NAA10 p.(D10G) and NAA10 p.(L11R) variants hamper formation of the NatA N-terminal acetyltransferase complex

The majority of the human proteome is subjected to N-terminal (Nt) acetylation catalysed by N-terminal acetyltransferases (NATs). The NatA complex is composed of two core subunits—the catalytic subunit NAA10 and the ribosomal anchor NAA15. Furthermore, NAA10 may also have catalytic and non-catalytic roles independent of NatA. Several inherited and de novo NAA10 variants have been associated with genetic disease in humans. In this study, we present a functional analysis of two de novo NAA10 variants, c.29A>G p.(D10G) and c.32T>G p.(L11R), previously identified in a male and a female, respectively. Both of these neighbouring amino acids are highly conserved in NAA10. Immunoprecipitation experiments revealed that both variants hamper complex formation with NAA15 and are thus likely to impair NatA-mediated Nt-acetylation in vivo. Despite their common impact on NatA formation, in vitro Nt-acetylation assays showed that the variants had opposing impacts on NAA10 catalytic activity. While NAA10 c.29A>G p.(D10G) exhibits normal intrinsic NatA activity and reduced monomeric NAA10 NAT activity, NAA10 c.32T>G p.(L11R) displays reduced NatA activity and normal NAA10 NAT activity. This study expands the scope of research into the functional consequences of NAA10 variants and underlines the importance of understanding the diverse cellular roles of NAA10 in disease mechanisms.publishedVersio

Functional characterization of N-terminal acetyltransferase 10 (NAA10) variants potentially causing disease

Approximately 80-90 % of all eukaryotic proteins are co- or post-translationally acetylated on their N-terminus by a group of enzymes called N-terminal acetyltransferases (NATs) (Arnesen et al., 2009). To date, eight NATs have been identified in eukaryotes, seven of which (NatA-NatF and NatH) are found in humans. Each of the NATs differ in subunit composition and have a distinct substrate specificity (Aksnes et al., 2019). The NatA complex is conserved from yeast to humans, acetylating approximately 40 % of the human proteome (Arnesen et al., 2009). NatA is composed of the catalytic subunit NAA10 and the auxiliary subunit NAA15 and has the broadest substrate specificity among the NATs (Arnesen et al., 2005a, Liszczak et al., 2013). In 2011, Rope et al., reported a NAA10 S37P missense mutation to be the cause of the lethal X-linked disorder named Ogden syndrome (Rope et al., 2011). Some years later, Esmailpour and colleagues reported that the genetically heterogeneous X-linked disorder Lenz microphtalmia syndrome (LMS) was caused by a splice mutation in NAA10 (Esmailpour et al., 2014). The last decade, several other NAA10 mutations have been reported to have pathological effects in the harboring patient. Intellectual disability, development delay, growth deficiency and cardiac and skeletal anomalies are among the most common phenotypes coupled to NAA10 deficiency. The focus of this thesis has been to functionally characterize two missense mutations in NAA10 suspected to cause disease in humans. These mutations are NAA10 L11R and NAA10 H16P, which were identified in female patients presenting with some symptoms typical of NAA10 deficiency. NatA complex formation and in vitro intrinsic catalytic activity, and cellular stability have been characterized, and bioinformatic analyses have been performed. The work presented in this thesis demonstrates that the NAA10 L11R variant and H16P variants have a reduced NatA complex formation and their NatA activity is functionally impaired. The L11R variant affects NatA activity to a smaller extent than the H16P mutation. In the cellular stability assay, the NAA10 L11R had a destabilizing effect, whereas NAA10 H16P appears stable and is unlikely to affect neither monomeric NAA10 nor NatA stability. The study presented in this thesis support that these variants are pathological, yet further studies are needed to define the detailed underlying mechanisms

NAA10 p.(D10G) and NAA10 p.(L11R) variants hamper formation of the NatA N-terminal acetyltransferase complex

The majority of the human proteome is subjected to N-terminal (Nt) acetylation catalysed by N-terminal acetyltransferases (NATs). The NatA complex is composed of two core subunits—the catalytic subunit NAA10 and the ribosomal anchor NAA15. Furthermore, NAA10 may also have catalytic and non-catalytic roles independent of NatA. Several inherited and de novo NAA10 variants have been associated with genetic disease in humans. In this study, we present a functional analysis of two de novo NAA10 variants, c.29A>G p.(D10G) and c.32T>G p.(L11R), previously identified in a male and a female, respectively. Both of these neighbouring amino acids are highly conserved in NAA10. Immunoprecipitation experiments revealed that both variants hamper complex formation with NAA15 and are thus likely to impair NatA-mediated Nt-acetylation in vivo. Despite their common impact on NatA formation, in vitro Nt-acetylation assays showed that the variants had opposing impacts on NAA10 catalytic activity. While NAA10 c.29A>G p.(D10G) exhibits normal intrinsic NatA activity and reduced monomeric NAA10 NAT activity, NAA10 c.32T>G p.(L11R) displays reduced NatA activity and normal NAA10 NAT activity. This study expands the scope of research into the functional consequences of NAA10 variants and underlines the importance of understanding the diverse cellular roles of NAA10 in disease mechanisms

Purification and Characterization of Native and Vaccine Candidate Mutant Enterotoxigenic Escherichia coli Heat-Stable Toxins

Enterotoxigenic Escherichia coli (ETEC), which secretes the heat-stable toxin (ST) is among the four most important enteropathogens that cause moderate-to-severe diarrhea in children in low- and middle-income countries. ST is an intestinal molecular antagonist causing diarrhea and hence an attractive vaccine target. A non-toxic and safe ST vaccine should include one or more detoxifying mutations, and rigorous characterization of such mutants requires structurally intact peptides. To this end, we established a system for purification of ST and ST mutants by fusing the sequence encoding the mature ST peptide to the disulfide isomerase DsbC. A Tobacco Etch Virus protease cleavage site facilitates the proteolytic release of free ST with no additional residues. The purified ST peptides have the expected molecular masses, the correct number of disulfide bridges, and have biological activities and antigenic properties comparable to ST isolated from ETEC. We also show that free DsbC can assist in refolding denatured and misfolded ST in vitro. Finally, we demonstrate that the purification system can be used to produce ST mutants with an intact neutralizing epitope, that two single mutations, L9S and A14T, reduce toxicity more than 100-fold, and that the L9S/A14T double mutant has no measurable residual toxicity