International Journal of Molecular Sciences

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9 pages, 1174 KiB

Open AccessArticle

Availability of Arg, but Not tRNA, Is a Rate-Limiting Factor for Intracellular Arginylation

by Irem Avcilar-Kucukgoze, Brittany MacTaggart and Anna Kashina

Int. J. Mol. Sci. 2022, 23(1), 314; https://doi.org/10.3390/ijms23010314 - 28 Dec 2021

Viewed by 1494

Abstract

Protein arginylation, mediated by arginyltransferase ATE1, is a posttranslational modification of emerging biological importance that consists of transfer of the amino acid Arg from tRNA to protein and peptide targets. ATE1 can bind tRNA and exhibits specificity toward particular tRNA types, but its [...] Read more.

Protein arginylation, mediated by arginyltransferase ATE1, is a posttranslational modification of emerging biological importance that consists of transfer of the amino acid Arg from tRNA to protein and peptide targets. ATE1 can bind tRNA and exhibits specificity toward particular tRNA types, but its dependence on the availability of the major components of the arginylation reaction has never been explored. Here we investigated key intracellular factors that can potentially regulate arginylation in vivo, including several tRNA types that show strong binding to ATE1, as well as availability of free Arg, in an attempt to identify intracellular rate limiting steps for this enzyme. Our results demonstrate that, while modulation of tRNA levels in cells does not lead to any changes in intracellular arginylation efficiency, availability of free Arg is a potentially rate-limiting factor that facilitates arginylation if added to the cultured cells. Our results broadly outline global pathways that may be involved in the regulation of arginylation in vivo. Full article

(This article belongs to the Special Issue Modifications of Protein Termini)

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14 pages, 2477 KiB

Open AccessArticle

MALDI-MS Analysis of Peptide Libraries Expands the Scope of Substrates for Farnesyltransferase

by Garrett L. Schey, Peter H. Buttery, Emily R. Hildebrandt, Sadie X. Novak, Walter K. Schmidt, James L. Hougland and Mark D. Distefano

Int. J. Mol. Sci. 2021, 22(21), 12042; https://doi.org/10.3390/ijms222112042 - 07 Nov 2021

Cited by 6 | Viewed by 2534

Abstract

Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This modification typically causes proteins to associate with the membrane and allows them to participate in signaling pathways. In the [...] Read more.

Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This modification typically causes proteins to associate with the membrane and allows them to participate in signaling pathways. In the canonical understanding of FTase, the isoprenoids are attached to the cysteine residue of a four-amino-acid CaaX box sequence. However, recent work has shown that five-amino-acid sequences can be recognized, including the pentapeptide CMIIM. This paper describes a new systematic approach to discover novel peptide substrates for FTase by combining the combinatorial power of solid-phase peptide synthesis (SPPS) with the ease of matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). The workflow consists of synthesizing focused libraries containing 10–20 sequences obtained by randomizing a synthetic peptide at a single position. Incubation of the library with FTase and farnesyl pyrophosphate (FPP) followed by mass spectrometric analysis allows the enzymatic products to be clearly resolved from starting peptides due to the increase in mass that occurs upon farnesylation. Using this method, 30 hits were obtained from a series of libraries containing a total of 80 members. Eight of the above peptides were selected for further evaluation, reflecting a mixture that represented a sampling of diverse substrate space. Six of these sequences were found to be bona fide substrates for FTase, with several meeting or surpassing the in vitro efficiency of the benchmark sequence CMIIM. Experiments in yeast demonstrated that proteins bearing these sequences can be efficiently farnesylated within live cells. Additionally, a bioinformatics search showed that a variety of pentapeptide CaaaX sequences can be found in the mammalian genome, and several of these sequences display excellent farnesylation in vitro and in yeast cells, suggesting that the number of farnesylated proteins within mammalian cells may be larger than previously thought. Full article

(This article belongs to the Special Issue Modifications of Protein Termini)

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16 pages, 1131 KiB

Open AccessArticle

Hydroxylation of the Acetyltransferase NAA10 Trp38 Is Not an Enzyme-Switch in Human Cells

by Rasmus Ree, Karoline Krogstad, Nina McTiernan, Magnus E. Jakobsson and Thomas Arnesen

Int. J. Mol. Sci. 2021, 22(21), 11805; https://doi.org/10.3390/ijms222111805 - 30 Oct 2021

Cited by 2 | Viewed by 2154

Abstract

NAA10 is a major N-terminal acetyltransferase (NAT) that catalyzes the cotranslational N-terminal (Nt-) acetylation of 40% of the human proteome. Several reports of lysine acetyltransferase (KAT) activity by NAA10 exist, but others have not been able to find any NAA10-derived KAT [...] Read more.

NAA10 is a major N-terminal acetyltransferase (NAT) that catalyzes the cotranslational N-terminal (Nt-) acetylation of 40% of the human proteome. Several reports of lysine acetyltransferase (KAT) activity by NAA10 exist, but others have not been able to find any NAA10-derived KAT activity, the latter of which is supported by structural studies. The KAT activity of NAA10 towards hypoxia-inducible factor 1α (HIF-1α) was recently found to depend on the hydroxylation at Trp38 of NAA10 by factor inhibiting HIF-1α (FIH). In contrast, we could not detect hydroxylation of Trp38 of NAA10 in several human cell lines and found no evidence that NAA10 interacts with or is regulated by FIH. Our data suggest that NAA10 Trp38 hydroxylation is not a switch in human cells and that it alters its catalytic activity from a NAT to a KAT. Full article

(This article belongs to the Special Issue Modifications of Protein Termini)

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17 pages, 1675 KiB

Open AccessArticle

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

by Petra Van Damme

Int. J. Mol. Sci. 2021, 22(19), 10692; https://doi.org/10.3390/ijms221910692 - 02 Oct 2021

Cited by 2 | Viewed by 1983

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Modifications of Protein Termini)

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25 pages, 6752 KiB

Open AccessArticle

N-Terminal Acetyltransferase Naa40p Whereabouts Put into N-Terminal Proteoform Perspective

by Veronique Jonckheere and Petra Van Damme

Int. J. Mol. Sci. 2021, 22(7), 3690; https://doi.org/10.3390/ijms22073690 - 01 Apr 2021

Cited by 11 | Viewed by 2649

Abstract

The evolutionary conserved N-alpha acetyltransferase Naa40p is among the most selective N-terminal acetyltransferases (NATs) identified to date. Here we identified a conserved N-terminally truncated Naa40p proteoform named Naa40p25 or short Naa40p (Naa40^S). Intriguingly, although upon ectopic expression in yeast, both Naa40p [...] Read more.

The evolutionary conserved N-alpha acetyltransferase Naa40p is among the most selective N-terminal acetyltransferases (NATs) identified to date. Here we identified a conserved N-terminally truncated Naa40p proteoform named Naa40p25 or short Naa40p (Naa40^S). Intriguingly, although upon ectopic expression in yeast, both Naa40p proteoforms were capable of restoring N-terminal acetylation of the characterized yeast histone H2A Naa40p substrate, the Naa40p histone H4 substrate remained N-terminally free in human haploid cells specifically deleted for canonical Naa40p27 or 237 amino acid long Naa40p (Naa40^L), but expressing Naa40^S. Interestingly, human Naa40^L and Naa40^S displayed differential expression and subcellular localization patterns by exhibiting a principal nuclear and cytoplasmic localization, respectively. Furthermore, Naa40^L was shown to be N-terminally myristoylated and to interact with N-myristoyltransferase 1 (NMT1), implicating NMT1 in steering Naa40^L nuclear import. Differential interactomics data obtained by biotin-dependent proximity labeling (BioID) further hints to context-dependent roles of Naa40p proteoforms. More specifically, with Naa40^S representing the main co-translationally acting actor, the interactome of Naa40^L was enriched for nucleolar proteins implicated in ribosome biogenesis and the assembly of ribonucleoprotein particles, overall indicating a proteoform-specific segregation of previously reported Naa40p activities. Finally, the yeast histone variant H2A.Z and the transcriptionally regulatory protein Lge1 were identified as novel Naa40p substrates, expanding the restricted substrate repertoire of Naa40p with two additional members and further confirming Lge1 as being the first redundant yNatA and yNatD substrate identified to date. Full article

(This article belongs to the Special Issue Modifications of Protein Termini)

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12 pages, 8199 KiB

Open AccessArticle

Development of A Continuous Fluorescence-Based Assay for N-Terminal Acetyltransferase D

by Yi-Hsun Ho, Lan Chen and Rong Huang

Int. J. Mol. Sci. 2021, 22(2), 594; https://doi.org/10.3390/ijms22020594 - 08 Jan 2021

Cited by 8 | Viewed by 3426

Abstract

N-terminal acetylation catalyzed by N-terminal acetyltransferases (NATs) has various biological functions in protein regulation. N-terminal acetyltransferase D (NatD) is one of the most specific NAT with only histone H4 and H2A proteins as the known substrates. Dysregulation of NatD has [...] Read more.

N-terminal acetylation catalyzed by N-terminal acetyltransferases (NATs) has various biological functions in protein regulation. N-terminal acetyltransferase D (NatD) is one of the most specific NAT with only histone H4 and H2A proteins as the known substrates. Dysregulation of NatD has been implicated in colorectal and lung cancer progression, implying its therapeutic potential in cancers. However, there is no reported inhibitor for NatD yet. To facilitate the discovery of small-molecule NatD inhibitors, we report the development of a fluorescence-based acetyltransferase assay in 384-well high-throughput screening (HTS) format through monitoring the formation of coenzyme A. The fluorescent signal is generated from the adduct in the reaction between coenzyme A and fluorescent probe ThioGlo4. The assay exhibited a Z′-factor of 0.77 and a coefficient of variation of 6%, indicating it is a robust assay for HTS. A pilot screen of 1280 pharmacologically active compounds and subsequent validation identified two hits, confirming the application of this fluorescence assay in HTS. Full article

(This article belongs to the Special Issue Modifications of Protein Termini)

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Review

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12 pages, 2097 KiB

Open AccessReview

Cellular Control of Protein Turnover via the Modification of the Amino Terminus

by Nikola Winter, Maria Novatchkova and Andreas Bachmair

Int. J. Mol. Sci. 2021, 22(7), 3545; https://doi.org/10.3390/ijms22073545 - 29 Mar 2021

Cited by 7 | Viewed by 2493

Abstract

The first amino acid of a protein has an important influence on its metabolic stability. A number of ubiquitin ligases contain binding domains for different amino-terminal residues of their substrates, also known as N-degrons, thereby mediating turnover. This review summarizes, in an exemplary [...] Read more.

The first amino acid of a protein has an important influence on its metabolic stability. A number of ubiquitin ligases contain binding domains for different amino-terminal residues of their substrates, also known as N-degrons, thereby mediating turnover. This review summarizes, in an exemplary way, both older and more recent findings that unveil how destabilizing amino termini are generated. In most cases, a step of proteolytic cleavage is involved. Among the over 500 proteases encoded in the genome of higher eukaryotes, only a few are known to contribute to the generation of N-degrons. It can, therefore, be expected that many processing paths remain to be discovered. Full article

(This article belongs to the Special Issue Modifications of Protein Termini)

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9 pages, 1061 KiB

Open AccessBrief Report

NAA10 p.(D10G) and NAA10 p.(L11R) Variants Hamper Formation of the NatA N-Terminal Acetyltransferase Complex

by Nina McTiernan, Christine Darbakk, Rasmus Ree and Thomas Arnesen

Int. J. Mol. Sci. 2020, 21(23), 8973; https://doi.org/10.3390/ijms21238973 - 26 Nov 2020

Cited by 3 | Viewed by 2286

Abstract

The majority of the human proteome is subjected to N-terminal (Nt) acetylation catalysed by N-terminal acetyltransferases (NATs). The NatA complex is composed of two core subunits—the catalytic subunit NAA10 and the ribosomal anchor NAA15. Furthermore, NAA10 may also have catalytic and non-catalytic roles [...] Read more.

The majority of the human proteome is subjected to N-terminal (Nt) acetylation catalysed by N-terminal acetyltransferases (NATs). The NatA complex is composed of two core subunits—the catalytic subunit NAA10 and the ribosomal anchor NAA15. Furthermore, NAA10 may also have catalytic and non-catalytic roles independent of NatA. Several inherited and de novo NAA10 variants have been associated with genetic disease in humans. In this study, we present a functional analysis of two de novo NAA10 variants, c.29A>G p.(D10G) and c.32T>G p.(L11R), previously identified in a male and a female, respectively. Both of these neighbouring amino acids are highly conserved in NAA10. Immunoprecipitation experiments revealed that both variants hamper complex formation with NAA15 and are thus likely to impair NatA-mediated Nt-acetylation in vivo. Despite their common impact on NatA formation, in vitro Nt-acetylation assays showed that the variants had opposing impacts on NAA10 catalytic activity. While NAA10 c.29A>G p.(D10G) exhibits normal intrinsic NatA activity and reduced monomeric NAA10 NAT activity, NAA10 c.32T>G p.(L11R) displays reduced NatA activity and normal NAA10 NAT activity. This study expands the scope of research into the functional consequences of NAA10 variants and underlines the importance of understanding the diverse cellular roles of NAA10 in disease mechanisms. Full article

(This article belongs to the Special Issue Modifications of Protein Termini)

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