WT KAT8 markedly promoted p53 acetylation, whereas the K145R mutant failed to produce a similar effect (Fig. 5J). In TIP60 KO cells, overexpressing either WT or K145R KAT8 failed to enhance p53 acetylation (Supplementary Fig. 9G), indicating that the interaction between KAT8 and TIP60 and KAT8 is crucial for p53 acetylation. Further supporting this, in KAT8 KO cells, reintroducing KAT8 increased p53 acetylation (Supplementary Fig. 9H, I), but this effect was significantly reduced when TIP60 was silenced (Supplementary Fig. 9H), while TIP60 overexpression further enhanced the acetylation of p53 (Supplementary Fig. 9I). Additionally, both recombinant and immunopurified TIP60 were able to directly and efficiently acetylate p53 (Fig. 5K). Immunoprecipitated WT KAT8, but not the K145R mutant, further boosted TIP60-mediated p53 acetylation (Fig. 5K, L). Notably, the interaction between TIP60 and KAT8 remained when the acetyltransferase activity of TIP60 was inactive (Q377E/G380E) (Supplementary Fig. 9J); however, catalytically inactive TIP60 mutant could not induce p53 acetylation (Fig. 5M, Supplementary Fig. 9J). Together, these findings demonstrate a relationship between K145 lactylation and the enhanced formation of the KAT8-TIP60 complex, which couples with p53 to facilitate its acetylation.

K145-lactylated KAT8 enhances BAX and PUMA transcription by promoting p53 acetylation at K120
Different acetylation sites on p53 are known to regulate distinct functions43. The next question we addressed was which specific acetylation site on p53 is affected by KAT8 lactylation at K145. Given previous findings showing that TIP60 acetylates p53 at K12044, we determined whether KAT8 lactylation at K145 influenced acetylation of p53 at K120. Our results revealed a significant increase in acetylation at p53 K120 in KAT8 WT cells compared to KAT8 K145R mutant cells (Fig. 6A). In contrast, acetylation levels at other p53 sites remained unchanged (Supplementary Fig. 10A). Furthermore, upregulation of GCN5 in KAT8 WT cells enhanced p53 K120 acetylation (Fig. 3E), while this effect was absent in KAT8 K145R (Fig. 3F) or TIP60 KO cells (Supplementary Fig. 10B). Similarly, downregulation of SIRT6 in KAT8 WT cells resulted in increased p53 K120 acetylation (Fig. 3K), but altering SIRT6 expression in KAT8 K145R (Supplementary Fig. 6H) or TIP60 KO (Supplementary Fig. 10C) cells did not produce significant changes. Collectively, these results indicate that KAT8 lactylation at K145 specifically promotes acetylation of p53 at K120.

Given the importance of acetylation at p53 K120, located within its DNA-binding domain (DBD), for p53-mediated transcription, we further explored the implications of our findings. Specifically, K120 acetylation version of p53 selectively functions at pro-apoptotic p53 target genes including BAX and PUMA35. Based on this understanding, we hypothesized that lactylation of KAT8 at K145 facilitated the transcription of BAX and PUMA by increasing the acetylation of p53 at K120. To test this hypothesis, chromatin immunoprecipitation (ChIP) assays were done to investigate p53 binding to the promoters of BAX and PUMA in both KAT8 WT and KAT8 K145R cells. The results revealed a significant increase in p53 binding affinity to the BAX and PUMA promoters in KAT8 WT cells compared to KAT8 K145R cells (Fig. 6B, C). Further supporting our hypothesis, upregulation of GCN5 in KAT8 WT cells enhanced p53 binding to these promoters (Fig. 6D, E). In contrast, in KAT8 K145R or TIP60 KO cells, GCN5 upregulation did not affect p53 binding (Supplementary Fig. 10D, E). Additionally, we found that downregulation of SIRT6 in KAT8 WT cells increased p53 binding to the BAX and PUMA promoters (Fig. 6F, G), while altering SIRT6 expression in KAT8 K145R or TIP60 KO cells had no significant effect (Supplementary Fig. 10F, G). Collectively, these findings underscore the crucial role of KAT8 lactylation at K145 in regulating p53's acetylation and its subsequent transcriptional activity.

To further investigate the biological implications of KAT8 K145 lactylation, we evaluated the expression levels of BAX and PUMA under various conditions. Our results demonstrated that both mRNA and protein levels of BAX and PUMA were significantly higher in KAT8 WT cells than in KAT8 K145R cells (Fig. 6H-J). Additionally, in KAT8 WT cells, increasing GCN5 expression led to elevated levels of both BAX and PUMA (Fig. 6K-M), while this upregulation did not affect their expression in KAT8 K145R or TIP60 KO cells (Supplementary Fig. 10H-K). Conversely, downregulating SIRT6 in KAT8 WT cells resulted in increased mRNA and protein levels of BAX and PUMA (Fig. 6N-P), an effect absent in KAT8 K145R or TIP60 KO cells (Supplementary Fig. 10L-O). Moreover, no notable differences were found in mRNA stability (Supplementary Fig. 10P), translation efficiency (Supplementary Fig. 10Q), or protein half-life (Supplementary Fig. 10R) for BAX and PUMA between the two cell types. Overall, these findings suggest that KAT8 lactylation at K145 enhances the transcriptional expression of BAX and PUMA.

Since p53 associates with the KAT8-TIP60 complex, we investigated whether this complex contributes to p53's acetylation-mediated transactivation of BAX and PUMA. To address this, we first performed ChIP assays on cells expressing either WT KAT8 or the K145R mutant, using TIP60-specific and Myc-tagged KAT8 antibodies. In cells expressing the K145R mutant form of KAT8, the BAX and PUMA promoters exhibited minimal enrichment on KAT8 and TIP60 (Fig. 6Q, R). Conversely, in cells expressing the WT form of KAT8, these promoters showed substantial enrichment on both TIP60 and KAT8 (Fig. 6Q, R). Further DNA pull-down assays revealed the recruitment of KAT8 and TIP60, along with K120-acetylated p53, to the promoter regions of BAX and PUMA in cells expressing WT KAT8 (Fig. 6S). Notably, the KAT8 protein at the BAX and PUMA promoters was identified as the K145 lactylated form (Fig. 6S). Conversely, this accumulation was absent in cells expressing the K145R mutant form of KAT8 (Fig. 6S). Consistent with previous reports35, p53 directly bound to the BAX and PUMA promoters (Fig. 6T). Interestingly, neither KAT8 nor TIP60 alone formed detectable complexes with these promoters (Fig. 6T). However, their combined presence significantly enhanced p53's DNA-binding activity at these sites (Fig. 6U), an effect that was absent in the presence of the K145R mutant of KAT8 (Fig. 6U). Collectively, these findings demonstrate that the KAT8-TIP60 complex facilitates p53 binding to the promoters of BAX and PUMA, thereby promoting its transcription activation.

KAT8 K145 lactylation mediates doxorubicin-induced cardiotoxicity
Previous research has demonstrated that the p53-mediated transcription of BAX and PUMA is critical in DIC45,46. This led us to hypothesize that KAT8 lactylation at K145, which enhances p53 acetylation at K120, may play a significant role in DIC. To investigate this hypothesis, we treated Ac16 cells with DOX, resulting in a significant increase in apoptotic cells (Fig. 7A). Concurrently, we observed a marked increase in KAT8 K145 lactylation, accompanied by increased interaction between KAT and TIP60, elevated acetylation of p53 at K120, and higher levels of BAX and PUMA (Fig. 7B). To further explore the implications of KAT8 lactylation, we reintroduced WT KAT8 and K145R mutant into KAT8 KO Ac16 cells. The results indicated that blocking KAT8 lactylation at K145 significantly reduce DOX-induced apoptosis (Fig. 7C), which was accompanied by a reduced interaction between KAT8 and TIP60, a substantial decrease in p53 K120 acetylation and lower levels of BAX and PUMA proteins (Fig. 7D). Collectively, these findings suggest that KAT8 lactylation at K145 may contribute to DIC by modulating p53 activity and its target genes.

Building upon these findings, we further investigated the contribution of KAT8 K145 lactylation to DIC by examining the effects of GCN5 overexpression and SIRT6 silencing. The results revealed that overexpression of GCN5 in KAT8 WT cells significantly promoted DOX-induced apoptosis (Supplementary Fig. 11A), accompanied by increased KAT8 K145 lactylation, elevated p53 K120 acetylation, and higher levels of BAX and PUMA Supplementary Fig. 11B). In contrast, GCN5 overexpression did not influence DOX-induced apoptosis or protein expression in KAT8 K145R (Supplementary Fig. 11C, D) or TIP60 KO cells (Supplementary Fig. 11E, F). Similarly, silencing SIRT6 in KAT8 WT cells led to increased DOX-induced apoptosis (Supplementary Fig. 11G), along with higher levels of KAT8 K145 lactylation, p53 K120 acetylation, and BAX and PUMA (Supplementary Fig. 11H). However, silencing SIRT6 did not affect DOX-induced apoptosis in KAT8 K145R or TIP60 KO cells, nor did it impact the levels of KAT8 K145 lactylation, p53 K120 acetylation, BAX, and PUMA (Supplementary Fig. 11I-L). To further validate our findings, we conducted in vivo experiments that confirmed significant increases in KAT8 K145 lactylation, p53 K120 acetylation, and BAX and PUMA levels in mice treated with DOX (Supplementary Fig. 11M). Additionally, mice treated with DOX exhibited significantly elevated plasma levels of cTnT and CK-MB compared to saline-treated controls, indicating increased cardiac injury (Supplementary Fig. 11N, O). To further investigate cardiotoxicity, cardiac atrophy was assessed as another critical phenotype47. Compared to saline-treated mice, DOX-treated mice demonstrated significantly reduced heart size and weight (Supplementary Fig. 11P, Q) and increased cardiomyocyte apoptosis (Supplementary Fig. 11R). These findings underscore the critical role of KAT8 K145 lactylation in the mechanisms underlying DIC.

An inhibitor blocking KAT8 K145 lactylation attenuates DIC
Given the critical role of KAT8 K145 lactylation in the development of DIC, it presents a promising target for drug discovery. To explore this potential, we conducted a virtual screening of small molecules designed to target KAT8 K145. From an initial pool of over 4,000 compounds in the DrugBank, we performed docking studies at the K145 site of KAT8 and selected the five compounds with the highest docking scores for further evaluation (Fig. 7E, Supplementary Fig. 11T-V). We subsequently tested these compounds for their effects on KAT8 K145 lactylation. Among the compounds evaluated, glimepiride (GM), a therapeutic agent used clinically for type 2 diabetes, was found to significantly inhibit KAT8 K145 lactylation without affecting overall KAT8 protein levels (Fig. 7F, Supplementary Fig. 11W). We next investigated the mechanism by which GM inhibits KAT8 K145 lactylation. Considering that GCN5 interacts with KAT8 and lactylates KAT8 at K145, we hypothesized that if the docking simulation between GM and KAT8 was accurate, GM could compete with GCN5 for binding to KAT8, thereby inhibiting KAT8 K145 lactylation. Supporting this hypothesis, BLI analysis showed that GM bound to KAT8 with a dissociation constant (KD) of 2.03±0.11 nM (Fig. 7G), which was significantly stronger than the GCN5-KAT8 interaction (KD = 13.97±0.05 nM) (Fig. 7H). In line with this, endogenous co-IP experiments revealed that GM disrupted the GCN5-KAT8 interaction (Fig. 7I), and GST pull-down assays confirmed that pre-incubation with GM reduced the binding affinity between KAT8 and GCN5 (Fig. 7J). Consistent with GM competing with GCN5 for KAT8 binding, GM significantly reversed the GCN5-induced increase in KAT8 K145 lactylation and concomitantly reduced the interaction between TIP60 and K145-lactylated KAT8 (Fig. 7K). Given the central role of KAT8 in catalyzing H4K16 acetylation35, it was critical to rule out the possibility that GM’s anti-apoptotic effect was mediated through an inhibition of this function. Our data show that GM treatment did not alter global H4K16 acetylation levels (Fig. 7L). Together, these findings demonstrate that GM does not act as a general inhibitor of KAT8's acetyltransferase activity, and establish that its pro-survival effect is independent of H4K16 acetylation. Collectively, these results indicate that GM inhibits KAT8 K145 lactylation by blocking the GCN5-KAT8 association, functioning like “molecular scissors,” a class of small molecules that break or disassemble protein-protein interactions.

Based on these findings, we further assessed the role of GM in DIC. To address this, Ac16 cells were treated with GM in the presence of DOX. As expected, GM significantly reduced the K145 lactylation of endogenous KAT8 (Supplementary Fig. 11W) and effectively prevented DOX-induced apoptosis (Fig. 7M). Additionally, cells treated with GM showed lower levels of p53 K120 acetylation, BAX, and PUMA (Supplementary Fig. 11W). Next, we explored the potential of GM to mitigate DIC in vivo. Mice treated with GM showed markedly reduced plasma levels of cTnT and CK-MB compared to DOX-treated mice (Fig. 7N, O), suggesting a strong cardioprotective effect. GM treatment significantly increased heart size and weight (Fig. 7P, Q) and decreased cardiomyocyte apoptosis (Fig. 7R). These findings indicate that GM effectively mitigates DOX-induced apoptosis and cardiotoxicity in a mouse model, highlighting its potential therapeutic value in preventing DIC. Moreover, following GM treatment, levels of KAT8 K145 lactylation, p53 K120 acetylation, BAX, and PUMA in cardiac tissues were significantly reduced (Fig. 7S). To determine whether GM affects the anti-tumor activity of DOX, we employed a syngeneic tumor model using 4T1 murine breast cancer cells in C57BL/6J mice. Mice were randomized into three groups and treated with DMSO, DOX alone, or the combination of DOX and GM. As expected, DOX treatment markedly suppressed tumor growth compared with DMSO (Supplementary Fig. 11X). Importantly, the addition of GM did not attenuate this tumor-suppressive effect, as tumor volumes in the DOX+GM group were comparable to those in the DOX-only group (Supplementary Fig. 11Y, Z). Thus, we concluded that GM preserves the anti-tumor efficacy of DOX while providing cardioprotection. Overall, these findings indicate that increased lactylation of KAT8 at K145 serves as a key driver of DIC, and its inhibition effectively prevents this condition, underscoring its potential as a therapeutic target.

Methods
Cell culture and treatment
Human embryonic kidney (HEK293T) and human cardiomyocyte (Ac16) cell lines were sourced from the American Type Culture Collection and cultured in Dulbecco's Modified Eagle Medium (DMEM) or DMEM/F-12 medium supplemented with 10% fetal bovine serum, maintained at 37°C with 5% CO₂ in a humidified incubator. All cell lines were authenticated via short tandem repeat fingerprinting and tested negative for mycoplasma contamination prior to experimentation. Ac16 cells were treated by DMSO, DOX (3 μM), DOX+Glimepiride (0.5 mM), Dihydroegotamine (10 μM), Naldemedine (30 μM), Glimepiride (0.5 mM), 3'-demethyletoposide (0.1 μM) and Digoxigenin bisdigitoxoside (15 μM) for 24h, then the cells were harvested for further experiments.

Generation of knockout cell lines using CRISPR-Cas9
Cells with KAT8, p53, or TIP60 knockout were generated using CRISPR-Cas9 technology. . After the KAT8, p53, or TIP60 Cas9/sgRNA vector was constructed, they were transfected using Lipofectamine 3000 for 24 hours. Afterwards, cells were selected with puromycin for 36 hours. Surviving cells were then diluted and seeded into 96-well plates for single-cell cloning to obtain colonies derived from individual cells. Successful knockout was confirmed by immunoblotting analysis. The two sgRNA targeting sites for KAT8, p53, and TIP60 are listed in Supplementary Table 1.

Plasmid constructs
Plasmids encoding wild-type KAT8-Myc, GCN5-HA, SIRT6-His, p53-Flag, p400, TRRAP, five acetyltransferases (p300, GCN5, CBP, TIP60 and PCAF), and seven deacetylases (SIRT1-7) were obtained from Guangzhou Bioyard Biotechnology Development Co., Ltd. KAT8 mutants K145R and K145E were generated using the MutExpress II Fast Mutagenesis Kit V2. The GCN5 BrD deletion mutant was generated using a chemically synthesized DNA fragment lacking the BrD region (amino acids 727-837), which was then inserted into the lentiviral expression vector pCDH-CMV-Puro and verified by Sanger sequencing. To knockdown the indicated gene expression, specific short hairpin RNAs (shRNAs) were designed and cloned into the pLKO.1-puro vector. All constructs and transfections were validated by sequencing or immunoblotting. The target sequences for shRNAs used in the present study are listed in Supplementary Table 1.

Transient plasmid overexpression
Cells at 70-80% confluence were transfected with the indicated expression plasmids using polyethylenimine (PEI) Transfection Reagent (MedChemExpress, Cat#HY-K2014) or Lipofectamine 3000 Transfection Reagent (Thermo Fisher, Cat#L3000015) according to the manufacturers’ instructions. Overexpression efficiency was confirmed by immunoblotting.

RNA interference
For gene knockdown, specific shRNA sequences targeting the indicated genes were cloned into the corresponding lentiviral vector. Lentiviral particles were produced in HEK293T cells by co-transfection of the shRNA plasmid together with the packaging plasmids psPAX2 and VSV-G using PEI Transfection Reagent. Viral supernatants were collected 72 h after transfection. Target cells at approximately 50% confluence were infected with recombinant lentivirus in the presence of 10 μg/mL polybrene for 8 h. Knockdown efficiency was confirmed by immunoblotting.

Lentiviral-mediated gene expression
For stable gene expression, lentiviral particles were generated in HEK293T cells by co-transfection of the respective lentiviral expression plasmid with psPAX2 and VSV-G using PEI Transfection Reagent. Viral supernatants were harvested 72 h post-transfection. Target cells at approximately 50% confluence were infected with recombinant lentivirus in the presence of 10 μg/mL polybrene for 8 h. Expression efficiency was verified by immunoblotting.

Nuclear, membrane and cytoplasmic separation
To analyze KAT8 in subcellular fractions, we used the Membrane, Cytosol, and Nuclear Protein Extraction Kit (KeyGEN BioTECH) according to the manufacturer's instructions. Briefly, cell pellets were suspended in 1 mL of Extraction Buffer A (supplemented with protease inhibitors) and lysed using a glass homogenizer. The lysates were incubated for 20 minutes at 4°C and then centrifuged at 15,000 g for 10 minutes at 4 °C. The supernatant was collected as the cytoplasmic fraction. The remaining pellets were washed twice with Extraction Buffer A, resuspended in 500 μL of Extraction Buffer B (supplemented with protease inhibitors), incubated for 10 minutes at 4 °C, and centrifuged at 15,000 g for 10 minutes at 4 °C. This supernatant was collected as the nuclear fraction. Subsequently, 500 μL of Extraction Buffer C (supplemented with protease inhibitors) was added to the remaining pellets, followed by incubation for 10 minutes at 4 °C and centrifugation at 15,000 g for 10 minutes at 4°C. The supernatant was collected as the membrane fraction. The fractions were quantified and analyzed by immunoblotting. Tubulin was used as the internal reference for the cytoplasmic fraction, ATP1A1 for the membrane fraction, and histone H3 for the nuclear fraction.

Immunoblotting analysis and antibodies
The indicated cells were lysed using RIPA buffer (10 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% Triton X-100; 0.1% SDS; 1 mM EDTA), supplemented with a protease inhibitor cocktail. Mouse tissues were homogenized in 1 mL of ice-cold tissue lysis buffer containing 25 mM Tris-HCl (pH 7.5), 10 mM Na3VO4, 100 mM NaF, 50 mM Na4P2O7, 5 mM EGTA, 5 mM EDTA, 0.5% SDS, 1% NP-40, and protease inhibitor cocktail. Following sonication, the lysates were centrifuged at 16,000 × g for 10 minutes, and the supernatants were carefully collected. Protein concentrations were determined using a Pierce BCA Protein Assay Kit. Protein samples were separated on 10% SDS-PAGE gels and subsequently transferred to polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% bovine serum albumin at room temperature for 1 hour and incubated with primary antibodies overnight at 4°C. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 hour. Signals were developed using enhanced chemiluminescence blotting detection reagents (Meilunbio). To facilitate the specific detection of KAT8 K145 lactylation, a custom antibody (referred as K145Lac) was generated and has been validated in this study. Other antibodies, listed in Supplementary Table 1, are from commercial sources and have been validated by the vendors and their validation data are available on the manufacturer's website.

Immunoprecipitation (IP) and co-immunoprecipitation (co-IP)
Cells were lysed for 30 minutes at 4°C using Immunoprecipitation Lysis Buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 5 mM EDTA) supplemented with a protease inhibitor cocktail (Beyotime). To preclear the cell extracts, Protein A/G magnetic beads were employed. The precleared extracts were then incubated overnight at 4°C with either IgG control antibody or the indicated antibodies. Subsequently, the immune complexes were incubated with Protein A/G beads for 2 hours. After washing, the immunoprecipitates were subjected to immunoblotting analysis using the indicated antibodies. For immunoblotting for the immunoprecipitates, conformation-specific secondary antibodies were used. These antibodies selectively recognize native IgG but do not bind denatured or reduced IgG heavy and light chains, thereby preventing interference from the immunoprecipitating antibody in immunoblotting detection.

mRNA half-life analysis
Cells were seeded into six-well plates and cultured for 24 hours before treatment with 5 μg/mL actinomycin D for various durations. Total RNA was subsequently extracted using TRIzol reagent and analyzed by reverse transcription quantitative PCR. An mRNA decay curve was established by setting the mRNA levels of untreated cells to 100%. The time point at which 50% of the mRNA remained was defined as the mRNA half-life.

Measurement of lactate levels
Lactate concentrations in intracellular cell lysates were measured using the L-Lactate Assay Kit (Beyotime). Each sample was determined in triplicate. The standard curves were generated, and all procedures were performed according to the manufacturer’s recommendations.

Measurement of Creatine Kinase MB Isoenzyme and cTnT levels
Creatine Kinase MB Isoenzyme and cTnT concentrations in mouse serum were measured using Mouse CKMB ELISA Kit and Mouse cTnT/TNNT2 ELISA Kit (Elabscience), respectively. Standard curves were generated using ELISACalc software, with all procedures performed in accordance with the manufacturer’s recommendations.

Chromatin immunoprecipitation (ChIP) assay
The specified HEK293T cells were cross-linked with 1% formaldehyde for 10 minutes at 37°C and then washed with cold phosphate-buffered saline (PBS). The cell pellets were resuspended in lysis buffer and subjected to sonication using a water-bath sonicator (Bioruptor Pico, Diagenode) to produce DNA fragments averaging 500 to 1,000 base pairs in length. The indicated antibodies were added to each sample, and the mixtures were incubated overnight at 4°C with rotation. Protein A/G Sepharose beads were then added to the complexes and incubated for 2 hours at 4°C. The beads were washed sequentially with low-salt buffer, high-salt buffer, LiCl buffer, and TE buffer, followed by elution with a buffer containing 1%SDS and 0.1 M NaHCO3. Cross-links were reversed by heating at 65°C overnight, and the DNA was dissolved in Tris-EDTA (TE) buffer and analyzed by real-time PCR. The used primers are listed in Supplementary Table 1.

RNA isolation and qRT-PCR
Total RNA was extracted from cells using TRIzol reagent according to the manufacturer's instructions. The isolated RNA was reverse-transcribed into complementary DNA using a TaqMan reverse transcription kit. Quantitative real-time PCR (qRT-PCR) was performed using a SuperScript III Platinum SYBR Green PCR Kit in a reaction volume of 10 μL. All reactions were conducted in triplicate, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the internal reference gene. Relative gene expression levels were calculated using the 2^-ΔΔCT method.

Apoptosis assay
Apoptosis was assessed by flow cytometric analysis of cells stained with annexin V-fluorescein isothiocyanate (FITC) and 7-aminoactinomycin D (7-AAD). Cells were seeded at a density of 5 × 10⁵ cells per well in six-well plates and incubated overnight. The following day, cells were treated with doxorubicin (DOX) or saline for 24 hours, followed by apoptosis analysis. Cells were collected by centrifugation, incubated with 5 μL Annexin V in 100 μL binding buffer for 10 min at room temperature, dyed with 7-AAD, and then immediately analyzed by flow cytometry. Fluorescence signals were detected and recorded via flow cytometry (BD Biosciences, New Jersey, USA), and obtained raw data were further analyzed and visualized using FlowJo v10.0.7. The percentages of apoptotic cells included the percentages of early (annexin V+7-AAD–) and late apoptotic cells (annexin V+7-AAD+).

Protein half-life assay
The specified cells were treated with 50 μg/mL cycloheximide to inhibit de novo protein synthesis for the indicated times. The relative levels of the indicated proteins were quantified by immunoblotting and normalized to β-actin levels.

In vitro lactylation assay
The indicated KAT8 proteins were incubated with HA-tagged GCN5 proteins or their mutants in a reaction buffer containing 50 mM HEPES, 30 mM KCl, 0.25 mM EDTA, 5.0 mM MgCl2, 5.0 mM sodium butyrate, 2.5 mM DTT (pH 7.8), and 20 mM lactyl-CoA. The reactions were incubated at 37°C for 30 minutes. Subsequently, 5×SDS loading buffer was added to the reactions, and the mixtures were boiled for 10 minutes at 100°C. Samples were then separated by SDS-PAGE and analyzed by immunoblotting using the indicated antibodies.

In vitro delactylation assay
The specified KAT8 proteins were incubated with HA-tagged GCN5 proteins, with or without His-tagged SIRT6 proteins. The incubation was performed in a reaction buffer containing 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 2 mM NAD+, and 20 mM lactyl-CoA. Reactions were carried out at 37°C for 30 minutes. Subsequently, 5×SDS loading buffer was added to each reaction, and the mixtures were boiled at 100 °C for 10 minutes. Samples were then separated by SDS-PAGE and analyzed by immunoblotting using the indicated antibodies.

In vitro acetylation assay
Immunopurified indicated proteins was incubated with immunopurified p53 in HAT buffer (Millipore) in a 30°C shaking incubator for 1 hour. The acetylation of p53 was assessed through immunoblotting using the Pan-Ac antibody.

Nascent protein synthesis
Newly synthesized proteins were labeled using the Click-iT AHA for Nascent Protein Synthesis Kit and the Click-iT Biotin Protein Analysis Detection Kit, following the manufacturer's instructions. Cells were incubated with 500 μM AHA for the indicated times and then lysed in lysis buffer. AHA-incorporated proteins were biotinylated. After precipitation and resuspension, biotinylated proteins were collected using Dynabeads M-280. Proteins were eluted by boiling in SDS sample buffer and quantified by immunoblotting using the indicated antibodies.

GST pull-down assay
For in vitro binding assays, the GST fusion proteins immobilized on Glutathione Sepharose 4B beads were incubated with purified proteins or cell lysates. After washing to remove unbound proteins, the bound proteins were separated by SDS-PAGE and analyzed by immunoblotting using the indicated antibodies. GST alone was used as a negative control.

DNA pull-down assay
The DNA pull-down assay was performed using a Biotinylated Protein Pull-Down Kit (Beyotime) according to manufacturer’s instructions. Briefly, the 5′-biotinylated DNA fragments were incubated with streptavidin magnetic beads and then purified proteins or cell lysates were added to the bead‐probe mixture. After washing with magnetic stand, the bound proteins were analyzed by immunoblotting using the indicated antibodies. The DNA fragment sequences were listed in Supplementary Table 1.

BLI assay
The BLI assay was conducted on an Octet RED96 (ForteBio, Menlo Park, CA, USA). Biosensors (ForteBio) were pre-equilibrated with buffer containing 50 mmol/L HEPES (pH 7.5), 300 mmol/L NaCl, 2 mmol/L MgCl2, and 2 mmol/L DTT for 10 min before each assay. To determine the dissociation constant (KD), at least four concentrations of the indicated analytes were tested to generate the binding curves. The data were analyzed using the global fitting algorithm in Octet Analysis Studio (ForteBio, Version: 12.2.2.26).

Peptide Dot Bolt
To determine the specificity of the anti-lactyl-K antibody, dot blot assays were performed. Synthetic peptides (lactylated, unmodified, and negative control) were dissolved in PBS and spotted onto a nitrocellulose membrane in the indicated amounts. After air-drying, the membrane was blocked with 5% BSA in TBST for 1 hour and incubated with the primary antibody overnight at 4°C, followed by HRP-conjugated secondary antibody incubation. Signals were detected using ECL reagent.

4D label-free lactylproteomics analysis
Cells were collected, and the 4D label-free quantitative lactylproteomics analysis was performed by Jingjie PTM BioLabs. Three independent HEK293T cell samples were analyzed as biological replicates. For protein extraction, cell sample was grinded with liquid nitrogen into cell powder and then transferred to a 5-mL centrifuge tube. After that, the powder was precipitated with cold 10% TCA acetone buffer for 4 h at -20 °C. After centrifugation at 4500 g 4 °C for 5 min, the supernatant was discarded. The remaining precipitate was washed with cold acetone for three times. The protein was redissolved in 8 M urea and the protein concentration was determined with BCA kit according to the manufacturer’s instructions.
The sample was slowly added to the final concentration of 20% (m/v) TCA to precipitate protein, then vortexed to mix and incubated for 2 h at 4 °C. The precipitate was collected by centrifugation at 4500 g for 5 min at 4 °C. The precipitated protein was washed with pre-cooled acetone for 3 times and dried for 1 min. The protein sample was then redissolved in 200 mM TEAB and ultrasonically dispersed. Trypsin was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight. The sample was reduced with 5 mM dithiothreitol for 60 min at 37 °C and alkylated with 11 mM iodoacetamide for 45 min at room temperature in darkness. Finally, the peptides were desalted by Strata X SPE column.
To enrich modified peptides, tryptic peptides dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0) were incubated with pre-washed antibody beads (PTM Bio) at 4°C overnight with gentle shaking. Then the beads were washed for four times with NETN buffer and twice with H2O. The bound peptides were eluted from the beads with 0.1% trifluoroacetic acid. Finally, the eluted fractions were combined and vacuum-dried. For LC-MS/MS analysis, the resulting peptides were desalted with C18 ZipTips (Millipore) according to the manufacturer’s instructions.
The tryptic peptides were dissolved in solvent A, directly loaded onto a home-made reversed-phase analytical column (25-cm length, 100 μm i.d.). The mobile phase consisted of solvent A (0.1% formic acid, 2% acetonitrile/in water) and solvent B (0.1% formic acid in acetonitrile). Peptides were separated with following gradient: 450 nl/min, and all at a constant flow rate of 0-18 min, 9%-24%B; 18-22 min, 24%-35%B; 22-26 min, 35%-90%B; 26-30 min, 90%Bon a Easy-nLC1000 UHPLC system (Bruker Daltonics). The peptides were subjected to capillary source followed by the 450 nl/min mass spectrometry. The electrospray voltage applied was 1.6 kV. Precursors and fragments were analyzed at the TOF detector. The timsTOF Pro was operated in data independent parallel accumulation serial fragmentation (dia-PASEF) mode. The full MS scan was set as100-1700 (MS/MS scan range) and 8PASEF (MS/MS mode)-MS/MS scans were acquired per cycle. The MS/MS scan range was set as 425-1025 and isolation window was set as 25 m/z.
For database search, the DIA data were processed using Spectronaut (v.17.0) software. Tandem mass spectra were searched against the Homo_sapiens_9606_SP_20230103.fasta (20389 entries) concatenated with reverse decoy database. Trypsin/P was specified as cleavage enzyme allowing up to 2 missing cleavages. The mass tolerance for precursor ions was set as 20 ppm in main search and 20 ppm in first search and the mass tolerance for fragment ions was set as 0.04 Da. Carbamidomethyl on Cys was specified as fixed modification, and lactylation on Lys and oxidation on Met were specified as variable modifications. False discovery rate (FDR) of protein, peptide and PSM was adjusted to <1%. The corresponding spectral library was imported into Spectronaut (v.17.0) software to predict the retention time by nonlinear correction and searched against with DIA data.

Compound Virtual Screening
Virtual screening to identify potential compounds targeting KAT8 was performed using AutoDock Vina. The three-dimensional structure of KAT8 with lysine 145 lactylation was predicted using AlphaFold3. The protein structure was prepared by adding hydrogen atoms, assigning bond orders, and optimizing the protonation states of residues within the active site. A compound library was obtained from DrugBank (Release Version 5.1.13). Each compound was docked into the lacetyl-CoA binding pocket of KAT8, and docking poses were evaluated on the basis of calculated binding affinity (ΔG) values. Top-ranking compounds were further prioritized according to docking scores, predicted binding interactions involving hydrogen bonding and hydrophobic contacts with critical residues, drug-like properties, and commercial availability. The selected compounds were subsequently advanced to experimental validation.

Animals and treatments
All animal experiments were performed in accordance with the National Institutes of Health (NIH) guidelines for the care and use of animals. Female C57BL/6J mice (6-8 weeks old) were bred and maintained under specific pathogen-free conditions at the Experimental Animal Center of GuangDong Zhiyuan Biopharmaceutical Technology. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of GuangDong Zhiyuan Biopharmaceutical Technology (Approval No. IAEC-2024092001).
To induce chronic doxorubicin-induced cardiotoxicity, female C57BL/6J mice aged 6-8 weeks were administered DOX (5 mg/kg) once weekly for four weeks via intraperitoneal injection. To evaluate the effect of GM on DOX-induced chronic cardiotoxicity, female C57BL/6J mice aged 6-8 weeks were administered DOX (5mg/kg) and GM (5mg/kg) once weekly for four weeks via intraperitoneal injection. One week after the final DOX or DOX+GM treatment, the mice were sacrificed for functional and molecular analyses. Tissues and organs were collected, fixed in 4% paraformaldehyde, or frozen in liquid nitrogen and stored at -80°C for subsequent experiments.
For the effects of GM on the anti-tumor activity of DOX, 1×10⁶ 4T1 murine breast cancer cells were resuspended in 100 μL PBS and subcutaneously injected into the left flanks of 6-8-week-old female C57BL/6J mice. When tumors reached approximately 100 mm³, mice were randomly assigned into three groups (n=5 per group): i) DMSO, ii) DOX (5 mg/kg, intraperitoneally, once weekly), and iii) DOX (5 mg/kg, intraperitoneally, once weekly) + GM (5mg/kg, intraperitoneally, once weekly). Tumor dimensions were measured once weekly using calipers, and volumes were calculated as V = (length × width²)/2. At the study endpoint, tumors were excised, weighed, and processed for subsequent analysis.
At the indicated time point, blood samples were collected from mice and centrifuged to separate plasma. Plasma cTnT and CK-MB levels were determined using commercial detection kits (Elabscience) according to the manufacturers’ instructions. After sacrifice, the hearts were harvested and weighed, and the tibial length of each mouse was measured. The heart weight-to-tibia length ratio was then calculated and expressed as mg/mm.

Histopathology and TUNEL assay
The heart samples from mouse were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections approximately 4 μm thick were cut and stained with hematoxylin and eosin (H&E) assess heart size. Then, TUNEL staining was performed on these heart sections according to the instructions of the TUNEL staining assay Kit (Beyotime). Fluorescence signals we acquired images using a confocal laser-scanning microscope (Zeiss LSM 880) with a core data acquisition system (Applied Precision).

Statistics and reproducibility
No statistical methods were used to predetermine sample size. Data were presented as mean ± standard deviation (SD) except were stated otherwise. Statistical analysis was conducted using SPSS statistical software (version 26.0; SPSS Inc., Chicago, Illinois). Detailed data processing, sample size and statistical methods for each result were shown in the corresponding figure legends. All p values were two-sided and p value ≤ 0.05 was considered as statistically significant.

Data availability
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the iProX partner repository68,69 with the dataset identifier PXD075302. The AlphaFold models corresponding to Fig. 2I, Fig. 4B, and Fig. 7E have been deposited in ModelArchive (www.modelarchive.org) and are available under the accession codes ma-amz8q, ma-9k082, and ma-tp0qr, respectively. Other data generated in this study are provided in the Article, its Supplementary Information and Source Data file. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript presents a well-conducted and insightful study revealing that lactylation at lysine 145 of KAT8 promotes the formation of the KAT8-TIP60 complex, thereby enhancing p53 K120 acetylation and its pro-apoptotic function. The authors have designed and performed comprehensive mechanistic investigations both in vitro and in vivo, and they further propose Glimepiride as a potential strategy to mitigate doxorubicin-induced cardiotoxicity. Overall, the study provides valuable mechanistic insights and suggests translational relevance.\nHowever, several points should be addressed to further strengthen the manuscript.\n1. KAT8 itself has been reported to possess lactyltransferase activity (Reference: KAT8-catalyzed lactylation promotes eEF1A2-mediated protein synthesis and colorectal carcinogenesis). Moreover, p53 has also been shown to undergo lactylation (Reference: Alanyl-tRNA synthetase, AARS1, is a lactate sensor and lactyltransferase that lactylates p53 and contributes to tumorigenesis). Thus, it is important to clarify whether K145 lactylation affects the intrinsic lactyltransferase activity of KAT8, and whether it influences the lactylation level of p53. Additional discussion or supporting data on this aspect would greatly improve the mechanistic completeness of the study.\n2. The key protein–protein interactions in this study are primarily supported by co-immunoprecipitation (co-IP) and pulldown assays. It is highly recommended to further validate some of these critical interactions (e.g., between KAT8 and TIP60, or KAT8 and GCN5) using orthogonal in vitro biophysical approaches, such as surface plasmon resonance (SPR), biolayer interferometry (BLI), or microscale thermophoresis (MST). This would provide more direct evidence and further strengthen the mechanistic conclusions.\n3. GLO1-mediated non-enzymatic lactylation typically generates D-lactyl-lysine modifications. If the pan-lactylation antibody used in this study specifically recognizes L-lactylation, it cannot detect D-Lac. This potential limitation should be clearly stated in the Methods section and appropriately discussed.\n4. Several figures contain both in vitro and in vivo protein data presented side by side. It is recommended to include only one representative set of protein data in the main figures and move the additional supporting data to the supplementary figures to improve clarity and conciseness.\n5. The section \"K145-lactylated KAT8 preferentially interacts with TIP60 to acetylate p53\" currently includes four main figure panels. It would be clearer to reduce this to two concise main figure panels and move the remaining results to the supplementary materials.\n6. The order of panels I–L in Figure 4 should be adjusted to ensure a logical and coherent flow for the reader.\n7. In the proposed model diagram, the label \"Eraser\" is incorrectly annotated; it should be corrected to \"SIRT6.\" Additionally, the overall layout of the figure could be further refined. The left side appears unnecessarily verbose and could be simplified to make the diagram more concise and easier to interpret.", "reviewer_2": "In this manuscript, the authors explore the role of the recently discovered post-translational modification, Lactylation, on KAT8. They demonstrate that lactylation of KAT8 at lysine 145 (K145) by GCN5 and SIRT6 enhances the assembly of the KAT8-TIP60 complex, which subsequently acetylates p53. This acetylation event promotes p53 binding to the promoters of pro-apoptotic genes such as BAX and PUMA, thereby enhances their transcription. Additionally, the study connects KAT8 lactylation to doxorubicin-induced cardiotoxicity (DIC), revealing that doxorubicin treatment increases K145 lactylation. This, in turn, increases the pro-apoptotic function of p53 and leads to increased cardiomyocyte apoptosis.\nThe data presented in the manuscript is detailed and convincing. The authors have done a very comprehensive experimental analysis to support their hypothesis. However, in my opinion, the following concerns to be addressed to improve the manuscript further:\n1. The authors speculate that KAT8-TIP60 complex promotes K120-acetylation of p53.In Figure 5F, it is shown that TIP60 co-sediments with K145-lactylated KAT8. Does this fraction also contain p53?\n2. The authors show that glucose-induced lactylation of KAT8 at K145 in catalytically inactive KAT8 mutant (C316S/E350Q) lead to increased p53 acetylation. Previous studies have shown that KAT8 could also acetylate p53 at K120 (Sykes et al, 2006), the same site acetylated by TIP60. In the context of a TIP60-HATm mutant, what is the status of p53 acetylation? Is acetylation at K120 completely abolished, or can KAT8 compensate for the loss of TIP60 activity?\n3. Immunoblotting was used to assess the localization of KAT8 wild-type and mutant proteins. Quantitative imaging of KAT8 and its mutants would provide more detailed information on their subcellular distribution and relative abundance.\n4. Treatment with glimepiride (GM) is shown to inhibit lactylation of K145 on KAT8. How does GM treatment affect the interaction between TIP60 and K145-lactylated KAT8?\n5. KAT8 is a known acetyltransferase, and acetylation of H4K16ac is most directly linked to the DNA repair versus apoptosis decision. It is important to check whether the inhibition of DOX-induced apoptosis in glimepiride (GM)-treated cells is due to changes in the acetyltransferase activity of KAT8.\n6. Whether K145 lactylation specifically affects p53-related pathways or exerts broader regulatory effects on other cellular processes. A comparative transcriptomic or proteomic analysis between WT and K145R mutant KAT8-expressing cells could further help to elucidate it.\n7. Since p53 is a central stress-responsive transcription factor, it is important to investigate whether activated p53 regulates the expression or activity of GCN5, SIRT6, or key genes involved in lactate metabolism, potentially revealing a feedback loop that connects cellular stress, metabolism, and epigenetic regulation.\n8. The discussion is quite lengthy and needs to be streamlined further.", "reviewer_3": "Liu H et al. describe a mechanism which apparently underlies p53 acetylation at K120, a PTM well known to be catalyzed by the TIP60 acetyltransferase and associated with enhanced p53 transcriptional activity. The authors claim that p50 K120 acetylation is promoted by lactylation of the lysine acetyltransferase KAT8 at the K145 residue. According to the authors, this last PTM promotes the interaction of KAT8 with TIP60, ultimately leading to enhanced recruitment of TIP60 to p53 and, consequently its acetylation at K120. The enzyme responsible for K145 KAT8 lactylation seems to be GCN5 acetyltransferase (writer), while the enzyme responsible for K145 delactylation is SIRT6 (eraser). Then, the authors go on claiming that K145 KAT8 lactylation enhances the pro-apoptotic p53 target genes BAX and PUMA and that this is responsible for doxorubicin cardiotoxicity. They were even able to identify a drug (Glimepiride, a safe drug used for type 2 diabetes) by in silico screening, which has amazing binding affinity to KAT8 and blocks K145 lactylation. Incredibly, this drug attenuates BAX and PUMA expression leading to reduced apoptosis and cardiotoxicity, and at the same time it does not reduce the efficacy of doxorubicin treatment on tumor regression in mice.\nAre the authors aware that a paper by the Fangfang Zhou lab published in Cell last year (DOI: 10.1016/j.cell.2024.04.002) shows that high lactose conditions associated with cellular transformation (Warburg effect) promote lactylation of p53 at K120 and, consequently, negative regulation of p53 transcriptional activities? How do the authors explain this discrepancy?\nIn addition to this, I have serious concerns with the data and methods description, which are listed below.\n1/ Western blot images, source data, background. The authors report an impressive number of Western blot data: over 950 Western blot images from about 170 different experiments using many different antibodies (about 40 antibodies in total). When looking at the source data file, none of the 950 images displays even the faintest background signal. This is very improbable especially in the case of the immunoprecipitation experiments on endogenous proteins or endogenous protein detection in whole cell lysate.\n2/ Western blot images, source data, immunoglobulin chains in IP experiments. The authors claim having performed many immunoprecipitation experiments on endogenous proteins using antibodies coupled to protein A/G magnetic beads. Then, in many cases, they perform Western blots with primary antibodies from the same species as the antibody used for the IP (based on the information provided in the resource table). However, the source data images do not show any immunoglobulin chains (or least the light chain, admitting that the authors have used a light chain specific secondary antibody…). Some examples:\n- IP with rabbit KAT8 antibody, IB with rabbit antibodies recognizing KAT8, Pan-Kla (Figures 1A, 1B, 1H, 1J, 2A …)\n- IP with mouse Pan-Ace antibody, IB with mouse antibodies recognizing p53, IRF1, WSTF, FASN (Figures 3A, 3F, 3I, 3K…)\n- IP with a rabbit anti TIP60 antibody, IB with rabbit antibodies recognizing KAT8, TIP60, p400 (Figures 5C, 5D, 5H …)\nDo the authors have an explanation for this?\n3/ Figure 2G. the legend says “Bacterially purified GST-KAT8, HA-GCN5, and its catalytically inactive mutant (Y621A/F622A, referred to as HA-GCN5-mut) were obtained from HEK293F cells.” What does this mean? What type of experiment is this?\n4/ Figure 3M. The GCN-GST constructs in this experiments migrate close to the 78 kDa marker like the GCN Del-GST construct lacking the bromodomain (see Figure 3K). In contrast the wt GCN-GST construct migrates close to the 113 KDa marker (see Figure 3K). Hence the GCN constructs used in the experiment of Figure 3M cannot contain the indicated bromodomain mutations because they not contain the bromodomain!\n5/ Figures 2A and 5A–H. Here the authors have analyzed high molecular weight proteins such as CBP/p300 and p400 which migrate very high on SDS-gels. What type of gels did the authors use? The images in the source data file, based on the molecular weight marker migration, I would say these are 10 or 12% gels.\n6/ Compound screening. There is no mention to the methods (software etc.) used for the screening of the compounds which bind to KAT8.\n7/ Effect of combined doxorubicin plus Glimepiride on tumor size. This “cherry on the cake” result is only shown in a little supplemental figure panel (Figure S9X). This experiment is explained in two lines of the Figure S9X legend. No method section, no description in the results section. I do not know what I am looking at here…It looks like the authors got tired of writing at the end of the paper…", "reviewer_4": "This manuscript presents a novel and mechanistically interesting study exploring how KAT8 (MOF) lactylation at lysine 145 modulates TIP60 (KAT5) complex formation and p53 acetylation, ultimately contributing to cardioprotective effects. The work is timely and addresses the emerging role of posttranslational lactylation in cardiovascular biology. The experimental design is generally solid, and the data are presented with a clear narrative. However, several key aspects remain insufficiently addressed and should be clarified or experimentally strengthened before the manuscript can be considered for publication.\n1. Unclear impact of the K145R mutation on KAT8 intrinsic acetyltransferase activity:\nAlthough the authors observe reduced p53 acetylation upon K145R overexpression, these data are derived from cellular systems and do not rule out the possibility that K145R directly impairs the catalytic activity of KAT8. This has been clearly demonstrated in the case of the K274R mutation, which significantly alters KAT8 activity.\nSuggestion: Perform an in vitro HAT assay comparing wild-type and K145R-mutant KAT8 proteins using histone or p53 as substrates, to determine whether the mutation affects acetyltransferase activity directly.\n2. Lack of direct evidence that lactylation promotes KAT8-TIP60 interaction:\nWhile co-immunoprecipitation and GST pulldown assays support the interaction between KAT8 and TIP60, they do not conclusively show whether K145 lactylation enhances this binding.\nSuggestion: Reconstitute an in vitro binding system using synthetically lactylated K145 peptides or in vitro lactylated KAT8 proteins to test TIP60 binding affinity and confirm a causal link.\n3. The physiological relevance of lactate treatment concentration remains unclear:\nThe experiments lack justification for the lactate concentrations used in cell culture, raising questions about the in vivo relevance of these doses.\nSuggestion: Provide literature support for the lactate levels observed in ischemic myocardium or measure endogenous lactate and protein lactylation levels in relevant cardiac tissues to establish biological plausibility.\n4. Absence of in vivo genetic validation of K145 lactylation’s role:\nThe animal experiments focus on KAT8 function but do not test whether the K145 site specifically is required for the observed cardioprotective effects.\nSuggestion: If technically feasible, introduce the KAT8-K145R mutant into mice or cardiomyocytes to assess whether disruption of K145 lactylation alone attenuates the protective effects of lactate treatment.\n5. TIP60 closely resembles KAT8. They share a similar domain structure, including chromo-like domain and a MYST domain. Is it possible that TIP60 is similarly lactylated and contributes to similar KAT8 functions?\nSuggestion: This point can be addressed in the manuscript’s discussion.\n6. Previous studies revealed that SIRT1 is a KAT8 deacetylase. In this current study, the authors suggest that SIRT1 could delactylate KAT8. This raises the interesting observation that SIRT1 may be a more promiscuous enzyme than previously thought.\nSuggestion: This point can be similarly addressed in the manuscript’s discussion.\nIn summary, the manuscript is promising but would benefit significantly from addressing several mechanistic gaps. I recommend major revision, with the expectation that the authors provide additional experimental clarification to support the central role of KAT8 K145 lactylation in cardioprotection.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "1 Point-by-point responses to the comments from the Reviewers\n2\n4 suggestions. We have addressed all of the raised issues at our best efforts, and hope that\n5 our revised manuscript now meets your expectations.\n6\n7 ===========================\n10 lactylation at lysine 145 of KAT8 promotes the formation of the KAT8-TIP60 complex,\n11 thereby enhancing p53 K120 acetylation and its pro-apoptotic function. The authors\n12 have designed and performed comprehensive mechanistic investigations both in vitro\n13 and in vivo, and they further propose Glimepiride as a potential strategy to mitigate\n14 doxorubicin-induced cardiotoxicity. Overall, the study provides valuable mechanistic\n15 insights and suggests translational relevance.\n17\n19 have addressed all of the raised issues at our best efforts. Please find our point-by-point\n20 responses to the comments as follows.\n21\n22\n24 (Reference: KAT8-catalyzed lactylation promotes eEF1A2-mediated protein synthesis\n25 and colorectal carcinogenesis). Moreover, p53 has also been shown to undergo\n26 lactylation (Reference: Alanyl-tRNA synthetase, AARS1, is a lactate sensor and\n27 lactyltransferase that lactylates p53 and contributes to tumorigenesis). Thus, it is\n28 important to clarify whether K145 lactylation affects the intrinsic lactyltransferase\n29 activity of KAT8, and whether it influences the lactylation level of p53. Additional\n30 discussion or supporting data on this aspect would greatly improve the mechanistic\n31 completeness of the study.\n32\n36 processes, we performed in vitro lactylation assays using purified WT, K145R\n37 (lactylation-deficient), and K145E (lactylation-mimic) KAT8 proteins. Our results\n38 demonstrated that the lactyltransferase activity of these variants was comparable when\n39 tested with eEF1A2 as substrates (see Figure 1A# below). Furthermore, analysis of\n40 cellular systems expressing KAT8 WT, K145R, or K145E revealed no appreciable\n41 differences in the lactylation level of p53 (see Figure 1B# below). Collectively, these\n42 findings indicate that K145 lactylation does not directly modulate the catalytic activity\n43 of KAT8 nor alter the lactylation status of p53.\n44\n45\n47 lactylation by WT, K145R, and K145E Myc-KAT8 proteins. (B) IP of p53 followed by\n49 histone H3 lactylation served as a positive control.\n50\n51\n53 by co-immunoprecipitation (co-IP) and pulldown assays. It is highly recommended to\n54 further validate some of these critical interactions (e.g., between KAT8 and TIP60, or\n56 plasmon resonance (SPR), biolayer interferometry (BLI), or microscale\n57 thermophoresis (MST). This would provide more direct evidence and further strengthen\n58 the mechanistic conclusions.\n59\n62 critical interactions between KAT8 and TIP60, as well as KAT8 and GCN5. The BLI\n63 results confirmed these interactions and were included in the revised manuscript\n64 (Figure 4H, and Figure 7H).\n65\n66\n68 lysine modifications. If the pan-lactylation antibody used in this study specifically\n69 recognizes L-lactylation, it cannot detect D-Lac. This potential limitation should be\n70 clearly stated in the Methods section and appropriately discussed.\n71\n73 that distinguishing between D- and L-lactylation is critical in this study. According to\n74 the manufacturer’s specifications, the Pan-Kla antibody used in our experiments (PTM-\n75 1401RM, PTM Bio) is highly specific for L-lactyl-lysine and shows minimal cross-\n76 reactivity with D-lactyl-lysine. To further clarify this point, we additionally employed\n77 a Pan-Kla antibody (PTM-1429RM, PTM Bio) that specifically recognizes D-\n78 lactylation. Using this antibody, we found no detectable signal for D-lactylation on\n80 than D-lactylation. Based on the Reviewer’s comments, the following sentences in the\n81 “Results” section have been rephrased, as follows:\n82\n83 (Manuscript, Results, Page 6, line 121-127)\n84 “To address this question, we performed immunoprecipitation of HEK293T cell lysates\n85 using a KAT8-specific antibody, followed by immunoblot (IB) with pan-anti-lactylated\n86 (Pan-Kla) antibodies that selectively recognize either L- or D-lactyl-lysine. The results\n87 demonstrated that endogenous KAT8 predominantly undergoes L-lactylation, but not\n90 (Figure 1B).”\n91\n92\n94 side by side. It is recommended to include only one representative set of protein data in\n95 the main figures and move the additional supporting data to the supplementary figures\n96 to improve clarity and conciseness.\n97\n99 conciseness of our figures. Accordingly, we have reorganized the data presentation by\n100 retaining one representative set of protein data in the main figures and moving the\n101 additional supporting data to the supplementary section. Specifically, the data\n102 previously presented in Figure 4G, Figure 5A-5H, Figure 6D, Figure 6F, Figure 6H,\n104 have been moved to the new Figure S7E, Figure S8A-S8H, Figure S9A, Figure S9B,\n107\n108\n110 to acetylate p53\" currently includes four main figure panels. It would be clearer to\n111 reduce this to two concise main figure panels and move the remaining results to the\n112 supplementary materials.\n113\n115 conciseness, we have reorganized the indicated section. In the revised version, only two\n116 representative figure panels are retained in main figure panels.\n117\n118\n120 and coherent flow for the reader.\n121\n123\n124\n126 annotated; it should be corrected to \"SIRT6.\" Additionally, the overall layout of the\n127 figure could be further refined. The left side appears unnecessarily verbose and could\n128 be simplified to make the diagram more concise and easier to interpret.\n129\n131 suggestions regarding the model diagram. In the revised version, the incorrect label\n132 “Eraser” has been corrected to “SIRT6”. In addition, we have refined the overall layout\n133 of the diagram by simplifying the left side and reducing unnecessary text.\n134\n135\n136 ===========================\n139 translational modification, Lactylation, on KAT8. They demonstrate that lactylation of\n142 p53 binding to the promoters of pro-apoptotic genes such as BAX and PUMA, thereby\n143 enhances their transcription. Additionally, the study connects KAT8 lactylation to\n144 doxorubicin-induced cardiotoxicity (DIC), revealing that doxorubicin treatment\n145 increases K145 lactylation. This, in turn, increases the pro-apoptotic function of p53\n146 and leads to increased cardiomyocyte apoptosis.\n148 a very comprehensive experimental analysis to support their hypothesis. However, in\n149 my opinion, the following concerns to be addressed to improve the manuscript further:\n150\n153\n155 acetylation of p53. In Figure 5F, it is shown that TIP60 co-sediments with K145-\n156 lactylated KAT8. Does this fraction also contain p53?\n157\n160 relevant fractions with anti-p53 antibody. The results confirmed that p53 co-sediments\n161 with the KAT8-TIP60 complex. These new findings have been incorporated into the\n162 revised manuscript (Figure S8F).\n163\n164\n166 catalytically inactive KAT8 mutant (C316S/E350Q) lead to increased p53 acetylation.\n168 2006), the same site acetylated by TIP60. In the context of a TIP60-HATm mutant, what\n169 is the status of p53 acetylation? Is acetylation at K120 completely abolished, or can\n171\n174 lane 1 vs. 3) and KAT8 (Figure 5J, lane 1 vs. 2) can acetylate p53 at K120.\n176 of KAT8 failed to enhance p53 acetylation in TIP60 knockout cells (Figure S9G) or in\n178 not completely abolish p53 K120 acetylation (Figure S9J), indicating the presence of\n180 possesses intrinsic acetyltransferase activity toward p53, but its effective action requires\n181 a functional TIP60 platform, which is further potentiated by K145 lactylation.\n182\n183\n186\n187\n189 and mutant proteins. Quantitative imaging of KAT8 and its mutants would provide more\n190 detailed information on their subcellular distribution and relative abundance.\n191\n193 wild-type and mutant KAT8, and these results were included in the revised manuscript\n194 (Figure S4C).\n195\n196\n198 on KAT8. How does GM treatment affect the interaction between TIP60 and K145-\n199 lactylated KAT8?\n200\n202 glimepiride (GM) treatment on the interaction between TIP60 and K145-lactylated\n204 which in turn diminished its association with TIP60 (Figure 7K). These findings\n205 indicate that GM interferes with the assembly of the KAT8-TIP60 complex by\n206 suppressing KAT8 lactylation. Based on the Reviewer’s comments, the following\n207 sentences in the “Results” section have been rephrased, as follows:\n208\n209 (Manuscript, Results, Page 20, line 535-538)\n210 “Consistent with GM competing with GCN5 for KAT8 binding, GM significantly\n211 reversed the GCN5-induced increase in KAT8 K145 lactylation and concomitantly\n212 reduced the interaction between TIP60 and K145-lactylated KAT8 (Figure 7K).”\n213\n214\n216 directly linked to the DNA repair versus apoptosis decision. It is important to check\n217 whether the inhibition of DOX-induced apoptosis in glimepiride (GM)-treated cells is\n218 due to changes in the acetyltransferase activity of KAT8.\n219\n221 examined the acetyltransferase activity of KAT8 toward histone H4K16 in GM-treated\n222 cells. GM treatment did not significantly alter H4K16 acetylation levels (Figure 7F),\n223 and KAT8 immunopurified from GM-treated cells retained full acetyltransferase\n224 activity toward recombinant histone H4 in vitro (Figure 7L). These findings\n225 demonstrate that the pro-survival effect of GM is not mediated via modulation of the\n228 revised manuscript, as follows:\n229\n230 (Manuscript, Results, Page 20, line 538-544)\n231 “Given the central role of KAT8 in catalyzing H4K16 acetylation36, it was critical to\n232 rule out the possibility that GM’s anti-apoptotic effect was mediated through an\n233 inhibition of this function. Our data show that GM treatment did not alter global H4K16\n234 acetylation levels (Figure 7L). Together, these findings demonstrate that GM does not\n235 act as a general inhibitor of KAT8's acetyltransferase activity, and establish that its pro-\n236 survival effect is independent of H4K16 acetylation.”\n237\n238\n240 exerts broader regulatory effects on other cellular processes. A comparative\n241 transcriptomic or proteomic analysis between WT and K145R mutant KAT8-expressing\n242 cells could further help to elucidate it.\n243\n245 global transcriptomic or proteomic analyses would be highly informative for\n246 elucidating the broader cellular processes regulated by KAT8 K145 lactylation and\n247 represent an important next step for this line of research. However, we think that future\n248 studies focusing on comprehensive omics studies, followed by rigorous validation\n249 might be able to answer this question more properly. In line with the Reviewer’s\n250 comment, we have revised the Discussion to highlight this as a key future direction, as\n251 follows:\n252\n253 (Manuscript, Discussion, Page 23, line 615-620)\n254 “While our study has delineated a linear pathway linking metabolic flux to apoptosis\n255 via KAT8 lactylation, it will be important for future studies to determine the global\n256 transcriptional landscape governed by this modification. Comparative\n257 transcriptomic/proteomic analyses in cells expressing WT versus K145R mutant KAT8\n258 will be essential to uncover potential roles beyond apoptosis, such as in metabolism,\n260\n261\n263 important to investigate whether activated p53 regulates the expression or activity of\n265 feedback loop that connects cellular stress, metabolism, and epigenetic regulation.\n266\n268 whereby activated p53 might regulate the expression or activity of GCN5, SIRT6, or\n269 key metabolic enzymes involved in lactate production, thus linking cellular stress,\n270 metabolism, and epigenetic regulation. To address this, we examined the protein\n271 expression levels of GCN5, SIRT6, and representative lactate metabolism enzymes\n272 (LDH, PKM, GAPDH, and ENO1) in Ac16 cells expressing either wild-type p53 or the\n273 acetylation-deficient p53 K120R mutant. Immunoblotting analysis revealed no\n274 significant differences in the expression levels of these proteins between the two\n275 cellular contexts (see Figure 2B# below). These findings suggest that p53 activation,\n276 at least under the conditions tested, does not directly regulate GCN5, SIRT6, or the\n277 major lactate metabolic enzymes examined, indicating that the KAT8 lactylation\n278 pathway operates largely independently of p53's K120 acetylation-mediated\n279 transactivation in this system. While our data do not support a direct feedback loop in\n280 this setting, we agree with the Reviewer that such regulatory crosstalk may emerge in\n281 other stress contexts or cell types, which represents an interesting avenue for future\n282 investigation. Furthermore, assessing whether p53 influences the enzymatic activity of\n283 these regulators, rather than their abundance, will be an important next step.\n284\n287 or the acetylation-deficient p53 K120R mutant. β-actin was used as a loading control.\n288\n289\n291\n294 this, we have carefully streamlined the text by condensing overlapping points, removing\n295 excessive background descriptions, and highlighting only the most essential\n296 interpretations of our findings. This revision ensures that the Discussion is now more\n297 concise and focused, while still retaining sufficient depth to convey the significance of\n298 our work.\n299\n300\n301 ===========================\n304 a PTM well known to be catalyzed by the TIP60 acetyltransferase and associated with\n305 enhanced p53 transcriptional activity. The authors claim that p50 K120 acetylation is\n306 promoted by lactylation of the lysine acetyltransferase KAT8 at the K145 residue.\n308 ultimately leading to enhanced recruitment of TIP60 to p53 and, consequently its\n309 acetylation at K120. The enzyme responsible for K145 KAT8 lactylation seems to be\n311 is SIRT6 (eraser). Then, the authors go on claiming that K145 KAT8 lactylation\n312 enhances the pro-apoptotic p53 target genes BAX and PUMA and that this is\n313 responsible for doxorubicin cardiotoxicity. They were even able to identify a drug\n314 (Glimepiride, a safe drug used for type 2 diabetes) by in silico screening, which has\n315 amazing binding affinity to KAT8 and blocks K145 lactylation. Incredibly, this drug\n316 attenuates BAX and PUMA expression leading to reduced apoptosis and cardiotoxicity,\n317 and at the same time it does not reduce the efficacy of doxorubicin treatment on tumor\n318 regression in mice.\n319\n321 work. Indeed, our study identifies K145 lactylation of KAT8 as a novel regulatory\n322 switch that promotes the interaction between KAT8 and TIP60, thereby enhancing\n324 genes such as BAX and PUMA. We further show that this mechanism contributes to\n325 doxorubicin-induced cardiotoxicity, and importantly, that Glimepiride effectively\n326 attenuates K145 lactylation, reduces cardiomyocyte apoptosis, and alleviates\n327 cardiotoxicity without compromising the anti-tumor efficacy of doxorubicin. We\n328 appreciate the Reviewer’s recognition of these findings, which we believe highlight\n329 both the mechanistic novelty of KAT8 lactylation in regulating p53 signaling and the\n330 translational potential of targeting this modification for cardioprotection during\n331 chemotherapy.\n332\n334 in Cell last year (DOI: 10.1016/j.cell.2024.04.002) shows that high lactose conditions\n335 associated with cellular transformation (Warburg effect) promote lactylation of p53 at\n337 the authors explain this discrepancy?\n338\n341 of p53 at K120 directly suppresses its transcriptional activity in proliferating cancer\n342 cells. At first glance, this appears to contrast with our findings and with prior reports\n343 showing that acetylation of p53 at K120 promotes its transcriptional activity (Mol Cell.\n344 2006;24:841-851; Cancer Res. 2013;73:3749-60; Mol Cell. 2013;52:406-20).\n345\n347 identified a direct lactylation of p53 at K120 that antagonizes its function, whereas our\n348 study describes an indirect mechanism in which K145 lactylation of KAT8 enhances\n349 its interaction with TIP60, thereby promoting TIP60-mediated acetylation of p53 at\n351 cardiomyocytes. The biological outcome is likely determined by (i) the specific\n352 modification involved (K120ac vs. K120lac), (ii) the distinct enzymatic machinery (the\n354 lactylation described by Zhou lab), and (iii) the cellular context.\n355\n357 instance, crotonylation versus acetylation of KAT7-K432 enhance versus diminish its\n358 histone acetyltransferase activity (Nat Commun. 2025;16:2379). Moreover, even the same\n360 lactylation, which has been reported to either activate (Genome Biol. 2021;22:85) or repress\n361 gene expression (J Biol Chem. 2025;301:110575). These precedents illustrate that PTM type\n362 and cellular context are critical determinants of functional outcome.\n363\n365 study suggests a framework in which metabolite-derived PTMs exert multi-layered and\n366 context-dependent regulation of p53 signaling. To address this point, we have revised\n367 the Discussion to clarify this distinction, as follows:\n368\n369 (Manuscript, Discussion, Page 22, line 590-602)\n370 “Recent work by Zhou and colleagues showed that direct lactylation of p53 at K120\n371 restrains its transcriptional activity in proliferating cancer cells9, whereas our study\n372 uncovers an upstream mechanism in which KAT8-K145 lactylation enhances TIP60-\n373 mediated K120 acetylation to drive pro-apoptotic gene expression in stressed\n374 cardiomyocytes. These findings reflect the multi-layered and highly dynamic nature of\n376 distinct enzymatic machineries and tuned by cellular state. This principle is echoed in\n377 other paradigms-acetylation versus crotonylation of KAT7-K432 exert opposite\n378 influences on histone acetyltransferase activity53, and H3K18 lactylation can either\n379 activate or repress transcription depending on cellular conditions54,55. Together, these\n380 precedents reinforce a broader concept: PTMs at the same lysine residue should be\n381 viewed not as binary marks but as versatile modulators whose functional outcome is\n382 dictated by modification type and context.”\n383\n384\n386 which are listed below.\n388 impressive number of Western blot data: over 950 Western blot images from about 170\n389 different experiments using many different antibodies (about 40 antibodies in total).\n391 background signal. This is very improbable especially in the case of the\n392 immunoprecipitation experiments on endogenous proteins or endogenous protein\n393 detection in whole cell lysate.\n394\n396 “clean” appearance of our Western blot images. As detailed in our Methods section, all\n398 well-established in the literature (Nat Commun. 2023;14(1):3815; Nature. 2024;631(8021):663-669)\n399 and is known to produce sharp bands with minimal visible background—especially\n400 when antibody dilutions, blocking conditions, and wash protocols are optimized.\n402 sensitivity for low-abundance targets while maintaining low background levels (J\n404 yields images with fewer background artifacts compared to digital CCD systems,\n405 provided exposure times are carefully controlled (Sci Rep. 2022;12(1):17599). In our\n406 laboratory, we rigorously optimized antibody titrations, blocking buffers, and washing\n407 steps in line with best practices to further minimize nonspecific signal (N Am J Med Sci.\n408 2012;4(9):429-434). The “source data” provided were the optimally exposed scans used for\n409 figure preparation, which account for their clean appearance. We sincerely thank the\n411 data even more transparent.\n412\n413\n415 experiments. The authors claim having performed many immunoprecipitation\n416 experiments on endogenous proteins using antibodies coupled to protein A/G magnetic\n417 beads. Then, in many cases, they perform Western blots with primary antibodies from\n418 the same species as the antibody used for the IP (based on the information provided in\n419 the resource table). However, the source data images do not show any immunoglobulin\n420 chains (or least the light chain, admitting that the authors have used a light chain\n421 specific secondary antibody…). Some examples:\n422 - IP with rabbit KAT8 antibody, IB with rabbit antibodies recognizing KAT8, Pan-Kla\n423 (Figures 1A, 1B, 1H, 1J, 2A …)\n424 - IP with mouse Pan-Ace antibody, IB with mouse antibodies recognizing p53, IRF1,\n426 - IP with a rabbit anti TIP60 antibody, IB with rabbit antibodies recognizing KAT8,\n429\n431 conventional IP-WB experiments using primary antibodies from the same species,\n432 immunoglobulin heavy (~55 kDa) and light (~25 kDa) chains are typically detected. To\n433 overcome this well-known issue, we deliberately employed conformation-specific\n434 secondary antibodies (Anti-rabbit IgG [Vazyme, Cat# RA1008-01] and Anti-mouse\n436 fail to bind denatured or reduced IgG chains after SDS-PAGE, because their\n437 conformational epitopes are lost during denaturation. As a result, antibody chain signals\n438 are effectively avoided, allowing clear and specific detection of the target proteins.\n439\n441 absence of heavy/light chain recognition by these secondary antibodies has been\n442 independently validated by the manufacturer (available on the official website,\n443 https://bio.vazyme.com). In addition, we have reproduced this property in our own\n444 laboratory by comparing conventional anti-IgG (H&L) versus conformation-specific\n445 secondaries, and observed consistent results (data not shown). Based on the Reviewer’s\n446 comments, we have explicitly described this methodological choice in the revised\n448\n449 (Manuscript, Methods and materials, Page 27, line 749-753)\n450 “For immunoblotting for the immunoprecipitates, conformation-specific secondary\n451 antibodies were used. These antibodies selectively recognize native IgG but do not bind\n452 denatured or reduced IgG heavy and light chains, thereby preventing interference from\n453 the immunoprecipitating antibody in immunoblotting detection.”\n454\n455\n457 and its catalytically inactive mutant (Y621A/F622A, referred to as HA-GCN5-mut)\n458 were obtained from HEK293F cells.” What does this mean? What type of experiment\n459 is this?\n460\n462 and we apologize for the confusion. The description was indeed imprecise. To clarify,\n463 in this experiment GST-tagged KAT8 was expressed and purified from E. coli, while\n466 using anti-HA antibodies. These proteins were then used in an in vitro enzymatic\n467 lactylation assay, in which GST-KAT8 was incubated with HA-GCN5 or HA-GCN5-\n468 mut in the presence or absence of lactyl-CoA, and KAT8 lactylation was subsequently\n469 assessed by immunoblotting with the Pan-Kla and K145Lac antibodies. Based on the\n471\n472 (Manuscript, Figure Legends, Page 45, line 1235-1241)\n473 “In vitro lactylation assay showing the effect of GCN5 on KAT8 lactylation. GST-\n474 tagged KAT8 was purified from E. coli, while HA-GCN5 and its catalytically inactive\n475 mutant (Y621A/F622A, referred to as HA-GCN5-mut) were expressed in HEK293F\n476 cells and immunopurified using anti-HA antibodies. Purified GST-KAT8 was then\n477 incubated with HA-GCN5 or HA-GCN5-mut in the presence or absence of lactyl-CoA,\n478 and KAT8 lactylation was subsequently assessed by immunoblotting with the Pan-Kla\n479 and K145Lac antibodies.”\n480\n481\n483 to the 78 kDa marker like the GCN Del-GST construct lacking the bromodomain (see\n485 marker (see Figure 3K). Hence the GCN constructs used in the experiment of Figure\n486 3M cannot contain the indicated bromodomain mutations because they not contain the\n487 bromodomain!\n488\n490 identifying the inconsistency. (For clarity, we believe the Reviewer is referring to\n492 relevant.) The Reviewer is correct that the band shown in the original Figure 2M\n493 migrated at approximately 78 kDa, corresponding to GST-KAT8 or a truncated GCN5\n494 construct lacking the bromodomain, rather than the full-length GST-GCN5 fusion\n495 protein (~120 kDa, near the 113 kDa marker). We deeply apologize for this oversight.\n496\n498 between GCN5 and KAT8 using in vitro GST pull-down assays. Four purified proteins\n499 were prepared: GST-KAT8 (~78 kDa), HA-GCN5 (~94 kDa), GST-GCN5 (~120 kDa),\n500 and Myc-KAT8 (~52 kDa). The pull-down experiments were conducted in two\n501 complementary formats: (i) GST-GCN5 with Myc-KAT8 (see Figure 3A# below), and\n502 (ii) GST-KAT8 with HA-GCN5 (see Figure 3B# below). Upon re-examining the raw\n503 images, we found that a panel from the first experiment (GST-KAT8, migrating at ~78\n504 kDa) was inadvertently assembled in place of the intended full-length GST-GCN5\n505 (~120 kDa) from the second experiment.\n506\n508 and bromodomain mutants), which now migrate at the expected molecular weight\n509 consistent with Figure 2K. The corrected figure, updated legend, and corresponding\n510 raw data are included in the revised manuscript. Importantly, this correction does not\n511 affect any results or conclusions. We sincerely appreciate the Reviewer’s careful\n512 examination, which has improved the accuracy and integrity of our work.\n513\n514\n516 kDa) and Myc-KAT8 (~52 kDa) were used for GST pull-down assays. (B) Purified\n518 down assays.\n519\n520\n522 weight proteins such as CBP/p300 and p400 which migrate very high on SDS-gels.\n524 molecular weight marker migration, I would say these are 10 or 12% gels.\n525\n527 specifying the gel conditions in the original submission. For the experiments analyzing\n528 high molecular weight proteins such as CBP/p300 and p400, we indeed used 10% SDS-\n530 system allowed us to reliably detect their presence and relative abundance, which was\n531 sufficient for the purpose of these assays. Our aim in these experiments was to assess\n532 protein expression and interactions rather than to achieve fine separation of\n533 proteins >250 kDa. To avoid confusion, we revised the Methods section to explicitly\n534 state the gel concentration used, as follows:\n535\n536 (Manuscript, Methods and materials, Page 27, line 730-732)\n537 “Protein samples were separated on 10% SDS-PAGE gels and subsequently transferred\n538 to polyvinylidene fluoride membranes (Millipore).”\n539\n540\n542 used for the screening of the compounds which bind to KAT8.\n543\n545 compound screening methodology. We apologize for this omission. In the revised\n546 manuscript, we have now added a detailed description of the virtual screening workflow,\n547 including the software (AutoDock Vina), structural preparation (AlphaFold3-based\n548 model of KAT8 K145 lactylation), compound library (DrugBank Release Version\n549 5.1.13), docking strategy, and criteria for compound selection. These details are now\n550 included in the Methods section as follows:\n551\n552 (Manuscript, Methods and materials, Page 31-32, line 863-875)\n553 “Compound Virtual Screening\n556 was predicted using AlphaFold3. The protein structure was prepared by adding\n557 hydrogen atoms, assigning bond orders, and optimizing the protonation states of\n558 residues within the active site. A compound library was obtained from DrugBank\n559 (Release Version 5.1.13). Each compound was docked into the lacetyl-CoA binding\n560 pocket of KAT8, and docking poses were evaluated on the basis of calculated binding\n561 affinity (ΔG) values. Top-ranking compounds were further prioritized according to\n562 docking scores, predicted binding interactions involving hydrogen bonding and\n563 hydrophobic contacts with critical residues, drug-like properties, and commercial\n564 availability. The selected compounds were subsequently advanced to experimental\n565 validation.”\n566\n567\n569 “cherry on the cake” result is only shown in a little supplemental figure panel (Figure\n571 section, no description in the results section. I do not know what I am looking at here…It\n572 looks like the authors got tired of writing at the end of the paper…\n573\n575 experiment combining doxorubicin (DOX) with Glimepiride (GM). We apologize that\n576 this important validation was presented only briefly in the Supplemental Figure S9X in\n577 the original submission. The intention of this experiment was to demonstrate that GM,\n578 while exerting cardioprotective effects by inhibiting KAT8 K145 lactylation, does not\n579 impair the established anti-tumor efficacy of DOX. We appreciate the Reviewer’s\n580 characterization of this finding as the “cherry on the cake,” as it indeed critically\n581 supports the translational potential of our study. We would like to emphasize that in our\n582 original experiment, both tumor growth curves (volume over time) and final tumor\n583 weights were systematically recorded. These data were available but not fully presented\n584 in the original submission due to space considerations and an oversight in emphasizing\n585 this key result.\n586\n589 design and treatment conditions – Tumor-bearing mice were treated with DOX alone\n590 or in combination with GM, and tumor growth was monitored over time. 2) Results\n591 presentation – Quantitative tumor size measurements are now included in the main text,\n592 showing that DOX robustly suppressed tumor growth and that the addition of GM did\n593 not attenuate this effect. 3) Interpretation – These results directly support our conclusion\n594 that GM protects against DOX-induced cardiotoxicity without compromising its anti-\n595 cancer efficacy. We appreciate the Reviewer’s comment, which allowed us to clarify\n596 this experiment more comprehensively. In accordance with the Reviewer’s comments,\n597 these points have been incorporated into the revised manuscript, as follows:\n598\n599 (Manuscript, Results, Page 21, line 562-570)\n600 “To determine whether GM affects the anti-tumor activity of DOX, we employed a\n601 syngeneic tumor model using 4T1 murine breast cancer cells in C57BL/6J mice. Mice\n602 were randomized into three groups and treated with DMSO, DOX alone, or the\n603 combination of DOX and GM. As expected, DOX treatment markedly suppressed\n604 tumor growth compared with DMSO (Figure 11X). Importantly, the addition of GM\n605 did not attenuate this tumor-suppressive effect, as tumor volumes in the DOX+GM\n606 group were comparable to those in the DOX-only group (Figure 11Y-S11Z). Thus, we\n607 concluded that GM preserves the anti-tumor efficacy of DOX while providing\n608 cardioprotection.”\n609\n610 (Manuscript, Methods and materials, Page 32, line 889-897)\n611 “For the effects of GM on the anti-tumor activity of DOX, 1×10⁶ 4T1 murine breast\n612 cancer cells were resuspended in 100 μL PBS and subcutaneously injected into the left\n613 flanks of 6–8-week-old female C57BL/6J mice. When tumors reached approximately\n614 100 mm³, mice were randomly assigned into three groups (n=5 per group): i) DMSO,\n615 ii) DOX (5 mg/kg, intraperitoneally, once weekly), and iii) DOX (5 mg/kg,\n616 intraperitoneally, once weekly) + GM (5mg/kg, intraperitoneally, once weekly). Tumor\n617 dimensions were measured once weekly using calipers, and volumes were calculated\n618 as V = (length × width²)/2. At the study endpoint, tumors were excised, weighed, and\n619 processed for subsequent analysis.”\n620\n621\n622 ===========================\n626 p53 acetylation, ultimately contributing to cardioprotective effects. The work is timely\n627 and addresses the emerging role of posttranslational lactylation in cardiovascular\n628 biology. The experimental design is generally solid, and the data are presented with a\n629 clear narrative. However, several key aspects remain insufficiently addressed and\n630 should be clarified or experimentally strengthened before the manuscript can be\n631 considered for publication.\n632\n634 suggestions on our work. We have provided point-by-point responses to your comments.\n635\n637 acetyltransferase activity:\n639 these data are derived from cellular systems and do not rule out the possibility that\n641 demonstrated in the case of the K274R mutation, which significantly alters KAT8\n642 activity.\n645 affects acetyltransferase activity directly.\n646\n648 possibility that K145R directly impairs KAT8 catalytic activity. To address this, we\n649 performed in vitro HAT assays using purified wild-type and K145R-mutant KAT8\n650 proteins with both histone H4 as substrate. The K145R mutant displayed\n651 acetyltransferase activity comparable to WT KAT8 on histone H4, indicating that the\n652 reduced p53 acetylation observed in cells is not attributable to impaired intrinsic\n653 enzymatic activity. These new results were added to the revised manuscript (Figure\n655\n656 (Manuscript, Results, Page 11, line 285-288)\n657 “Consistent with its known role as a histone H4 acetyltransferase36, WT KAT8\n658 acetylated histone H4 in vitro (Figure S7A). Notably, the K145R mutant displayed\n659 enzymatic activity comparable to WT KAT8 on histone H4 (Figure S7A).”\n660\n661\n664 between KAT8 and TIP60, they do not conclusively show whether K145 lactylation\n665 enhances this binding.\n667 peptides or in vitro lactylated KAT8 proteins to test TIP60 binding affinity and confirm\n668 a causal link.\n669\n671 an in vitro reconstitution assay was performed using synthetic peptides according to an\n672 established methodology (Nat Commun. 2019; 10:5800). To this end, we synthesized biotin-\n673 labeled peptides corresponding to the K145-containing region of KAT8 in three forms:\n674 unmodified (K), K145-lactylated (Kla), and K145-acetylated (Kac). These peptides\n675 were immobilized on streptavidin beads and incubated with purified recombinant\n677 the unmodified or acetylated control peptides (Figure S7N). This result provides direct\n678 biochemical evidence that K145 lactylation is sufficient to promote a stronger\n679 interaction with TIP60 and that this enhancement is specific to the lactylation\n680 modification. These data were added to the \"Results\" section accordingly, as follows:\n681\n682 (Manuscript, Results, Page 12-13, line 318-325)\n683 “To directly test whether K145 lactylation enhances TIP60 binding, we performed an\n684 in vitro peptide pull-down assay. Biotin-labeled peptides encompassing the K145\n685 residue of KAT8 were synthesized in unmodified (K), K145-lactylated (Kla), or K145-\n686 acetylated (Kac) forms. Strikingly, the Kla peptide, but not the K or Kac controls, pulled\n687 down substantially more TIP60 protein (Figure S7N). These findings provide direct\n688 evidence that K145 lactylation is sufficient and specific for enhancing the KAT8-TIP60\n689 interaction.”\n690\n691\n693 unclear: The experiments lack justification for the lactate concentrations used in cell\n694 culture, raising questions about the in vivo relevance of these doses.\n696 myocardium or measure endogenous lactate and protein lactylation levels in relevant\n697 cardiac tissues to establish biological plausibility.\n698\n700 elevated lactate levels correlate with worse outcomes in heart failure patients (Eur J Heart\n702 lactylation research (Nature. 2019; 574:575-580), we used ~5-10 mM lactate in cell culture.\n704 mice subjected to doxorubicin-induced cardiotoxicity and observed a significant\n705 increase to ~10-15 mM compared with controls (see Figure 4A# below), consistent\n706 with reports of doxorubicin-induced upregulation of lactate dehydrogenase (Egypt Heart J.\n707 2023;75:73). These values align well with our in vitro conditions and were sufficient to\n708 induce protein lactylation. Notably, even higher pathological levels (up to 40 mM) have\n709 been reported in certain tissues (Cancer Res. 2000;60:916-921), further underscoring the\n710 biological relevance of our experimental design.\n711\n712\n714 saline-treated control mice and doxorubicin-treated mice (DOX) (n = 5 mice per group).\n716 as mean ± SD. ***p < 0.001 by two-tailed Student’s t-test.\n717\n718\n720 animal experiments focus on KAT8 function but do not test whether the K145 site\n721 specifically is required for the observed cardioprotective effects.\n723 cardiomyocytes to assess whether disruption of K145 lactylation alone attenuates the\n724 protective effects of lactate treatment.\n725\n727 optimal set-up to establish causality in vivo. We fully agree that generating a KAT8-\n729 specific contribution of K145 lactylation to cardioprotection. However, developing and\n730 characterizing such a genetic model is technically demanding and beyond the scope of\n731 the current study. Importantly, our cellular K145R mutant analyses, together with in\n732 vitro biochemical assays, consistently demonstrate that K145 lactylation is\n733 indispensable for KAT8-TIP60 complex formation and p53 acetylation, supporting its\n734 functional importance. We acknowledged this limitation in the revised “Discussion”\n735 and emphasized that generating a KAT8-K145R knock-in model will be a key future\n736 direction to rigorously establish the in vivo relevance of this modification, as follows:\n737\n738 (Manuscript, Discussion, Page 24, line 645-652)\n739 “A limitation of our study is the absence of in vivo genetic validation to directly test\n740 the role of K145 lactylation. While our cellular K145R mutant analyses and in vitro\n741 binding assays consistently demonstrate that this residue is indispensable for KAT8-\n743 possibility of additional context-dependent effects in vivo. Generating a KAT8-K145R\n744 knock-in mouse model will therefore be an important future direction to establish the\n745 physiological relevance of K145 lactylation.”\n746\n747\n749 including chromo-like domain and a MYST domain. Is it possible that TIP60 is similarly\n750 lactylated and contributes to similar KAT8 functions?\n752\n754 highly similar domain organization with KAT8, including a chromo-like domain and a\n756 lactylation and thereby contribute to functions analogous to those of KAT8. At present,\n757 however, there is no direct evidence from our data or from the literature to support\n759 a direction for future investigation, as follows:\n760\n761 (Manuscript, Discussion, Page 23-24, line 638-645)\n762 “Given the high structural similarity between KAT8 and TIP60, including their\n763 conserved chromo-like and MYST domains, it is conceivable that TIP60 may also\n764 undergo lysine lactylation and participate in analogous regulatory functions. Such a\n765 modification could potentially influence TIP60’s role in p53 acetylation or other\n766 chromatin-associated processes, thereby contributing to stress-responsive\n767 transcriptional programs and cardioprotective pathways. Although our current study\n768 and available literature provide no direct evidence for TIP60 lactylation, this remains a\n769 possibility and an important avenue for future investigation.”\n770\n771\n773 current study, the authors suggest that SIRT1 could delactylate KAT8. This raises the\n774 interesting observation that SIRT1 may be a more promiscuous enzyme than previously\n775 thought.\n777\n779 like to clarify a possible source of confusion: in our study, we identified SIRT6, rather\n780 than SIRT1, as the delactylase for KAT8. This finding is consistent with previous work\n781 (Cell Death Dis. 2023;14(2):164), which reported that SIRT6 functions as a deacetylase for\n783 act as a delactylase, suggesting that it may serve as a broader regulator of lysine acyl\n784 modifications on KAT8. We have now revised the Discussion to emphasize this point\n785 and to highlight the potential implications of SIRT6’s dual enzymatic activities.\n786\n787 (Manuscript, Discussion, Page 22, line 586-590)\n788 “our findings demonstrate that SIRT6, previously recognized as a deacetylase for\n790 may serve as a versatile regulator of lysine acylations, underscoring its broader role in\n791 coordinating diverse PTMs.”\n792\n793\n795 from addressing several mechanistic gaps. I recommend major revision, with the\n796 expectation that the authors provide additional experimental clarification to support\n797 the central role of KAT8 K145 lactylation in cardioprotection.\n798\n800 our manuscript and for providing these constructive comments. We appreciate the\n801 positive assessment of our work as “promising” and fully agree that addressing the\n802 mechanistic details is crucial to firmly establish the central role of KAT8 K145\n803 lactylation in cardioprotection. We performed extensive additional experiments and\n804 substantially revised the manuscript to directly address each of the points raised. As\n805 detailed in our point-by-point responses, these revisions include:\n806\n807 1) In vitro biochemical assays to confirm that the K145R mutation does not affect\n808 intrinsic catalytic activity.\n809 2) A peptide-based binding assay to establish a direct causal link between K145\n810 lactylation and enhanced TIP60 interaction.\n811 3) Measurement of endogenous lactate levels in our disease model to validate the\n812 physiological relevance of our lactate treatment concentrations.\n813 4) Additional discussion points to address the potential roles of TIP60 lactylation and\n815\n817 study. We believe the manuscript is now greatly improved and hope it is deemed\n818 suitable for publication.\n819\n820\n821 ===========================\n825 and to provide appropriate recognition for Early Career Researchers who co-review\n826 manuscripts.\n827\n829 manuscript. We greatly appreciate the initiative by Nature Communications to provide\n830 training and recognition for Early Career Researchers in the peer review process, and\n831 we are grateful for the valuable input provided from this perspective. The comments\n832 have been instrumental in helping us improve our work.\nPoint-by-point responses to the comments from the Reviewers\nWe sincerely thank all of the Reviewers for their constructive comments and helpful\nsuggestions. We have addressed all of the raised issues at our best efforts, and hope that\nour revised manuscript now meets your expectations.\n===========================\nResponses to comments (Reviewer #1)\nMost of the issues have been addressed, but one issue remains as follows:\nThe graphical abstract is not accurate, as it predominantly features inhibition arrows,\nwhich make it quite confusing. It would be better to place this figure later in the\nmanuscript or in the supplementary figures, with an appropriate figure legend added.\nReply: We thank the Reviewer for this suggestion. We have redesigned the schematic\nto improve accuracy and reduce confusion, and moved it to the Supplementary\nInformation (Fig S12) with an appropriate figure legend added. These changes address\nthe concern that the original graphical abstract contained too many inhibition arrows\nand was difficult to interpret.\n===========================\nResponses to comments (Reviewer #3)\nThe authors have not convincedly answered to the issues on Western blot data (see\nremarks to the answers of comments 2, 5, 6 reported in the rebuttal letter). Since most\nof the work is based on Western blot analysis, I cannot recommend acceptance of this\nmanuscript.\nReply: We respectfully acknowledge the Reviewer’s concern regarding the Western\nblot data, and we agree that these issues must be addressed rigorously given the central\nrole of immunoblotting in this study. Please find our point-by-point responses to the\ncomments as follows.\nComment 1： Remark to Reply 2 (lines 396-412 of rebuttal letter). Digital Western\nblot imaging is well known to yield images with a better signal to noise ratio compared\nto film exposure. The arguments put forward by the authors are not convincing.\nBackground noise is essential to judge data integrity. The authors should have provided\nimages of Ponceau S stained membranes and the raw data from the first film in the\ncases they did stacked film exposure.\nReply 1: We sincerely thank the Reviewer for raising this important concern. At the\ntime these experiments were conducted, Ponceau S staining and routine archiving of\nthe first short-exposure film were not part of our laboratory’s standard film-based\nWestern blot workflow, a practice that was also common in many laboratories using\n1 / 11\nstacked film exposure. Nevertheless, we fully acknowledge that more comprehensive\ndocumentation is preferable for publication in high-impact journals.\nIn direct response to the Reviewer’s concern, we performed additional validation\nexperiments using stacked film exposure of the same blot. Specifically, multiple films\nwere exposed simultaneously for the same duration, allowing direct comparison of\nsignals recorded on films positioned at different layers within the stack. Comparison of\nmolecular weight marker patterns and band morphology confirmed a common blot\norigin, with identical marker migration and band positions and differences confined to\nsignal intensity and background (see Figure 1A-1B, Figure 2A-2B, Figure 2D-2E\nbelow).\nThis approach is conceptually equivalent to the widely accepted practice of varying\nexposure duration (short versus long exposure) to optimize the signal-to-noise ratio,\nunder which absolute signal intensity and background levels may vary while the relative\ntrends between experimental conditions remain unchanged (Nature 2024;631:663-669; Nat\nCancer 2023;4:382-400). For ease of visualization, we provide an external example from the\npublished literature (Nature.2024;631(8021):663-669) to illustrate the general difference in\nbackground appearance between short and long exposure conditions (see Figure 1C\nbelow). Consistent with this principle, in stacked film exposure, films closer to the\nmembrane receive stronger chemiluminescent signals and may display higher\nbackground (see Figure 1A below), whereas films deeper in the stack experience signal\nattenuation, resulting in reduced background (see Figure 1B below). These paired films\ntherefore provide a direct, same-blot comparison of higher-background versus lower-\nbackground images obtained under identical exposure conditions. The images\npresented in our manuscript were selected from deeper films that provided optimal\nsignal-to-noise ratios, consistent with exposure optimization practices widely used in\nwestern blot analysis.\n2 / 11\nFigure 1. Technical validation of exposure optimization in film-based Western blot\nanalysis. (A-B) Western blot images obtained from HEK293T cell lysates under\ncontrol and siRNA-mediated TIP60 knockdown conditions, probed with a TIP60\nspecific antibody, and derived from the same membrane using simultaneous stacked\nfilm exposure. Across different exposure conditions, the relative extent of TIP60\nknockdown between control and siRNA-treated samples remains consistent. (A) A film\npositioned closer to the membrane exhibits stronger signal with higher background,\nwhereas (B) a film positioned deeper in the stack shows attenuated signal and reduced\nbackground. (C)Image reproduced from Figure 1K of Chen et al. (Nature 2024;631:663-669),\ndirectly extracted from the published article without modification, and included solely\nfor illustrative purposes as an external reference example to facilitate visualization of\nbackground differences between short and long exposure conditions.\nWe further note that background and non-specific bands are antibody-associated. For\nsome antibodies, minimal background was observed even on films positioned closer to\nthe membrane (see Figure 2A-2B below). In contrast, for antibodies known to exhibit\nnon-specific signals under certain conditions, such as the NSUN2 antibody (PTM BIO,\nCat#PTM-6001) as documented by the manufacturer (see Figure 2C below),\nbackground and non-specific bands were apparent on films closer to the membrane (see\nFigure 2D below). Importantly, these non-specific signals were markedly attenuated\non deeper films within the same stacked exposure (see Figure 2E below), reflecting\nimproved signal-to-noise ratio.\nCritically, exposure optimization did not affect data interpretation. Across all\ncomparisons, relative band intensity trends between experimental conditions remained\nfully consistent regardless of film position within the stack (see Figure 1A-1B, Figure\n2A-2B, and Figure 2D-2E below). This is directly analogous to the well-established\nshort- versus long-exposure paradigm, a widely accepted and routinely used practice in\nwestern blot analysis for optimizing signal-to-noise ratio without altering relative band\nrelationships or biological interpretation (Nat Cancer. 2023;4:382-400; Nature.2024;631:663-669.).\nAccordingly, stacked film exposure enhances image clarity through exposure\noptimization while faithfully preserving the underlying biological conclusions.\nTogether, these additional data provide direct experimental evidence that our film-\nbased workflow yields clean images without compromising data integrity.\n3 / 11\nFigure 2. Antibody-associated background characteristics revealed by stacked\nfilm exposure. (A-B) Western blot images obtained from HEK293T cell lysates under\ncontrol and siRNA-mediated KAT8 or P53 knockdown conditions, probed with a\nKAT8- or P53-specific antibody exhibiting minimal background. Clean signals are\nobserved on films positioned closer to the membrane and deeper in the stack during\nstacked film exposure. Across different exposure conditions, the relative extent of\nKAT8 or P53 knockdown between control and siRNA-treated samples remains\nconsistent. (C) Manufacturer-provided documentation for the NSUN2 antibody\nindicating the presence of non-specific bands or elevated background under certain\nexperimental conditions. (D-E) Western blot images obtained from HEK293T cell\nlysates under control and siRNA-mediated NSUN2 knockdown conditions, probed with\nthe NSUN2-specific antibody, derived from the same membrane using simultaneous\nstacked film exposure. Across different exposure conditions, the relative extent of\nNSUN2 knockdown between control and siRNA-treated samples remains consistent. A\nfilm positioned closer to the membrane (D) displays stronger signal accompanied by\nhigher background and non-specific bands, whereas a film positioned deeper in the\nstack (E) shows marked attenuation of background and non-specific signals.\nComment 2： Remark to Reply 5 (lines 490-519 of rebuttal letter). Please refer to\nFigure 3# of rebuttal letter on GST pulldowns. Based on the protein ladder migration\npatterns, the IB:Myc and IB:GST images of panels A and B do not come from the same\nmembrane! What are the authors doing here?\nReply 2: Regarding Reviewer’s concern that the IB: Myc and IB: GST blots do not\noriginate from the same membrane, we respectfully clarify that this interpretation does\nnot reflect the actual experimental design. Panels A and B represent two independent\nreciprocal GST pull-down assays performed using different constructs, tags, and\npurified protein samples, and therefore they were neither expected nor intended to\noriginate from the same gel or membrane.\n4 / 11\nComment 3：Remark to Reply 6 (lines 527-539 of rebuttal letter). The migration\npatterns of p300/CBP, TRRAP, p400 (between 300 and 437 kDa) are inconsistent with\nprotein ladder migration and with a 10% SDS-PAGE gel. See for example Fig. 5B, IB\np400 in the original data file. Here, the band indicated as p400 migrates just above the\n154 kDa marker. In the case of a 10% SDS-PAGE gel, such a 400 kDA protein would\njust enter the running gel and the band would look very compressed. A similar\nmigration pattern can only be obtained with a gradient gel. The authors are again\ninconsistent.\nReply 3: We thank the Reviewer for raising this point. We confirm that all blots\ninvolving p300/CBP, TRRAP, and p400 were performed using 10% SDS-PAGE gels,\nand that complete uncropped raw membranes have been provided in the manuscript.\nThe Reviewer’s concern is based on the assumption that high-molecular-weight\nproteins must obligatorily remain at the stacking-resolving gel interface or display\nseverely compressed band morphology when separated on non-gradient or 10% SDS-\nPAGE gels. While this expectation is understandable as a commonly cited theoretical\nguideline, it does not represent a universal electrophoretic rule.\nAt the level of empirical evidence, examination of the published literature indicates that,\nalthough severely compressed band patterns of high-molecular-weight proteins have\nindeed been reported under certain electrophoretic conditions, such compression is not\nan obligatory outcome of non-gradient SDS-PAGE. In practice, apparent migration\nbehavior and band morphology vary substantially across experimental contexts.\nAlthough the SDS-PAGE gel percentage is not explicitly reported in some studies (Nat\nCommun. 2019;10(1):5792; Nat Cell Biol. 2020;22(9):1064-1075; Cell. 2024;187(2):294-311.e21; Nucleic Acids\nRes. 2022;50(7):3817-3834; Cell Metab. 2024;36(8):1696-1710.e10), these reports are cited not to infer\nexact gel composition, but to illustrate observed migration behavior of large proteins\nunder non-gradient SDS-PAGE conditions. In these studies, the spacing of molecular-\nweight markers and overall migration patterns are consistent with non-gradient gels,\nand high-molecular-weight proteins are repeatedly observed to migrate well into the\nresolving gel and to form clearly distinguishable bands rather than remaining at the gel\nentry point or appearing obligatorily compressed.\nFor contextual reference, representative examples from the published literature\nillustrating the migration of some high-molecular-weight proteins on non-gradient\nSDS-PAGE are summarized in Figure 3. Specifically, You et al. (Nat Commun.\n2019;10(1):5792) showed that p300 and CBP, both well-recognized high-molecular-weight\nproteins, migrated slightly above the ~170 kDa marker without pronounced band\ncompression (see Figure 3A below). Similarly, Gao et al. (Nat Cell Biol. 2020;22(9):1064-1075)\nreported that p300 migrates slightly above the ~170 kDa marker and displays a well-\nresolved band profile (see Figure 3B-3C below). Comparable migration behavior of\np300 and CBP was also observed by Chen et al. (Cell. 2024;187(2):294-311.e21), where these\nproteins migrate slightly above the ~180 kDa marker without severe compression (see\nFigure 3D-3G below). Together, these independent reports demonstrate that large\nproteins can migrate substantially into the resolving region of non-gradient gels and do\nnot obligatorily exhibit compressed band morphology.\n5 / 11\nIndependent evidence comes from Coomassie Brilliant Blue staining of purified\nproteins resolved on non-gradient SDS-PAGE gels, a setting that eliminates antibody-\ndependent effects, membrane transfer variability, and exposure-related artifacts (Nucleic\nAcids Res. 2022;50(7):3817-3834). Under these controlled conditions, the large protein CBP\ndoes not exhibit a more compressed band morphology than the substantially smaller\nprotein MOB1 when resolved on the same gel (see Figure 3H below), indicating that\nband width is not intrinsically dictated by molecular weight. Consistent with this, Li et\nal. (Cell Metab. 2024;36(8):1696-1710.e10) showed that PKM2 monomers and tetramers, despite\na four-fold difference in apparent molecular weight, can both be resolved on non-\ngradient SDS-PAGE gels without pronounced compression of the tetrameric species\n(see Figure 3I below). Similar observations have been reported across broader protein\nsets (Nat Commun. 2019;10(1):5792), where relatively large proteins display band widths\ncomparable to or even broader than smaller proteins (see Figure 3J-3K below).\nMoreover, even within a single study (Nat Cell Biol. 2020;22(9):1064-1075), p300 band\nmorphology varies across experimental contexts (see Figure 3L-3M below),\nunderscoring that SDS-PAGE band compression is not solely determined by molecular\nweight or gel percentage, but is instead influenced by multiple experimental and\nsample-dependent factors.\n6 / 11\nFigure 3. Representative literature examples of some high-molecular-weight\nprotein migration on non-gradient SDS-PAGE. (A) Image reproduced from Figure\n2A of You et al. (Nature Communications 2019;10:5792), showing p300 and CBP migrating\nslightly above the ~170 kDa marker without pronounced band compression. (B-C)\nImages reproduced from Figures 1B and 1F of Gao et al. (Nature Cell Biology 2020;22:1064-\n1075), showing p300 migrating slightly above the ~170 kDa marker with a well-resolved\nband profile. (D-G) Images reproduced from Figures S4B and S4E–S4G of Chen et al.\n(Cell 2024;187:294-311.e21), showing p300 and CBP migrating slightly above the ~180 kDa\nmarker without severe band compression. (H) Image reproduced from Figure 1J of Jin\net al. (Nucleic Acids Res. 2022;50(7):3817-3834), illustrating Coomassie Brilliant Blue staining\nof purified CBP and MOB1 separated on non-gradient SDS-PAGE, in which the larger\nprotein CBP does not exhibit a more compressed band profile than the smaller protein\nMOB1. (I) Image reproduced from Figure 4I of Li et al. (Cell Metab. 2024;36(8):1696-1710.e10),\nshowing that tetrameric PKM2 resolved on 10% SDS-PAGE does not display\npronounced band compression relative to the monomeric form. (J-K) Images\nreproduced from Figure 4A-4B of You et al. (Nature Communications 2019;10:5792), showing\nthat under non-gradient SDS-PAGE conditions, bands of relatively large proteins may\nappear comparable in width to, or broader than, those of smaller proteins. (L-M) Images\nreproduced from Figure 2A and Figure 2D of Gao et al. (Nature Cell Biology 2020;22:1064-\n1075), illustrating variability in p300 band morphology under different experimental or\nsample contexts within the same study. As shown, p300 bands appear substantially\nbroader under certain conditions (L) but relatively more compact under others (M). All\nimages are reproduced from previously published studies and are provided as external\nreference examples (A-M). For ease of reading, red boxes indicate the regions relevant\nto the points discussed here (A-M).\nTo directly address the Reviewer’s concern, we next examined published studies in\nwhich the SDS-PAGE gel composition is explicitly reported. Multiple independent\nreports demonstrate that some high-molecular-weight proteins can migrate well into 10%\nor even 12% SDS-PAGE gels, rather than remaining at the gel entry point or exhibiting\nextreme compression (J Biol Chem. 2005;280:30604-30610; Transl Cancer Res. 2025;14(10):6919-6939;\nExp Cell Res. 2025;444(2):114399; Commun Biol. 2024;7(1):1504; J Cell Sci. 2009;122(Pt 8):1091-9; EMBO J.\n2002;21(13):3516-25; Phytomedicine. 2025:140:156591; Science. 2014;343(6169):428-32; Adv Sci.\n7 / 11\n2025;12(28):e2412698; J Lipid Res. 2012 Apr;53(4):755-66; Oncol Lett. 2019;18(6):6909-6916; iScience.\n2024;27(6):109850; PLoS One. 2012;7(6):e39679). Taken together, these reports establish that\npronounced band compression is not an inherent or obligatory feature of high-\nmolecular-weight proteins resolved on 10% SDS-PAGE gels.\nRepresentative examples from the published literature illustrating the behavior of some\nhigh-molecular-weight proteins on 10% SDS-PAGE are summarized in Figure 4.\nSpecifically, MacPartlin et al. (J Biol Chem. 2005;280:30604-30610) reported that p300\nmigrates into 10% SDS-PAGE gels (see Figure 4A below) and even 12% SDS-PAGE\ngels (see Figure 4B below). Similarly, Wu et al. (Transl Cancer Res. 2025;14(10):6919-6939)\nshowed that p300 migrates into a 10% SDS-PAGE gel and forms a clearly detectable\nband within the resolving region (see Figure 4C below). Consistent findings were\nreported by Cai et al. (Exp Cell Res. 2025;444(2):114399) and Cao et al. (Commun Biol.\n2024;7(1):1504), both of whom showed that p300 migrates into 10% SDS-PAGE gels\nwithout evidence of severe band compression (see Figure 4D-4F below).\nThis observation extends beyond p300 to other high-molecular-weight proteins.\nKang et al. (J Cell Sci. 2009;122(Pt 8):1091-9) showed that other high-molecular-weight proteins,\nincluding ZO-1 and ZO-2, migrate into 10% SDS-PAGE gels without extreme\ncompression (see Figure 4G below). Ustrell et al. (EMBO J. 2002;21(13):3516-25) reported\ncomparable migration behavior for the ~200 kDa protein PA200 on 10% SDS-PAGE\ngels (see Figure 4H below). Even proteins substantially larger than p300, such as the\n~469 kDa DNA-PKcs, have been shown by Li et al. (Phytomedicine. 2025:140:156591) to\nmigrate into 10% SDS-PAGE gels without severe compression (see Figure 4I below),\nwhile Monroe et al. (Science. 2014;343(6169):428–432) reported the migration of ~469 kDa\nDNPK-1 on 12.5% SDS-PAGE gels, with the corresponding band appearing just above\nthe ~148 kDa marker and without obvious band compression (see Figure 4J below).\nMore recent studies further reinforce this conclusion. A study by Wang et al. (Adv Sci.\n2025;12(28):e2412698) employed 10% SDS-PAGE to resolve several high-molecular-\nweight proteins, including MDC1, 53BP1, ATM, and DNA-PKcs, none of which\nexhibited severe band compression (see Figure 4K below). Importantly, the same study\nanalyzed the full-length DNA-PKcs and its truncated fragments within a single\nexperimental framework using identical sample preparation, electrophoretic conditions,\nand immunoblotting procedures (see Figure 4L below). Under these matched\nconditions, the band corresponding to full-length DNA-PKcs did not exhibit a\ncompressed morphology relative to its lower-molecular-weight truncated forms (see\nFigure 4L below). Rather, the DNA-PKcs band migrated into the resolving gel and\ndisplayed a band width comparable to that of the smaller fragments (see Figure 4L\nbelow).\nComparable observations have also been reported for other large proteins. Jeon et al. (J\nLipid Res. 2012 Apr;53(4):755-66) resolved the high-molecular-weight protein FASN on 12%\nSDS-PAGE gels without severe band compression (see Figure 4M below), while\nSchcolnik-Cabrera et al. (Oncol Lett. 2019;18(6):6909-6916) reported similar results for FASN\non 10% SDS-PAGE gels (see Figure 4N below). In addition, Wu et al. (iScience.\n2024;27(6):109850) demonstrated that both FASN and mTOR migrate into 10% SDS-\nPAGE gels without pronounced compression (see Figure 4O below), a finding\nconsistent with earlier observations of mTOR migration on 10% SDS-PAGE reported\nby Calderwood et al. (PLoS One. 2012;7(6):e39679) (see Figure 4P below). Collectively,\n8 / 11\nthese convergent lines of evidence demonstrate that high-molecular-weight proteins\ncan, and frequently do, migrate into 10% SDS-PAGE gels without exhibiting obligatory\nband compression.\nTaken together, although gradient gels may offer advantages for resolving very large\nproteins, the absence of a gradient gel does not invalidate the migration patterns\nobserved here. Both the available literature and our experimental data indicate that the\nReviewer’s concern reflects a theoretical expectation rather than a universally\napplicable rule, and therefore does not undermine the validity of the data presented in\nthis study.\n9 / 11\nFigure 4. Representative literature examples showing that some high-molecular-\nweight proteins can be resolved on 10% SDS-PAGE gels without pronounced\nband compression. (A-B) Images reproduced from Figures 2A and 4A of MacPartlin\net al. (J Biol Chem. 2005;280:30604-30610), showing that p300 can be resolved on both 10%\n(A) and 12% (B) SDS-PAGE gels. (C) Image reproduced from Figure 9A of Wu et al.\n(Transl Cancer Res. 2025;14(10):6919-6939), showing p300 resolved within the separating region\nof a 10% SDS-PAGE gel. (D-E) Image reproduced from Figures 5G and 6G of Cai et\nal. (Exp Cell Res. 2025;444(2):114399), and (F) Image reproduced from Figure 6D of Cao et al.\n(Commun Biol. 2024;7(1):1504), both showing p300 resolved within the separating region of\na 10% SDS-PAGE gel. (G) Image reproduced from Figures 10C-10D of Kang et al. (J\nCell Sci. 2009;122(Pt 8):1091-9), showing that ZO-1 and ZO-2 are resolved on a 10% SDS-\nPAGE gel. (H) Image reproduced from Figure 4 of Ustrell et al. (EMBO J. 2002;21(13):3516-\n25), showing that PA200 is resolved on a 10% SDS-PAGE gel. (I) Image reproduced\nfrom Figure 4E of Li et al. (Phytomedicine. 2025:140:156591), showing that ~469 kDa DNA-\nPKcs is resolved on a 10% SDS-PAGE gel. (J)Image reproduced from Figures 1A and\n1D of Monroe et al. (Science. 2014;343(6169):428-432), showing that the ~469 kDa DNPK-1\ncan be resolved on a 12.5% SDS-PAGE gel, with the corresponding band migrating\njust above the ~148 kDa marker. (K) Images reproduced from Figures 2B and 2G of\nWang et al. (Adv Sci. 2025;12(28):e2412698), showing that MDC1, 53BP1, ATM, and DNA-\nPKcs are resolved on a 10% SDS-PAGE gel. (L) Image reproduced from Figure 7Q of\nWang et al. (Adv Sci. 2025;12(28):e2412698), showing full-length DNA-PKcs and its truncated\nfragments resolved on the 10% SDS-PAGE gel. (M) Image reproduced from Figure 1H\nof Jeon et al. (J Lipid Res. 2012 Apr;53(4):755-66), showing that FASN is resolved on a 12%\nSDS-PAGE gel. (N) Image reproduced from Figure 3C of Schcolnik-Cabrera et al.\n(Oncol Lett. 2019;18(6):6909-6916), showing FASN resolved on a 10% SDS-PAGE gel. (O)\nImage reproduced from Figure 4A of Wu et al. (iScience. 2024;27(6):109850), showing that\nFASN and mTOR are resolved on a 10% SDS-PAGE gel. (P) Image reproduced from\nFigure 3A of Calderwood et al. (PLoS One. 2012;7(6):e39679), showing that mTOR is\nresolved on a 10% SDS-PAGE gel. All images are reproduced from previously\npublished studies and are provided as external reference examples (A-P). For ease of\nreading, red boxes indicate the regions relevant to the points discussed here (A-P).\n===========================\n10 / 11\nResponses to comments (Reviewer #4)\nI have read the revised manuscript and the authors' point-by-point response. Overall,\nthe authors have addressed the major concerns comprehensively, and as a result, the\nrevised paper is vastly improved.\nSpecifically, the authors have:\n1. Added an in vitro HAT assay (Fig. S7A) demonstrating that the K145R mutation does\nnot impair KAT8 catalytic activity.\n2. Performed a peptide-based pull-down assay (Fig. S7) providing direct evidence that\nK145 lactylation enhances the KAT8–TIP60 interaction.\n3. Measured cardiac lactate levels (≈10–15 mM) to demonstrate the physiological\nrelevance of their lactate treatment.\n4. Expanded the Discussion to address the potential roles of TIP60 lactylation and\nSIRT6's dual enzymatic functions.\n5. Acknowledged the lack of a K145R knock-in model and appropriately identified this\nas a future direction.\nOverall, the additional data and textual revisions adequately resolve my previous\nconcerns.\nReply: We sincerely thank the Reviewer for the positive evaluation of our revised\nmanuscript. We appreciate the Reviewer’s recognition that the added experiments and\ntextual revisions have addressed the major concerns. We are grateful for the\nconstructive feedback, which has significantly improved the quality and clarity of the\nmanuscript.\n===========================\nResponses to comments (Reviewer #5)\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nReply: We thank the Reviewer for co-reviewing our manuscript as part of the Nature\nCommunications peer-review training initiative. We appreciate the time and effort\ninvested by the reviewer team, and we are grateful for the constructive reports, which\nhave helped improve the quality and clarity of our work.\n11 / 11"}]

Decision letter