Functional assessment of N-terminal acetyltransferase variants

N-terminal (Nt)-acetylation is a key protein modification within cells, catalyzed by a family of N-terminal acetyltransferases (NATs). Nt-acetylation can affect various cellular processes by influencing protein interactions, subcellular targeting, protein folding and stability. Pathogenic variants in genes encoding NATs have been linked to rare genetic disorders in humans. To evaluate the pathogenicity of NAT variants, understanding their impact on NAT functionality is essential. This chapter presents protocols for two methods to screen the Nt-acetylation capacity of NAT variants in cells or in vitro. The Nt-specific antibody-based assay utilizes antibodies that specifically target Nt-acetylated proteins to determine their Nt-acetylation level in cells. The immunoprecipitation-based acetylation assay measures Nt-acetylation activity in vitro using enzymes immunoprecipitated from human cells. Together, these methods can provide complementary insights into the functional implications of NAT variants.