Arylamine N-Acetyltransferase 1 Activity is Regulated by the Protein Acetylation Status

Abstract

Arylamine N-acetyltransferase 1 (NAT1) is a drug metabolizing enzyme that influences cancer cell proliferation and survival, especially in breast cancer. Lysine-acetylation is an important Post-Translational Modification (PTM) in the regulation of diverse cellular processes. Histone deacetylases (HDACs) and Sirtuins (SIRT) may have an important role on the NAT1 acetylation status, affecting its catalytic capacity and having an impact on the downstream functions of this protein. The aim of the present work is to investigate the acetylation status of NAT1 in human breast cancer. Breast cancer cell lines MDA-MB-231 (ER-, PR-, HER2-) and ZR-75-1 (estrogen receptor+, PR+, HER2+) were cultured in the presence of HDAC inhibitors (SAHA, TSA) or Sirtuin inhibitors (AGK2, EX527, Sirtinol). Under these conditions, NAT1 protein and gene expression as well as enzymatic activity were quantified. Acetylation of NAT1 protein was evaluated following an immunoprecipitation protocol and acetyl-Lysine quantification. Sirt1 and Sirt2 knockdown were performed and NAT1 protein and NAT1 mRNA expression and catalytic activity were quantified. The treatment of MDA-MB-231 or ZR-75-1 cells with increasing HDAC inhibitors resulted in 2 to 15-fold upregulation in NAT1 message expression. Finally, the catalytic activity of NAT1 in the presence of HDAC inhibition increased 2-fold. Conversely, the inhibition of Sirtuin activity did not cause significant changes in NAT1 message but produced a significant decrease in NAT1 catalytic activity. NAT1 acetylation was higher in the cells treated with HDAC inhibitors, as well as Sirtuin inhibitors. Finally, silencing of Sirt1 and Sirt2 genes by siRNA transient knockdown of each or both genes resulted in reduction of NAT1 protein expression and catalytic activity. The use of HDAC and Sirtuin inhibitors has been demonstrated as a promising powerful therapeutic alternative in various cancers. These inhibitors can significantly attenuate tumor burden by limiting tumor growth and metastasis. These compounds can also induce DNA damage, cell cycle arrest, apoptosis, and autophagy to promote cancer cell death. Several studies have shown that NAT1 is upregulated in cancer cells. The results of the present study show that the acetylation status of NAT1 is an important factor that might have a relevant role in the progression of cancer.

The Arylamine N-Acetyltransferases as Therapeutic Targets in Metabolic Diseases Associated with Mitochondrial Dysfunction

“…Cells treated with TSA increase NAT1 transcription by recruiting Sp1 to the proximal promoter suggesting the NAT1 gene is partially repressed by chromatin condensation. Both TSA and the pan histone deacetylase inhibitor suberoylanilide hydroxamic acid induced NAT1 expression in both MDA-MB-231 and ZR-75-1 cells (Salazar-González et al, 2022). These results are supported by observations in blood cells from patients with acute lymphoblastic leukemia, who had significantly lower NAT1 activity compared to controls and lower acetylated histones associated with the NAT1 promoter (Hernandez-Gonzalez et al, 2023).…”

“…When peripheral blood mononuclear cells were treated with nicotinamide for 3 hours, NAT2 activity increased (Turiján-Espinoza et al, 2018). Moreover, up-regulation of SIRT1 with SRT1720 decreased NAT2 activity whereas activity increased in cells treated with SIRT1 siRNA (Salazar-González et al, 2022). Like NAT1, NAT2 is acetylated, although the specific lysine residues modified remain unknown.…”

“…As noted above, SIRT1 deacetylates NAT1 and NAT2 but with opposing effects on their activities (Section 1C). While SIRT1 activates NAT1 (Butcher et al, 2020;Salazar-González et al, 2018), it inactivates NAT2 (Salazar-González et al, 2022).…”

β-Nicotinamide mononucleotide activates NAD+/SIRT1 pathway and attenuates inflammatory and oxidative responses in the hippocampus regions of septic mice

“…BV-2 cells were pretreated with EX-527 (10 μM) for 1 h, and then stimulated with 1 μg/ml lipopolysaccharide (LPS, Sigma Corporation, USA) and/or NMN (1 mM) for 24 h to establish the inflammatory model in vitro [ 34 ]. The dose of NMN and EX-527 were referred to previous studies [ [35] , [36] , [37] ]. All reagents and procedures were strictly sterile, and all operations were completed in a biological safety cabin.…”

Small Molecule Inhibitors of Arylamine N-Acetyltransferase 1 Attenuate Cellular Respiration

“…29 Increasing protein acetylation using sirtuin inhibitors such as trichostatin A or vorinostat also increases NAT1 expression and activity. 30,31 Aside from their role as experimental tools to study NAT1 function, NAT1 inhibitors may be useful in the future for the treatment of various hypermetabolic diseases. For example, high NAT1 expression in patients with amyotrophic lateral sclerosis correlates with a hypermetabolic state, 32 which predicts poorer outcomes.…”