A Saccharomyces cerevisiae Model Reveals In Vivo Functional Impairment of the Ogden Syndrome N-Terminal Acetyltransferase NAA10 Ser37Pro Mutant

Abstract

N-terminal acetylation (Nt-acetylation) occurs on the majority of eukaryotic proteins and is catalyzed by N-terminal acetyltransferases (NATs). Nt-acetylation is increasingly recognized as a vital modification with functional implications ranging from protein degradation to protein localization. Although early genetic studies in yeast demonstrated that NAT-deletion strains displayed a variety of phenotypes, only recently, the first human genetic disorder caused by a mutation in a NAT gene was reported; boys diagnosed with the X-linked Ogden syndrome harbor a p.Ser37Pro (S37P) mutation in the gene encoding Naa10, the catalytic subunit of the NatA complex, and suffer from global developmental delays and lethality during infancy. Here, we describe a Saccharomyces cerevisiae model developed by introducing the human wild-type or mutant NatA complex into yeast lacking NatA (NatA-⌬). The wild-type human NatA complex phenotypically complemented the NatA-⌬ strain, whereas only a partial rescue was observed for the Ogden mutant NatA complex suggesting that hNaa10 S37P is only partially functional in vivo. Immunoprecipitation experiments revealed a reduced subunit complexation for the mutant hNatA S37P next to a reduced in vitro catalytic activity. We performed quantitative Nt-acetylome analyses on a control yeast strain (yNatA), a yeast NatA deletion strain (yNatA-⌬), a yeast NatA deletion strain expressing wild-type human NatA (hNatA), and a yeast NatA deletion strain expressing mutant human NatA (hNatA S37P). Interestingly, a generally reduced degree of Nt-acetylation was observed among a large group of NatA substrates in the yeast expressing mutant hNatA as compared with yeast expressing wild-type hNatA. Combined, these data provide strong support for the functional impairment of hNaa10 S37P in vivo and suggest that reduced Nt-acetylation of one or more target substrates contributes to the pathogenesis of the Ogden syndrome. Comparative analysis between human and yeast NatA also provided new insights into the co-evolution of the NatA complexes and their substrates. For instance, (Met-)Ala-

The world of protein acetylation

“…In yeast, the Naa10 S37P mutation displayed impaired functionality in vivo. Yeast cells expressing the mutant human Naa10-Naa15 complex displayed a reduced degree of Nt-acetylation of NatA substrates as compared to yeast cells expressing the wildtype human NatA complex [124]. Another exome sequence study identified a NAA10 missense mutation, p. R116W (exon 5), in an intellectual disability syndrome [125].…”

The biological functions of Naa10 — From amino-terminal acetylation to human disease

“…Today it is believed that the majority of the proteome of higher organisms is fully or partially acetylated. In fact, recent large-scale proteomics analyses have identified peptides that were fully or partially acetylated at their designated N-terminus in the following percentages: 13–19% in Halobacterium salinarum and Natronomonas pharaonis (Falb et al, 2006; Aivaliotis et al, 2007), 29% in Haloferax volcanii (Kirkland et al, 2008), about 16% in 45 tested bacteria (Bonissone et al, 2013), 60–70% in Saccharomyces cerevisiae (Arnesen et al, 2009b; Van Damme et al, 2011c, 2014; Bonissone et al, 2013), 75% in Drosophila melanogaster (Goetze et al, 2009), 90% in Arabidopsis thaliana (Bienvenut et al, 2012), at least 4% in Caenorhadbitis elegans (Mawuenyega et al, 2003), 83% in mouse (Lange and Overall, 2011), 90% in human erythrocytes (Lange et al, 2014) and 85% in HeLa cells (Arnesen et al, 2009b; Van Damme et al, 2011c). However, these values do not necessarily reflect the whole proteomes.…”

“…Furthermore, proteomic analyses identified NTA of internal peptides, further supporting the idea of post-translational acetylation (Helbig et al, 2010; Helsens et al, 2011). This is especially interesting for many proteins that are imported into organelles, after which the cleaved mature N-terminus of the protein (now missing its target/transit peptide) is acetylated by dedicated NATs that reside in the respective target organelle as shown for yeast mitochondrial localized proteins (Van Damme et al, 2014) or chloroplast proteins in Chlamydomonas reinhardtii and A. thaliana (Zybailov et al, 2008; Bienvenut et al, 2011, 2012). …”

“…(Met)-Ala-N-termini are more prevalent in the human proteome, whereas (Met)-Ser-N-termini are more abundant in the yeast proteome (Van Damme et al, 2011c). Accordingly, hNatA displays a preference towards these Ala-N-termini whereas yNatA seems to be the more efficient in acetylating Ser-starting N-termini, indicating that NatA substrate specificity/efficiency of NTA has co-evolved with the repertoire of NatA type substrates expressed (Van Damme et al, 2014). …”

Protein amino-terminal modifications and proteomic approaches for N-terminal profiling

“…COFRADIC is widely applied. Recent studies utilizing the COFRADIC strategy include label free quantitation of the N-acetylome in yeast expressing human wild type and mutant N-acetyltransferase [46], as well as the annotation and validation of 534 protein N-termini in the marine bacterium Roseobacter denitrificans OCh114 [47] as well as the interrogation of differential cleavage specificity of human and murine granzyme A [48]. In addition, COFRADIC can be combined with stable isotope labeling for quantitative investigations [49,50].…”