Proteome-derived Peptide Libraries Allow Detailed Analysis of the Substrate Specificities of Nα-acetyltransferases and Point to hNaa10p as the Post-translational Actin Nα-acetyltransferase

Abstract

The impact of N ␣ -terminal acetylation on protein stability and protein function in general recently acquired renewed and increasing attention. Although the substrate specificity profile of the conserved enzymes responsible for N ␣ -terminal acetylation in yeast has been well documented, the lack of higher eukaryotic models has hampered the specificity profile determination of N ␣ -acetyltransferases (NATs) of higher eukaryotes. The fact that several types of protein N termini are acetylated by so far unknown NATs stresses the importance of developing tools for analyzing NAT specificities. Here, we report on a method that implies the use of natural, proteome-derived modified peptide libraries, which, when used in combination with two strong cation exchange separation steps, allows for the delineation of the in vitro specificity profiles of NATs. The human NatA complex, composed of the auxiliary hNaa15p (NATH/hNat1) subunit and the catalytic hNaa10p (hArd1) and hNaa50p (hNat5) subunits, cotranslationally acetylates protein N termini initiating with Ser, Ala, Thr, Val, and Gly following the removal of the initial Met. In our studies, purified hNaa50p preferred Met-Xaa starting N termini (Xaa mainly being a hydrophobic amino acid) in agreement with previous data. Surprisingly, purified hNaa10p preferred acidic N termini, representing a group of in vivo acetylated proteins for which there are currently no NAT(s) identified. The most prominent representatives of the group of acidic N termini are ␥-and ␤-actin. Indeed, by using an independent quantitative assay, hNaa10p strongly acetylated peptides representing the N termini of both ␥-and ␤-actin, and only to a lesser extent, its previously characterized substrate motifs. The immunoprecipitated NatA complex also acetylated the actin N termini efficiently, though displaying a strong shift in specificity toward its known Ser-starting type of substrates. Thus, complex formation of NatA might alter the substrate specificity profile as compared with its isolated catalytic subunits, and, furthermore,

NAA10 controls osteoblast differentiation and bone formation as a feedback regulator of Runx2

“…A growing body of evidence has suggested that NAA10 has diverse functions beyond the cotranslational modification. NAA10 can catalyse amino-terminal a-acetylation or lysine e-acetylation in many proteins as post-translational modification [17][18][19]30 , and our results provide additional support for this role of NAA10. Moreover, given our result showing that recombinant NAA10 catalyses lysyl acetylation without auxiliary partners, NAA10 is likely to have its enzymatic activity as a monomer.…”

N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB

“…Conversely, the immature MEEE-variant of γ-actin, of which the Nt-acetylation level was unaffected by NatB knockdown, was found to be significantly up-regulated (P ≤ 0.01) in both sihNatB setups-4-to 10-fold when going from 80-95% of knockdown-but expression of its mature EEE-variant remained unaffected. hNaa10p and hNatA were recently found to Nt-acetylate the mature actin N termini (30) and, to complete the current view on actin Nt- acetylation, we pursued if hNatB can Nt-acetylate the immature β-and γ-actin N termini. Quantitative HPLC-based in vitro acetylation assays indeed demonstrated Nt-acetylation of the immature β-actin N-terminal peptide (MDDD-) by immunoprecipitated hNatB but not of the mature β-and γ-actin N termini (Fig.…”

The N-terminal Acetyltransferase Naa10/ARD1 Does Not Acetylate Lysine Residues

“…These peptides were ϳ20 residues long and contained the reported lysine in the center of the peptide. We assayed three reported substrates: MLCK, MSRA, and Runx2, and a peptide corresponding to the N terminus of actin, a known Naa10 substrate, as a positive control (9,10). We carried out acetyltransferase assays with a saturating concentration of peptide (1 mM) and radiolabeled acetyl-CoA and measured the signal by scintillation counting.…”

“…Structural and enzymatic studies reveal that the enzymatic subunit Naa10 has a different substrate profile when it is not in complex with the auxiliary subunit Naa15. As a monomer, Naa10 acetylates N termini in which the N-terminal residues are acidic, such as actin, whose first three N-terminal residues are glutamates (9,10).…”