A pilot study of the modulation of sirtuins on arylamine N-acetyltransferase 1 and 2 enzymatic activity

Abstract

Arylamine N-acetyltransferase (NAT; E.C. 2.3.1.5) enzymes are responsible for the biotransformation of several arylamine and hydrazine drugs by acetylation. In this process, the acetyl group transferred to the acceptor substrate produces NAT deacetylation and, in consequence, it is susceptible of degradation. Sirtuins are protein deacetylases, dependent on nicotine adenine dinucleotide, which perform post-translational modifications on cytosolic proteins. To explore possible sirtuin participation in the enzymatic activity of arylamine NATs, the expression levels of NAT1, NAT2, SIRT1 and SIRT6 in peripheral blood mononuclear cells (PBMC) from healthy subjects were examined by flow cytometry and Western blot. The in situ activity of the sirtuins on NAT enzymatic activity was analyzed by HPLC, in the presence or absence of an agonist (resveratrol) and inhibitor (nicotinamide) of sirtuins. We detected a higher percentage of positive cells for NAT2 in comparison with NAT1, and higher numbers of SIRT1+ cells compared to SIRT6 in lymphocytes. In situ NAT2 activity in the presence of NAM inhibitors was higher than in the presence of its substrate, but not in the presence of resveratrol. In contrast, the activity of NAT1 was not affected by sirtuins. These results showed that NAT2 activity might be modified by sirtuins.

Synergistic antitumor activity of artesunate and HDAC inhibitors through elevating heme synthesis via synergistic upregulation of ALAS1 expression

“…After treated with the compounds for the indicated times, cells were lysed by RIPA buffer (150 mmol/L NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mmol/L Tris-HCl, pH 7.5) containing protease and phosphatase inhibitor cocktail (Merck Millipore, Billerica, MA, USA) with freshly added 1 mmol/L of PMSF. After centrifugation at 12,000 rpm (MicroCL 17R, Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C for 20 min, supernatants were collected and equal amounts of protein were subjected to Western blot assay as described before37, 38. After being visualized using an electrochemiluminescence kit (Sudgen, China), luminescence was assessed by chemiluminescent imaging system (Tanon, Shanghai, China).…”

The function of histone acetylation in cervical cancer development

“…The mechanism behind this phenomenon is H3K56 acetylation [36]. Using HeLa cells as positive control researchers found that once deacetylation function of sirtuins was suppressed, they were impossible to remove the acetyl groups from arylamine N -acetyltransferase 2 (NAT2), making this protein more stable, thereby increasing the activity of NAT2 [37]. The acetylation of P53 increased in Sirt1 silenced HeLa cells.…”

Expression and genotype-dependent catalytic activity of N-acetyltransferase 2 (NAT2) in human peripheral blood mononuclear cells and its modulation by Sirtuin 1

“…A regulation mechanism proposed for N-acetyltransferase 1 suggests a role for acetylation/deacetylation equilibrium that confers stability to the protein and prevents lysine deacetylation and ubiquitin degradation [23]. A recent report provided preliminary data in support of Sirtuin modulation of NAT2 [24]. …”

“…NAT2 and SIRT1 expression were evaluated on PBMC using different combinations of monoclonal antibodies on the T, B or NK cell subpopulations as previously reported [24, 33, 34]. Briefly, to detect CD3, CD19 or CD56 molecules on plasmatic membrane an incubation step using the corresponding monoclonal antibodies, anti-CD3-PE, anti-CD19-PE or anti-CD56-PE was performed (BD Biosciences, San Jose, CA, USA).…”

“…The N -acetylation capacity of PBMC was evaluated as previously reported [24, 33]. Briefly, 2×10 5 previously purified PBMC were plated on Corning® Costar® 96-Well Cell Culture Plates (Millipore-Sigma, St. Louis, MO, USA), and cultured on pre-warmed DMEM media supplemented with 50 U/mL penicillin and 50 μg/mL streptomycin (Lonza, Basel, SUI).…”