Influence of N‐acetyltransferase polymorphism in the N‐acetylation of asparagine and putrescine
Authors: Salazar‐Gonzalez Raul A. , Hein David W. 
Published: May 2022

Amino acids play central roles in cancer progression beyond their function as building blocks for protein synthesis. Because of this, targeting amino acid acquisition and uptake has been proved to be a potential therapeutic strategy to limit cancer cell growth while leaving the normal tissues largely intact. Polyamines like Putrescine (Put) are low molecular weight, aliphatic polycations found in the cells of all living organisms. Recent findings have described that abundance of N‐acetylated Asparagine (Asp) and Put are highly correlated with N‐acetylation capacity of N‐acetyltransferase 1 (NAT1). Here we tested the N‐acetylation of Asp and Put by recombinant human NAT1 and NAT2 as well as the effect of N‐acetylation polymorphisms in the metabolism of these substrates. Using genetically engineered CHO cells expressing either NAT1*4, NAT1*14, NAT2*4 or NAT2*5B alleles, in vitro N‐acetylation activity reactions were quantified using HPLC. Our results show that Asp is metabolized by both NAT1 and NAT2; enzymatic activity was quantified at different time points up to 60 min, as well as, increasing concentrations of the cofactor AcCoA. Using 100 μM of AcCoA, the formation of N‐acetyl Asp was 1.7 ±0.32 nmoles Ac‐Asp/mg protein in the reference allele (NAT1*4); on the other hand, NAT1*14 (slow) allele produced 1.12 ±0.32 nmoles Ac‐Asp/mg protein (p< 0.01); increasing AcCoA to 300 μM resulted in an increase 3.98 ±0.58 and 2.93 ±0.62 nmoles Ac‐Asp/mg protein, respectively (p< 0.01), Finally, Increasing the availability of AcCoA to 1 mM, produced an N‐acetylation rate of 9.82 ±3.73 and 4.64 ±0.61 nmoles Ac‐Asp/mg protein (p< 0.001). For NAT2, in the presence of 100 μM of AcCoA, the reference allele (NAT2*4) produced 1.43 ±0.6, whereas NAT2*5B (slow) produced 1.05 ±0.19 nmoles Ac‐Asp/mg protein (p< 0.05). 300 μM of AcCoA increased the rate to 3.1 ±0.93 vs 2.4 ±0.52 nmoles Ac‐Asp/mg protein (p< 0.05). Finally, using 1 mM AcCoA, N‐acetylation increased to 7.23 ±2.69 vs 5.11 ±0.79 nmoles Ac‐Asp/mg protein (p< 0.01). Both NAT1 and NAT2 catalyzed the Put N‐acetylation. For NAT1 in the presence of 100 μM of AcCoA, we observed the formation of 0.19 ±0.03 nmoles Ac‐Put/mg protein in NAT1*4, and 0.108 ±0.02 nmoles Ac‐Put/mg protein in NAT1*14 allele (p< 0.05). Using 300 μM of AcCoA, 0.27 ±0.02 in NAT1*4 vs 0.14 ±0.3 nmoles Ac‐Put/mg protein in NAT1*14 (p< 0.05). Finally, 1 mM of AcCoA yielded 0.43 ±0.03 vs 0.23 ±0.03 nmoles Ac‐Put/mg protein, respectively (p< 0.05). Finally, for NAT2 and Put N‐acetylation, our results show that the reference allele produces 0.26 ±0.03 vs 0.16 ±0.04 nmoles Ac‐Put/mg protein in the slow (NAT2*5B) allele when we used 100 μM of AcCoa (p< 0.05), 0.3 ±0.03 and 0.22 ±0.02 nmoles Ac‐Put/mg protein respectively when we used 300 μM of AcCoA; and 0.4 ±0.03 vs 0.3 ±0.02 nmoles Ac‐Put/mg protein respectively when we used 1 mM AcCoA. In conclusion, these results show that NAT1 and NAT2 are capable of the N‐acetylation of endogenous compounds like Asparagine and Putrescine. Moreover, this metabolic reaction is dependent of the AcCoA present and influenced by the genetic polymorphisms. Future studies will help us understand the implications of the N‐acetylation of these compounds in cellular processes like cancer cell progression.