Human Naa50 Protein Displays Broad Substrate Specificity for Amino-terminal Acetylation: DETAILED STRUCTURAL AND BIOCHEMICAL ANALYSIS USING TETRAPEPTIDE LIBRARY (original) (raw)
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Human Naa50 Protein Displays Broad Substrate Specificity for Amino-terminal Acetylation
Journal of Biological Chemistry, 2016
Amino-terminal acetylation is a critical co-translational modification of the newly synthesized proteins in a eukaryotic cell carried out by six amino-terminal acetyltransferases (NATs). All NATs contain at least one catalytic subunit, and some contain one or two additional auxiliary subunits. For example, NatE is a complex of Naa10, Naa50, and Naa15 (auxiliary). In the present study, the crystal structure of human Naa50 suggested the presence of CoA and acetylated tetrapeptide (AcMMXX) that have co-purified with the protein. Biochemical and thermal stability studies on the tetrapeptide library with variations in the first and second positions confirm our results from the crystal structure that a peptide with Met-Met in the first two positions is the best substrate for this enzyme. In addition, Naa50 acetylated all MXAA peptides except for MPAA. Transcriptome analysis of 10 genes that make up six NATs in humans from eight different cell lines suggests that components of NatE are transcribed in all cell lines, whereas others are variable. Because Naa10 is reported to acetylate all amino termini that are devoid of methionine and Naa50 acetylates all other peptides that are followed by methionine, we believe that NatE complex can be a major contributor for amino-terminal acetylation at the ribosome exit tunnel. Co-translational protein modifications are important biochemical events. Prominent among them are amino-terminal modifications such as methionine cleavage, acetylation, and myristoylation (1-3). Initiator methionine in eukaryotic proteins is removed co-translationally by the enzyme methionine aminopeptidase (MetAP) 3 when the second amino acid is small and uncharged (1). About 70% of the matured proteins do not retain their amino-terminal methionine (4). Similarly, about 90% of all newly synthesized cytosolic proteins undergo aminoterminal acetylation (5). Protein amino-terminal acetylation is carried out by six amino-terminal acetyltransferases (NATs) named sequentially NatA to NatF, classified based on substrate preference (6-8). NatA acetylates the peptides with serine, alanine, threonine, cysteine, and valine on the amino termini that are formed after MetAP action at the ribosome exit tunnel (5, 9), whereas NatB acts on methionine followed by acidic residues in the second position (10, 11). NatC, NatE, and NatF act on methionine that is followed by hydrophobic amino acids like leucine, phenylalanine, isoleucine, and tryptophan (8, 10, 12, 13). Additionally, NatF also acetylates methionine that is followed specifically by a lysine (14). NatD acetylates the amino termini of Ser-Gly of histones H2A and H4 (15). Each of the NATs (NatA-NatF) is a complex of one or two of the enzymes labeled serially as Naa10, Naa20, Naa30, Naa40, Naa50, and Naa60 attached to one or two of the auxiliary proteins named Naa15, Naa25, Naa35, and Naa38 (7, 9). Some NATs are known to bind to the ribosome at the exit tunnel through their auxiliary subunits (11, 16). Protein amino-terminal acetylation has potential effects on cell metabolism. A single nucleotide polymorphism observed in Naa10 results in Ogden syndrome, a lethal X-linked disorder of infancy whose symptoms include an aged appearance, craniofacial anomalies, hypotonia, global developmental delays, cryptorchidism, cardiac arrhythmias, and large eyes leading to death within a year after birth (17). N-Acetylation was shown as a degradation signal for individual proteins and inhibits the targeting of proteins to the endoplasmic reticulum (18, 19). Knockdown of Naa10 results in decreased cell proliferation and induction of apoptosis (20). Naa50 knockdown impaired sister chromatid cohesion and chromosome condensation (21, 22), emphasizing the important role of NATs in human health (23). Liszczak et al. (9) reported the first structure of a heterodimeric NatA complex that consists of Naa10 and Naa15 with an inhibitor that is the conjugate of coenzyme A (CoA) and the substrate-like peptide. Naa50 associates with NatA, and the complex is named as NatE because of differences in the activity compared with NatA (24). Several studies aiming to understand substrate specificities of NATs have been reported (5, 10, 13-15, 25, 26). Understanding substrate specificity of multiple
A novel human NatA Nα-terminal acetyltransferase complex: hNaa16p-hNaa10p (hNat2-hArd1)
BMC Biochemistry, 2009
Background: Protein acetylation is among the most common protein modifications. The two major types are post-translational N ε -lysine acetylation catalyzed by KATs (Lysine acetyltransferases, previously named HATs (histone acetyltransferases) and co-translational N αterminal acetylation catalyzed by NATs (N-terminal acetyltransferases). The major NAT complex in yeast, NatA, is composed of the catalytic subunit Naa10p (N alpha acetyltransferase 10 protein) (Ard1p) and the auxiliary subunit Naa15p (Nat1p). The NatA complex potentially acetylates Ser-, Ala-, Thr-, Gly-, Val-and Cys-N-termini after Met-cleavage. In humans, the homologues hNaa15p (hNat1) and hNaa10p (hArd1) were demonstrated to form a stable ribosome associated NAT complex acetylating NatA type N-termini in vitro and in vivo.
ACS Medicinal Chemistry Letters, 2020
Two novel compounds were identified as Naa50 binders/inhibitors using DNA-encoded technology screening. Biophysical and biochemical data as well as cocrystal structures were obtained for both compounds (3a and 4a) to understand their mechanism of action. These data were also used to rationalize the binding affinity differences observed between the two compounds and a MLGP peptide-containing substrate. Cellular target engagement experiments further confirm the Naa50 binding of 4a and demonstrate its selectivity toward related enzymes (Naa10 and Naa60). Additional analogs of inhibitor 4a were also evaluated to study the binding mode observed in the cocrystal structures.
Composition and biological significance of the human Nalpha-terminal acetyltransferases
2009
Protein N -terminal acetylation is one of the most common protein modifications in eukaryotic cells, occurring on approximately 80% of soluble human proteins. An increasing number of studies links N -terminal acetylation to cell differentiation, cell cycle, cell survival, and cancer. Thus, N terminal acetylation is an essential modification for normal cell function in humans. Still, little is known about the functional role of N -terminal acetylation. Recently, the three major human Nacetyltransferase complexes, hNatA, hNatB and hNatC, were identified and characterized. We here summarize the identified N-terminal acetyltransferase complexes in humans, and we review the biological studies on N -terminal acetylation in humans and other higher eukaryotes.
Molecular determinants of the N-terminal acetyltransferase Naa60 anchoring to the Golgi membrane
The Journal of biological chemistry, 2017
Nα-Acetyltransferase 60 (Naa60 or NatF) was recently identified as an unconventional N-terminal acetyltransferase (NAT) because it localizes to organelles, in particular the Golgi apparatus, and has a preference for acetylating N termini of the transmembrane proteins. This knowledge challenged the prevailing view of N-terminal acetylation as a co-translational ribosome-associated process and suggested a new mechanistic functioning for the enzymes responsible for this increasingly recognized protein modification. Crystallography studies on Naa60 were unable to resolve the C-terminal tail of Naa60, which is responsible for the organellar localization. Here, we combined modeling, in vitro assays, and cellular localization studies to investigate the secondary structure and membrane interacting capacity of Naa60. The results show that Naa60 is a peripheral membrane protein. Two amphipathic helices within the Naa60 C terminus bind the membrane directly in a parallel position relative to t...
Characterization of the human Nα-terminal acetyltransferase B enzymatic complex
BMC Proceedings, 2009
Background: Human N -acetyltransferase complex B (hNatB) is integrated by hNaa20p (hNAT5/ hNAT3) and hNaa25p (hMDM20) proteins. Previous data have shown that this enzymatic complex is implicated in cell cycle progression and carcinogenesis. In yeast this enzyme acetylates peptides composed by methionine and aspartic acid or glutamic acid in their first two positions respectively and it has been shown the same specificity in human cells.
Human Genetics
NAA10 is the catalytic subunit of the N-terminal acetyltransferase complex, NatA, which is responsible for N-terminal acetylation of nearly half the human proteome. Since 2011, at least 21 different NAA10 missense variants have been reported as pathogenic in humans. The clinical features associated with this X-linked condition vary, but commonly described features include developmental delay, intellectual disability, cardiac anomalies, brain abnormalities, facial dysmorphism and/or visual impairment. Here, we present eight individuals from five families with five different de novo or inherited NAA10 variants. In order to determine their pathogenicity, we have performed biochemical characterisation of the four novel variants c.16G>C p.(A6P), c.235C>T p.(R79C), c.386A>C p.(Q129P) and c.469G>A p.(E157K). Additionally, we clinically describe one new case with a previously identified pathogenic variant, c.384T>G p.(F128L). Our study provides important insight into how diff...
Proceedings of the National Academy of Sciences, 2009
N ␣ -terminal acetylation is one of the most common protein modifications in eukaryotes. The COmbined FRActional DIagonal Chromatography (COFRADIC) proteomics technology that can be specifically used to isolate N-terminal peptides was used to determine the N-terminal acetylation status of 742 human and 379 yeast protein N termini, representing the largest eukaryotic dataset of N-terminal acetylation. The major N-terminal acetyltransferase (NAT), NatA, acts on subclasses of proteins with Ser-, Ala-, Thr-, Gly-, Cys-and Val-N termini. NatA is composed of subunits encoded by yARD1 and yNAT1 in yeast and hARD1 and hNAT1 in humans. A yeast ard1-⌬ nat1-⌬ strain was phenotypically complemented by hARD1 hNAT1, suggesting that yNatA and hNatA are similar. However, heterologous combinations, hARD1 yNAT1 and yARD1 hNAT1, were not functional in yeast, suggesting significant structural subunit differences between the species. Proteomics of a yeast ard1-⌬ nat1-⌬ strain expressing hNatA demonstrated that hNatA acts on nearly the same set of yeast proteins as yNatA, further revealing that NatA from humans and yeast have identical or nearly identical specificities. Nevertheless, all NatA substrates in yeast were only partially N-acetylated, whereas the corresponding NatA substrates in HeLa cells were mainly completely N-acetylated. Overall, we observed a higher proportion of N-terminally acetylated proteins in humans (84%) as compared with yeast (57%). N-acetylation occurred on approximately one-half of the human proteins with Met-Lys-termini, but did not occur on yeast proteins with such termini. Thus, although we revealed different N-acetylation patterns in yeast and humans, the major NAT, NatA, acetylates the same substrates in both species.
N-terminal acetylation and other functions of Nα-acetyltransferases
Biological Chemistry, 2012
Protein N-terminal acetylation by N α-acetyltransferases (NATs) is an omnipresent protein modifi cation that affects a large number of proteins. The exact biological role of N-terminal acetylation has, however, remained enigmatic for the overall majority of affected proteins, and only for a rather small number of proteins, N-terminal acetylation was linked to various protein features including stability, localization, and interactions. This minireview tries to summarize the recent progress made in understanding the functionality of N-terminal protein acetylation and also focuses on noncanonical functions of the NATs subunits.
N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB
Proceedings of the National Academy of Sciences, 2012
Protein N-terminal acetylation (Nt-acetylation) is an important mediator of protein function, stability, sorting, and localization. Although the responsible enzymes are thought to be fairly well characterized, the lack of identified in vivo substrates, the occurrence of Nt-acetylation substrates displaying yet uncharacterized N-terminal acetyltransferase (NAT) specificities, and emerging evidence of posttranslational Nt-acetylation, necessitate the use of genetic models and quantitative proteomics. NatB, which targets Met-Glu-, Met-Asp-, and Met-Asn-starting protein N termini, is presumed to Nt-acetylate 15% of all yeast and 18% of all human proteins. We here report on the evolutionary traits of NatB from yeast to human and demonstrate that ectopically expressed hNatB in a yNatB-Δ yeast strain partially complements the natB -Δ phenotypes and partially restores the yNatB Nt-acetylome. Overall, combining quantitative N-terminomics with yeast studies and knockdown of hNatB in human cel...