Abstract
N-terminal acetylation, catalysed by N-terminal acetyltransferases (NATs), is among the most common protein modifications in eukaryotes and involves the transfer of an acetyl group from acetyl-CoA to the α-amino group of the first amino acid. Functions of N-terminal acetylation include protein degradation and sub-cellular targeting. Recent findings in humans indicate that a dysfunctional Nα-acetyltransferase (Naa) 10, the catalytic subunit of NatA, the major NAT, is associated with lethality during infancy. In the present study, we identified the Danio rerio orthologue zebrafish Naa 10 (zNaa10). In vitro N-terminal acetylation assays revealed that zNaa10 has NAT activity with substrate specificity highly similar to that of human Naa10. Spatiotemporal expression pattern was determined by in situ hybridization, showing ubiquitous expression with especially strong staining in brain and eye. By morpholino-mediated knockdown, we demonstrated that naa10 morphants displayed increased lethality, growth retardation and developmental abnormalities like bent axis, abnormal eyes and bent tails. In conclusion, we identified the zebrafish Naa10 orthologue and revealed that it is essential for normal development and viability of zebrafish.
Highlights
Nα-terminal acetylation (Nt-acetylation), the process in which an acetyl group from acetyl-CoA is transferred on to the α amino group of a protein’s first amino acid residue, occurs on the majority of eukaryotic proteins [1,2,3,4]
Whereas earlier work suggested that Nt-acetylation protects proteins from degradation [5,6], possibly by blocking ubiquitination of the N-terminus [7], more recent studies demonstrate that this modification is functionally implicated in targeting proteins for degradation via a novel branch of the N-end rule pathway, probably as a way of degrading misfolded proteins or maintaining appropriate stoichiometries of protein subunits [8,9,10]
The putative zebrafish Naa10 is highly similar to Naa10 of other eukaryotes The amino acid sequences of the known human N-terminal acetyltransferase (NAT) (Naa10, Naa11, Naa20, Naa30, Naa40, Naa50 and Naa60) were entered into the pBLAST search engine, searching the zebrafish proteome
Summary
Nα-terminal acetylation (Nt-acetylation), the process in which an acetyl group from acetyl-CoA is transferred on to the α amino group of a protein’s first amino acid residue, occurs on the majority of eukaryotic proteins [1,2,3,4]. The number of affected substrates, the severity of knockdown phenotypes across several model systems and implications in disease and development all point to a critical role of Naa in normal cell function. We identified a zebrafish Naa (zNaa10) candidate and confirmed a high degree of similarity between its sequence and those of characterized Naa10-proteins from other species. The zNaa10-candidate was recombinantly expressed and purified and the substrate specificity, as assessed by in vitro Nt-acetylation assays, revealed that this protein was zNaa. The acetyltransferase assay was performed by incubating purified MBP– zNaa (120 nM) or MBP–hNaa (50 nM) in acetylation buffer (50 mM Tris/HCl, 1 mM DTT, 0.2 mM EDTA, pH 8.5) with 300 μM substrate peptide (Biogenes) and 300 μM acetyl-CoA (Sigma–Aldrich). PCR validation of naa splice inhibition RNA was isolated from 1 dpf embryos and used to make a cDNA library via reverse transcriptase, as described above. Embryos were cleared using Murray’s clear and imaged using a Leica m420 microscope with a CoolSNAP-Pro digital camera (Media cybernetics)
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