Characterization of NAA10 mutations in patients exhibiting phenotypes associated with N-terminal acetylation deficiency
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Approximately 80% of the proteins found in human cells are acetylated on their N-terminus, either partially or completely, by a group of enzymes called N-terminal acetyltransferases (NATs)^1. To date, seven NATs, NatA-NatF^2–7 and NatH^8, have been identified in humans; NatA-NatE associate with ribosomes and acetylate their substrates co-translationally, NatF associate with the Golgi apparatus membrane and acetylate membrane proteins posttranslationally, and NatH is found in the cytosol where it acetylates actins, also posttranslationally^8,9. The NatA complex, consisting of catalytic subunit NAA10 and auxiliary subunit NAA15 facilitating the association of the complex with the ribosome, has the most protein N-termini targets among the NATs, and is also the most studied complex^2,9. The function of NatA and uncomplexed NAA10 in the cell is gradually being characterized by an increasing number of studies on patient identified NAA10 mutations^10–14. The patients share a wide spectrum of phenotypes, most commonly developmental delay, growth deficiency, post-natal growth failure, and cardiac and skeletal anomalies^13. This thesis’ main focus is to functionally characterize two novel NAA10 missense mutations, C21G and R83H, identified in three male patients presenting with typical NAA10 deficiency. Characterization involves in vitro acetylation assay of uncomplexed recombinant MBPNAA10, cellular stability assay of NAA10 and NatA, and bioinformatic assessments of structural functions as well as conservation and in silico prediction of disease. The work presented here, demonstrates that both mutations are found in highly conserved regions in NAA10 involved with Ac-CoA and substrate binding as well as NatA complex formation. The acetylation assay show that while the R83H mutant has profoundly reduced catalytic activity, the C21G mutant show an increased activity. From the cellular stability assay, none of the mutations show any apparent destabilizing effects. However, to draw conclusions about the functional impact of these mutations on the NAA10 and NatA and their link to human health, more research is needed than is presented here in this thesis.