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Charting the N-Terminal Acetylome: A Comprehensive Map of Human NatA Substrates.

ABSTRACT: N-terminal acetylation (Nt-acetylation) catalyzed by conserved N-terminal acetyltransferases or NATs embodies a modification with one of the highest stoichiometries reported for eukaryotic protein modifications to date. Comprising the catalytic N-alpha acetyltransferase (NAA) subunit NAA10 plus the ribosome anchoring regulatory subunit NAA15, NatA represents the major acetyltransferase complex with up to 50% of all mammalian proteins representing potential substrates. Largely in consequence of the essential nature of NatA and its high enzymatic activity, its experimentally confirmed mammalian substrate repertoire remained poorly charted. In this study, human NatA knockdown conditions achieving near complete depletion of NAA10 and NAA15 expression resulted in lowered Nt-acetylation of over 25% out of all putative NatA targets identified, representing an up to 10-fold increase in the reported number of substrate N-termini affected upon human NatA perturbation. Besides pointing to less efficient NatA substrates being prime targets, several putative NatE substrates were shown to be affected upon human NatA knockdown. Intriguingly, next to a lowered expression of ribosomal proteins and proteins constituting the eukaryotic 48S preinitiation complex, steady-state levels of protein N-termini additionally point to NatA Nt-acetylation deficiency directly impacting protein stability of knockdown affected targets.

SUBMITTER: Van Damme P

PROVIDER: S-EPMC8509067 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A novel human NatA Nalpha-terminal acetyltransferase complex: hNaa16p-hNaa10p (hNat2-hArd1).

Project description:BACKGROUND:Protein acetylation is among the most common protein modifications. The two major types are post-translational Nepsilon-lysine acetylation catalyzed by KATs (Lysine acetyltransferases, previously named HATs (histone acetyltransferases) and co-translational Nalpha-terminal 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. RESULTS:We here describe a novel human protein, hNaa16p (hNat2), with 70% sequence identity to hNaa15p (hNat1). The gene encoding hNaa16p originates from an early vertebrate duplication event from the common ancestor of hNAA15 and hNAA16. Immunoprecipitation coupled to mass spectrometry identified both endogenous hNaa15p and hNaa16p as distinct interaction partners of hNaa10p in HEK293 cells, thus demonstrating the presence of both hNaa15p-hNaa10p and hNaa16p-hNaa10p complexes. The hNaa16p-hNaa10p complex acetylates NatA type N-termini in vitro. hNaa16p is ribosome associated, supporting its potential role in cotranslational Nalpha-terminal acetylation. hNAA16 is expressed in a variety of human cell lines, but is generally less abundant as compared to hNAA15. Specific knockdown of hNAA16 induces cell death, suggesting an essential role for hNaa16p in human cells. CONCLUSION:At least two distinct NatA protein Nalpha-terminal acetyltransferases coexist in human cells potentially creating a more complex and flexible system for Nalpha-terminal acetylation as compared to lower eukaryotes.

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Overlap of NatA and IAP substrates implicates N-terminal acetyltion in protein stabilization

Project description:SMAC/DIABLO and HTRA2 are mitochondrial proteins whose N-termina sequences, known as inhibitor of apoptosis binding motifs (IBMs), bind and activate ubiquitin ligases knwon as inhibitor of apoptosis proteins (IAPs), unleashing a cell´s poteintial.IBMs comprise a 4-residue, loose consensus sequence, and binding to IAPs requires an unmodified N-terminus. Closely related, IBM-like N-termini, are present in approximately 5% of human proteins. We show that suppression of the N-alpha-acetytransferase NatA truns these cryptic IBM-like sequences into very efficient IAP binders in cell lysates and in vitro, and ultimately also triggers apoptosis. Thus, N-terminal acetylation of IBM-like motifs in NatA substrates shields them from IAPs. This previously unrecognized relatonship suggests that N-terminal acetylation is generally protective against protein degradation in human cells. It also identifies IAPs as agents of a general quality control mechanism targeting unacetylated rogues in metazoans

2021-09-09 | PXD021815 | Pride

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Project description:The study of the human urinary proteome has the potential to offer significant insights into normal physiology as well as disease pathology. The information obtained from such studies could be applied to the diagnosis of various diseases. The high sensitivity, resolution, and mass accuracy of the latest generation of mass spectrometers provides an opportunity to accurately catalog the proteins present in human urine, including those present at low levels. To this end, we carried out a comprehensive analysis of human urinary proteome from healthy individuals using high-resolution Fourier transform mass spectrometry. Importantly, we used the Orbitrap for detecting ions in both MS (resolution 60 000) and MS/MS (resolution 15 000) modes. To increase the depth of our analysis, we characterized both unfractionated as well as lectin-enriched proteins in our experiments. In all, we identified 1,823 proteins with less than 1% false discovery rate, of which 671 proteins have not previously been reported as constituents of human urine. This data set should serve as a comprehensive reference list for future studies aimed at identification and characterization of urinary biomarkers for various diseases.

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Project description:In eukaryotes, N-terminal acetylation is one of the most common protein modifications involved in a wide range of biological processes. Most N-acetyltransferase complexes (NATs) act co-translationally, with the heterodimeric NatA complex modifying the majority of substrate proteins. Here we show that the Huntingtin yeast two-hybrid protein K (HypK) binds tightly to the NatA complex comprising the auxiliary subunit Naa15 and the catalytic subunit Naa10. The crystal structures of NatA bound to HypK or to a N-terminal deletion variant of HypK were determined without or with a bi-substrate analogue, respectively. The HypK C-terminal region is responsible for high-affinity interaction with the C-terminal part of Naa15. In combination with acetylation assays, the HypK N-terminal region is identified as a negative regulator of the NatA acetylation activity. Our study provides mechanistic insights into the regulation of this pivotal protein modification.

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Project description:N-terminal acetylation is a prominent protein modification, and inactivation of N-terminal acetyltransferases (NATs) cause protein homeostasis stress. Using multiplexed protein stability profiling with linear ubiquitin fusions as reporters for the activity of the ubiquitin proteasome system, we observed increased ubiquitin proteasome system activity in NatA, but not NatB or NatC mutants. We find several mechanisms contributing to this behavior. First, NatA-mediated acetylation of the N-terminal ubiquitin-independent degron regulates the abundance of Rpn4, the master regulator of the expression of proteasomal genes. Second, the abundance of several E3 ligases involved in degradation of UFD substrates is increased in cells lacking NatA. Finally, we identify the E3 ligase Tom1 as a novel chain-elongating enzyme (E4) involved in the degradation of linear ubiquitin fusions via the formation of branched K11, K29, and K48 ubiquitin chains, independently of the known E4 ligases involved in UFD, leading to enhanced ubiquitination of the UFD substrates.

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