Acetylation is a dynamic and reversible post-translational modification of proteins, which is regulated by acetyltransferases and modulated by deacetylases, and is closely related to the biological functions of DNA damage repair, cell division, signaling, and inflammatory response, etc. Two research results on the involvement of acetylation modification regulatory enzymes in the regulation of inflammatory response have been published consecutively in Nature Communications. In Nature Communications, two consecutive publications on the regulation of acetylation modification regulatory enzymes in the inflammatory response have been published, respectively revealing the mechanism by which KAT5 regulates the activation of NLRP3 inflammatory vesicles, and the mechanism by which the deacetylase SIRT6 regulates the release of IL-17A and drives the inflammation and remodeling of allergic airways, which have once again deepened our understanding of the biological functions of acetylation modification.
Case 1 Acetylation of NLRP3 K24 Promotes Inflammasome Formation (1)
Aberrant activation of the NLRP3 (Nucleotide-binding domain, leucine-rich repeat, and pyrin domain–containing protein 3) inflammasome is implicated in the pathogenesis of inflammatory diseases including sepsis, gout, diabetes, neurodegenerative disorders, etc. Full activation of the NLRP3 inflammasome requires two consecutive signals: a priming signal followed by a second assembly signal. Previous studies have shown that post-translational modifications (phosphorylation/ubiquitination/sumoylation, etc.) play crucial roles in regulating the activity of NLRP3 inflammasome during the priming stage. However, little is known about the mechanisms regulating NLRP3 activity during the assembly stage.
To address the scientific question of whether acetylation modification is involved in the activation of NLRP3 inflammasome, researchers first stimulated the activation of NLRP3 inflammasome in mouse macrophages and examined the levels of NLRP3 acetylation at different stages. They found that NLRP3 acetylation occurred during the activation stage rather than the priming stage, suggesting the involvement of acetylation in the activation of NLRP3 inflammasome. Furthermore, using acetyl-proteomics approach, researchers identified potential acetylation sites in NLRP3 and confirmed three potential acetylation modification sites (Lys24/234/875) on NLRP3 protein. Among them, Lys24 acetylation specifically occurred during the activation stage, indicating acetylation modification at the Lys24 site of NLRP3 during activation.
Researchers further developed a specific antibody recognizing acetylation at the K24 site and detected acetylation at this site in primary macrophages stimulated to activate the NLRP3 inflammasome. They also generated NLRP3-K24R point mutant mice and found that Lys24 acetylation promoted the activation of NLRP3 inflammasome. These mutant mice exhibited improved systemic inflammatory response induced by bacterial endotoxin and reduced acute lung injury induced by LPS. Through SDD-AGE, BLUE-native-PAGE, and live-cell imaging, it was found that acetylation at the NLRP3-K24 site promoted NLRP3 oligomerization. These experiments collectively suggest that acetylation modification at Lys24 regulates NLRP3 oligomerization, thereby participating in inflammasome activation.
NLRP3 Lys24 acetylation promotes activation of inflammatory vesicles
So which class of acetyltransferases regulates NLRP3 acetylation? To this end, the researchers explored the effects of all known acetyltransferase inhibitors on NLRP3 inflammatory vesicle activation. The results showed that only NU9056 (a KAT5 inhibitor) had an inhibitory effect on NLRP3 inflammatory vesicles. Next, the researchers confirmed the existence of a direct interaction between KAT5 and NLRP3 using immunoprecipitation, immunofluorescence and GST pull-down assays. Further, the researchers found that KAT5 mediated acetylation at the NLRP3-K24 site by in vitro protein kinase assays using KAT5 myeloid-specific knockout (Kat5fl/fl lyz2-Cre) mice and mice with attenuated functional activity of KAT5 acetylation (KAT5S86A/S86A).
KAT5 mediates NLRP3 acetylation
Finally, the researchers further explored whether NLRP3 acetylation could serve as a therapeutic target for NLRP3 inflammatory vesicle-associated diseases. LPS (Lipopolysaccharide)-induced endotoxin shock model and MSU (Monosodium urate)-induced peritonitis model were used to evaluate the effect of KAT5-mediated acetylation of NLRP3-K24 site on inflammatory vesicle activation in vivo, and the results showed that myeloid-specific knockdown of KAT5 and specific inhibitors significantly reduced activation of NLRP3 inflammatory vesicles in vitro, and that in vivo it has a protective effect against inflammatory responses induced by bacterial endotoxin or uric acid crystals. These results suggest that targeted NLRP3 acetylation modification provides a new strategy for the treatment of diseases associated with NLRP3 inflammatory vesicle activation.
NU 9056 effectively inhibits NLRP3 inflammatory vesicles in vivo
Case 2 Deacetylase SIRT6 Regulates IL-17A Release and Drives Allergic Airway Inflammation and Remodeling (1)
The dysregulation of IL-17A is closely associated with airway inflammation and remodeling in severe asthma patients. However, the molecular mechanisms regulating IL-17A remain unclear. To investigate whether members of the sirtuin family are involved in asthma airway remodeling, researchers first examined the expression of sirtuin family members in lung tissues of mice with acute severe asthma. The results showed that only the mRNA and protein levels of SIRT6 were significantly upregulated. Combining with clinical samples, it was found that the expression of SIRT6 was also significantly increased in asthmatic patients, participating in the process of airway remodeling, and positively correlated with disease severity, suggesting its potential as a biomarker for severe asthma.
The expression level of SIRT6 is positively correlated with the severity of asthma.
Early studies have found that IL-17A is closely associated with airway inflammation and remodeling in severe asthma. Therefore, to further investigate whether SIRT6 regulates IL-17A in the process of airway remodeling and its regulatory mechanism, researchers used immunoprecipitation and mass spectrometry analysis to identify 168 potential SIRT6 binding proteins, among which RORγt is necessary for Th17 program initiation and induction of IL-17A expression. Subsequently, using Co-IP experiments, glutathione S-transferase (GST) pull-down assays, and proximity ligation assay (PLA) experiments, it was validated that SIRT6 and RORγt directly interact and co-localize in the nucleus.
Furthermore, researchers constructed a RORγt deletion mutant (RORγt-ΔPY) and found that RORγt-ΔPY failed to interact with SIRT6. Three fragments, SIRT6-Core (43-276aa), SIRT6-ΔN (43-355aa), and SIRT6-ΔC (1-276aa), were also constructed, and the results showed that SIRT6-ΔC was the main structural domain interacting with RORγt. Additionally, the study found that SIRT6 can regulate the expression of RORγt in airway epithelium both in vivo and in vitro.
SIRT6 Directly Binds to RORγt and Regulates RORγt Expression
Furthermore, considering SIRT6 is a deacetylase, researchers explored whether SIRT6 targets RORγt for deacetylation to suppress IL-17A expression. Through cell transfection and luciferase reporter assays, the authors found that SIRT6 can promote the deacetylation of HDM/LPS-induced RORγt through its deacetyltransferase activity. Additionally, researchers identified acetylated lysine (K) residues on RORγt using mass spectrometry-based acetylation proteomics approach and identified three acetylated lysine modification sites (K456, K120, and K192). Experimental validation revealed that K192 is the primary acetylation site of RORγt targeted by SIRT6. Therefore, the above experiments indicate that SIRT6 reduces the acetylation of RORγt at the K192 site, playing a crucial role in controlling IL-17A production.
SIRT6 Targets Deacetylation of RORγt K192 Site
Lastly, researchers further explored the feasibility of using the SIRT6 inhibitor OSS_128167 (OSS) for clinical treatment of severe asthma patients. Combining mouse models of severe asthma and cell line experiments, the therapeutic and preventive effects of OSS on asthma mice were systematically evaluated. The results showed that after inhibiting the activity of SIRT6, OSS effectively alleviated the inflammation levels of severe asthma and promoted airway remodeling.
SIRT6 Inhibitor OSS_128167 (OSS) Prevents Airway Remodeling in Asthma Mice
In summary, the two studies mentioned above revealed that acetylation modification is a crucial mechanism for the oligomerization and full activation of NLRP3, as well as an important mechanism for regulating asthma airway remodeling. On the other hand, based on the mechanisms of KAT5-NLRP3 and SIRT6-RORγt-IL17A interactions, the potential application value of targeting acetylation modifier enzyme (KAT5) or deacetylase (SIRT6) for the treatment of clinically relevant diseases was explored. These studies suggest that proteomics and post-translational modification proteomics are of significant importance in revealing cellular biology and disease mechanisms, as well as in developing new therapeutic strategies.
References
- Zhang, Yening, et al. "Acetylation is required for full activation of the NLRP3 inflammasome." Nature Communications 14.1 (2023): 8396.
- Quan, Jingyun, et al. "Epithelial SIRT6 governs IL-17A pathogenicity and drives allergic airway inflammation and remodeling." Nature Communications 14.1 (2023): 8525.
