N-terminal Acetyltransferase Research Articles

Physical and Biochemical Properties of N-Acetyl Transferase RimL of the Hyperthermophilic Bacteria Thermus thermophilus

Bacterial N-terminal acetyltransferases (NATs) are involved in the biosynthesis/degradation of antibiotics. The RimL enzyme from E. coli provides it with resistance to the antibiotic microcin C. To date, the NATs of pathogenic bacteria have been well studied, but there is no data on the NATs of thermophilic bacteria. The purpose of the work is to study the physicochemical properties and specificity of a new NAT — RimL from Thermus thermophilus. We cloned the RimL ORF (TTHA1799) and developed a method for purifying the enzyme. The stability of RimL to pH, high temperatures and denaturing agents was studied using the protein intrinsic fluorescence method. We have obtained a thermophilic enzyme that can be used in biotechnology for the acetylation of proteins and peptides under non-standard conditions.

Yeast Nat4 regulates DNA damage checkpoint signaling through its N-terminal acetyltransferase activity on histone H4.

The DNA damage response (DDR) constitutes a vital cellular process that safeguards genome integrity. This biological process involves substantial alterations in chromatin structure, commonly orchestrated by epigenetic enzymes. Here, we show that the epigenetic modifier N-terminal acetyltransferase 4 (Nat4), known to acetylate the alpha-amino group of serine 1 on histones H4 and H2A, is implicated in the response to DNA damage in S. cerevisiae. Initially, we demonstrate that yeast cells lacking Nat4 have an increased sensitivity to DNA damage and accumulate more DNA breaks than wild-type cells. Accordingly, upon DNA damage, NAT4 gene expression is elevated, and the enzyme is specifically recruited at double-strand breaks. Delving deeper into its effects on the DNA damage signaling cascade, nat4-deleted cells exhibit lower levels of the damage-induced modification H2AS129ph (γH2A), accompanied by diminished binding of the checkpoint control protein Rad9 surrounding the double-strand break. Consistently, Mec1 kinase recruitment at double-strand breaks, critical for H2AS129ph deposition and Rad9 retention, is significantly impaired in nat4Δ cells. Consequently, Mec1-dependent phosphorylation of downstream effector kinase Rad53, indicative of DNA damage checkpoint activation, is reduced. Importantly, we found that the effects of Nat4 in regulating the checkpoint signaling cascade are mediated by its N-terminal acetyltransferase activity targeted specifically towards histone H4. Overall, this study points towards a novel functional link between histone N-terminal acetyltransferase Nat4 and the DDR, associating a new histone-modifying activity in the maintenance of genome integrity.

A repository of Ogden syndrome patient derived iPSC lines and isogenic pairs by X-chromosome screening and genome-editing.

Amino-terminal (Nt-) acetylation (NTA) is a common protein modification, affecting 80% of cytosolic proteins in humans. The human essential gene, NAA10, encodes the enzyme NAA10, as the catalytic subunit for the N-terminal acetyltransferase A (NatA) complex, including the accessory protein, NAA15. The first human disease directly involving NAA10 was discovered in 2011, and it was named Ogden syndrome (OS), after the location of the first affected family residing in Ogden, Utah, USA. Since that time, other variants have been found in NAA10 and NAA15. Here we describe the generation of 31 iPSC lines, with 16 from females and 15 from males. This cohort includes CRISPR-mediated correction to the wild-type genotype in 4 male lines, along with editing one female line to generate homozygous wild-type or mutant clones. Following the monoclonalizaiton and screening for X-chromosome activation status in female lines, 3 additional pairs of female lines, in which either the wild type allele is on the active X chromosome (Xa) or the pathogenic variant allele is on Xa, have been generated. Subsets of this cohort have been successfully used to make cardiomyocytes and neural progenitor cells (NPCs). These cell lines are made available to the community via the NYSCF Repository.

Biochemical Characterisation of the Short Isoform of Histone N-Terminal Acetyltransferase NAA40.

N-alpha-acetyltransferase 40 (NAA40) is an evolutionarily conserved N-terminal acetyltransferase (NAT) linked to oncogenesis and chemoresistance. A recent study reported the generation of a second, shorter NAA40 isoform (NAA40S) through alternative translation, which we proceeded to further characterise. Notably, recombinant NAA40S had a greater in vitro enzymatic activity and affinity towards its histone H2A/H4 substrates compared to full-length NAA40 (NAA40L). Within cells, NAA40S was enzymatically active, based on its ability to suppress the H2A/H4S1Ph antagonistic mark in CRISPR-generated NAA40 knockout cells. Finally, we show that in addition to alternative translation, the NAA40S isoform could be derived from a primate and testis-specific transcript, which may align with the "out-of-testis" origin of recently evolved genes and isoforms. To summarise, our data reveal an even greater functional divergence between the two NAA40 isoforms than had been previously recognised.

Light Changes Promote Distinct Responses of Plastid Protein Acetylation Marks

Protein acetylation is a key co- and post-translational modification. However, how different types of acetylation respond to environmental stress is still unknown. To address this, we investigated the role of a member of the newly discovered family of plastid acetyltransferases (GNAT2), which features both lysine- and N-terminal acetyltransferase activities. Our study aimed to provide a holistic multi-omics acetylation-dependent view of plant acclimation to short-term light changes. We found that both the yield and coverage of the N-terminal acetylome remained unchanged in wild-type and gnat2-knockout backgrounds after two hours of exposure to high light or darkness. Similarly, no differences in transcriptome or adenylate energy charge were observed between the genotypes under the tested light conditions. In contrast, the lysine acetylome proved to be sensitive to the changes in light conditions, especially in the gnat2 background. This suggests unique strategies of plant acclimation for quick responses to environmental changes involving lysine, but not N-terminal, GNAT2-mediated acetylation activity.

NatB Protects Procaspase-8 from UBR4-Mediated Degradation and Is Required for Full Induction of the Extrinsic Apoptosis Pathway.

N-terminal acetyltransferase B (NatB) is a major contributor to the N-terminal acetylome and is implicated in several key cellular processes including apoptosis and proteostasis. However, the molecular mechanisms linking NatB-mediated N-terminal acetylation to apoptosis and its relationship with protein homeostasis remain elusive. In this study, we generated mouse embryonic fibroblasts (MEFs) with an inactivated catalytic subunit of NatB (Naa20-/-) to investigate the impact of NatB deficiency on apoptosis regulation. Through quantitative N-terminomics, label-free quantification, and targeted proteomics, we demonstrated that NatB does not influence the proteostasis of all its substrates. Instead, our focus on putative NatB-dependent apoptotic factors revealed that NatB serves as a protective shield against UBR4 and UBR1 Arg/N-recognin-mediated degradation. Notably, Naa20-/- MEFs exhibited reduced responsiveness to an extrinsic pro-apoptotic stimulus, a phenotype that was partially reversible upon UBR4 Arg/N-recognin silencing and consequent inhibition of procaspase-8 degradation. Collectively, our results shed light on how the interplay between NatB-mediated acetylation and the Arg/N-degron pathway appears to impact apoptosis regulation, providing new perspectives in the field including in therapeutic interventions.

Assessing N-terminal acetylation status of cellular proteins via an antibody specific for acetylated methionine

N-terminal acetylation is being recognized as a factor affecting protein lifetime and proteostasis. It is a modification where an acetyl group is added to the N-terminus of proteins, and this occurs in 80 % of the human proteome. N-terminal acetylation is catalyzed by enzymes called N-terminal acetyltransferases (NATs). The various NATs acetylate different N-terminal amino acids, and methionine is a known target for some of the NATs. Currently, the acetylation status of most proteins can only be assessed with a limited number of methods, including mass spectrometry, which although powerful and robust, remains laborious and can only survey a fraction of the proteome. We here present testing of an antibody that was developed to specifically recognize Nt-acetylated methionine-starting proteins. We have used dot blots with synthetic acetylated and non-acetylated peptides in addition to protein analysis of lysates from NAT KO cell lines to assess the specificity and application of this anti-Nt-acetylated methionine antibody (anti-NtAc-Met). Our results demonstrate that this antibody is indeed NtAc-specific and further show that it has selectivity for some subtypes of methionine-starting N-termini, specifically potential substrates of the NatC, NatE and NatF enzymes. We propose that this antibody may be a powerful tool to identify NAT substrates or to analyse changes in N-terminal acetylation for specific cellular proteins of interest.

N-terminal acetylation of Set1-COMPASS fine-tunes H3K4 methylation patterns.

H3K4 methylation by Set1-COMPASS (complex of proteins associated with Set1) is a conserved histone modification. Although it is critical for gene regulation, the posttranslational modifications of this complex that affect its function are largely unexplored. This study showed that N-terminal acetylation of Set1-COMPASS proteins by N-terminal acetyltransferases (NATs) can modulate H3K4 methylation patterns. Specifically, deleting NatA substantially decreased global H3K4me3 levels and caused the H3K4me2 peak in the 5' transcribed regions to shift to the promoters. NatA was required for N-terminal acetylation of three subunits of Set1-COMPASS: Shg1, Spp1, and Swd2. Moreover, deleting Shg1 or blocking its N-terminal acetylation via proline mutation of the target residue drastically reduced H3K4 methylation. Thus, NatA-mediated N-terminal acetylation of Shg1 shapes H3K4 methylation patterns. NatB also regulates H3K4 methylation, likely via N-terminal acetylation of the Set1-COMPASS protein Swd1. Thus, N-terminal acetylation of Set1-COMPASS proteins can directly fine-tune the functions of this complex, thereby substantially shaping H3K4 methylation patterns.

N-terminal acetylation orchestrates glycolate-mediated ROS homeostasis to promote rice thermoresponsive growth.

Climate warming poses a significant threat to global crop production and food security. However, our understanding of the molecular mechanisms governing thermoresponsive development in crops remains limited. Here we report that the auxiliary subunit of N-terminal acetyltransferase A (NatA) in rice OsNAA15 is a prerequisite for rice thermoresponsive growth. OsNAA15 produces two isoforms OsNAA15.1 and OsNAA15.2, via temperature-dependent alternative splicing. Among the two, OsNAA15.1 is more likely to form a stable and functional NatA complex with the potential catalytic subunit OsNAA10, leading to a thermoresponsive N-terminal acetylome. Intriguingly, while OsNAA15.1 promotes plant growth under elevated temperatures, OsNAA15.2 exhibits an inhibitory effect. We identified two glycolate oxidases (GLO1/5) as major substrates from the thermoresponsive acetylome. These enzymes are involved in hydrogen peroxide (H2O2) biosynthesis via glycolate oxidation. N-terminally acetylated GLO1/5 undergo their degradation through the ubiquitin-proteasome system. This leads to reduced reactive oxygen species (ROS) production, thereby promoting plant growth, particularly under high ambient temperatures. Conclusively, our findings highlight the pivotal role of N-terminal acetylation in orchestrating the glycolate-mediated ROS homeostasis to facilitate thermoresponsive growth in rice.

Bioinformatic Analysis Reveals the Association of Human N-Terminal Acetyltransferase Complexes with Distinct Transcriptional and Post-Transcriptional Processes.

N-terminal acetyltransferases (NAT) are the protein complexes that deposit the abundant N-terminal acetylation (Nt-Ac) on eukaryotic proteins, with seven human complexes currently identified. Despite the increasing recognition of their biological and clinical importance, NAT regulation remains elusive. In this study, we performed a bioinformatic investigation to identify transcriptional and post-transcriptional processes that could be involved in the regulation of human NAT complexes. First, co-expression analysis of independent transcriptomic datasets revealed divergent pathway associations for human NAT, which are potentially connected to their distinct cellular functions. One interesting connection uncovered was the coordinated regulation of the NatA and proteasomal genes in cancer and immune cells, confirmed by analysis of multiple datasets and in isolated primary T cells. Another distinctive association was of NAA40 (NatD) with DNA replication, in cancer and non-cancer settings. The link between NAA40 transcription and DNA replication is potentially mediated through E2F1, which we have experimentally shown to bind the promoter of this NAT. Second, the coupled examination of transcriptomic and proteomic datasets revealed a much greater intra-complex concordance of NAT subunits at the protein compared to the transcript level, indicating the predominance of post-transcriptional processes for achieving their coordination. In agreement with this concept, we also found that the effects of somatic copy number alterations affecting NAT genes are attenuated post-transcriptionally. In conclusion, this study provides novel insights into the regulation of human NAT complexes.

Evaluating possible maternal effect lethality and genetic background effects in Naa10 knockout mice.

Amino-terminal (Nt-) acetylation (NTA) is a common protein modification, affecting approximately 80% of all human proteins. The human essential X-linked gene, NAA10, encodes for the enzyme NAA10, which is the catalytic subunit in the N-terminal acetyltransferase A (NatA) complex. There is extensive genetic variation in humans with missense, splice-site, and C-terminal frameshift variants in NAA10. In mice, Naa10 is not an essential gene, as there exists a paralogous gene, Naa12, that substantially rescues Naa10 knockout mice from embryonic lethality, whereas double knockouts (Naa10-/Y Naa12-/-) are embryonic lethal. However, the phenotypic variability in the mice is nonetheless quite extensive, including piebaldism, skeletal defects, small size, hydrocephaly, hydronephrosis, and neonatal lethality. Here we replicate these phenotypes with new genetic alleles in mice, but we demonstrate their modulation by genetic background and environmental effects. We cannot replicate a prior report of "maternal effect lethality" for heterozygous Naa10-/X female mice, but we do observe a small amount of embryonic lethality in the Naa10-/y male mice on the inbred genetic background in this different animal facility.

Diverging co-translational protein complex assembly pathways are governed by interface energy distribution

Protein-protein interactions are at the heart of all cellular processes, with the ribosome emerging as a platform, orchestrating the nascent-chain interplay dynamics. Here, to study the characteristics governing co-translational protein folding and complex assembly, we combine selective ribosome profiling, imaging, and N-terminomics with all-atoms molecular dynamics. Focusing on conserved N-terminal acetyltransferases (NATs), we uncover diverging co-translational assembly pathways, where highly homologous subunits serve opposite functions. We find that only a few residues serve as “hotspots,” initiating co-translational assembly interactions upon exposure at the ribosome exit tunnel. These hotspots are characterized by high binding energy, anchoring the entire interface assembly. Alpha-helices harboring hotspots are highly thermolabile, folding and unfolding during simulations, depending on their partner subunit to avoid misfolding. In vivo hotspot mutations disrupted co-translational complexation, leading to aggregation. Accordingly, conservation analysis reveals that missense NATs variants, causing neurodevelopmental and neurodegenerative diseases, disrupt putative hotspot clusters. Expanding our study to include phosphofructokinase, anthranilate synthase, and nucleoporin subcomplex, we employ AlphaFold-Multimer to model the complexes’ complete structures. Computing MD-derived interface energy profiles, we find similar trends. Here, we propose a model based on the distribution of interface energy as a strong predictor of co-translational assembly.

N-terminal acetyltransferase 6 facilitates enterovirus 71 replication by regulating PI4KB expression and replication organelle biogenesis.

Enterovirus 71 (EV71) is one of the major pathogens causing hand, foot, and mouth disease in children under 5 years old, which can result in severe neurological complications and even death. Due to limited treatments for EV71 infection, the identification of novel host factors and elucidation of mechanisms involved will help to counter this viral infection. N-terminal acetyltransferase 6 (NAT6) was identified as an essential host factor for EV71 infection with genome-wide CRISPR/Cas9 screening. NAT6 facilitates EV71 viral replication depending on its acetyltransferase activity but has little effect on viral release. In addition, NAT6 is also required for Echovirus 7 and coxsackievirus B5 infection, suggesting it might be a pan-enterovirus host factor. We further demonstrated that NAT6 is required for Golgi integrity and viral replication organelle (RO) biogenesis. NAT6 knockout significantly inhibited phosphatidylinositol 4-kinase IIIβ (PI4KB) expression and PI4P production, both of which are key host factors for enterovirus infection and RO biogenesis. Further mechanism studies confirmed that NAT6 formed a complex with its substrate actin and one of the PI4KB recruiters-acyl-coenzyme A binding domain containing 3 (ACBD3). Through modulating actin dynamics, NAT6 maintained the integrity of the Golgi and the stability of ACBD3, thereby enhancing EV71 infection. Collectively, these results uncovered a novel mechanism of N-acetyltransferase supporting EV71 infection.IMPORTANCEEnterovirus 71 (EV71) is an important pathogen for children under the age of five, and currently, no effective treatment is available. Elucidating the mechanism of novel host factors supporting viral infection will reveal potential antiviral targets and aid antiviral development. Here, we demonstrated that a novel N-acetyltransferase, NAT6, is an essential host factor for EV71 replication. NAT6 could promote viral replication organelle (RO) formation to enhance viral replication. The formation of enterovirus ROs requires numerous host factors, including acyl-coenzyme A binding domain containing 3 (ACBD3) and phosphatidylinositol 4-kinase IIIβ (PI4KB). NAT6 could stabilize the PI4KB recruiter, ACBD3, by inhibiting the autophagy degradation pathway. This study provides a fresh insight into the relationship between N-acetyltransferase and viral infection.

Development of an Algorithm for Identification of N-terminal Acetyltransferases and Verification of Their Functional Activity

N-terminal acetyltransferases (NATs) of bacteria acetylate the alpha-amino group in amino acids and proteins, participate in the biosynthesis of lantibiotics, and inactivate a number of antibiotics. NATs are used in biotechnology for targeted acetylation of recombinant proteins and peptides. In this regard, the search for NATs that differ in terms of substrate specificity and are also capable of functioning in the reaction at elevated temperatures, a wide pH range, etc., seems relevant. In this work, we develop specific characteristics and a search algorithm for the identification of N-terminal acetyltransferases using the Thermus thermophilus thermophilic bacterium as an example. Out of 14 Abs annotated in the genome, we selected six «putative» NATs. Some of the genes encoding the selected NATs were successfully cloned, generated, and purified from E. coli cells. The specific enzymatic activity of a number of enzymes was confirmed.

N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevity

Most eukaryotic proteins are N-terminally acetylated, but the functional impact on a global scale has remained obscure. Using genome-wide CRISPR knockout screens in human cells, we reveal a strong genetic dependency between a major N-terminal acetyltransferase and specific ubiquitin ligases. Biochemical analyses uncover that both the ubiquitin ligase complex UBR4-KCMF1 and the acetyltransferase NatC recognize proteins bearing an unacetylated N-terminal methionine followed by a hydrophobic residue. NatC KO-induced protein degradation and phenotypes are reversed by UBR knockdown, demonstrating the central cellular role of this interplay. We reveal that loss of Drosophila NatC is associated with male sterility, reduced longevity, and age-dependent loss of motility due to developmental muscle defects. Remarkably, muscle-specific overexpression of UbcE2M, one of the proteins targeted for NatC KO-mediated degradation, suppresses defects of NatC deletion. In conclusion, NatC-mediated N-terminal acetylation acts as a protective mechanism against protein degradation, which is relevant for increased longevity and motility.

NAA20 recruits Rin2 and promotes triple-negative breast cancer progression by regulating Rab5A-mediated activation of EGFR signaling

Triple-negative breast cancer (TNBC) is the most aggressive subtype with poor prognosis and high mortality. To improve the prognosis and survival of TNBC patients, it is necessary to explore new targets and signaling pathways to develop novel therapies for TNBC treatment. N-α-acetyltransferase 20 (NAA20) is one of the catalytic subunits of N-terminal acetyltransferase (NatB). It has been reported that NAA20 played a critical role in cancer progression. In this study, we found that NAA20 expression was markedly higher in TNBC tissues than in paracancerous normal tissues using The Cancer Genome Atlas (TCGA) analysis. This result was further confirmed by qRT-PCR and immunohistochemistry (IHC). Knockdown of NAA20 significantly inhibited TNBC cell viability by CCK8 and colony formation assays and cell migration and invasion by Transwell assays. Additionally, NAA20 knockdown decreased the expression of EGFR in TNBC cells. Upon stimulation with EGF and knockdown of NAA20, EGFR internalization and degradation were observed by confocal microscopy. The western blot results showed that NAA20 knockdown down-regulated PI3K, AKT, and mTOR phosphorylation. Next, we further explored the underlying molecular mechanisms of NAA20 by co-immunoprecipitation (Co-IP). The results suggested that there was an interacting relationship between NAA20 and Rab5A. Over-expression of NAA20 could potentiate the expression of Rab5A. Furthermore, the knockdown of Rab5A inhibited EGFR expression and the phosphorylation of downstream signaling targets. NAA20 over-expression offset the knockdown effect of Rab5A and activated EGFR signaling. Finally, we constructed a xenograft mouse model transfected TNBC cells to investigate the role of NAA20 in vivo. NAA20 knockdown markedly suppressed tumor growth and decreased tumor volume and weight. In conclusion, our study demonstrated that NAA20, a novel target of TNBC, could promote TNBC progression by regulating Rab5A-mediated activation of EGFR signaling.

Functional mapping of N-terminal residues in the yeast proteome uncovers novel determinants for mitochondrial protein import.

N-terminal ends of polypeptides are critical for the selective co-translational recruitment of N-terminal modification enzymes. However, it is unknown whether specific N-terminal signatures differentially regulate protein fate according to their cellular functions. In this work, we developed an in-silico approach to detect functional preferences in cellular N-terminomes, and identified in S. cerevisiae more than 200 Gene Ontology terms with specific N-terminal signatures. In particular, we discovered that Mitochondrial Targeting Sequences (MTS) show a strong and specific over-representation at position 2 of hydrophobic residues known to define potential substrates of the N-terminal acetyltransferase NatC. We validated mitochondrial precursors as co-translational targets of NatC by selective purification of translating ribosomes, and found that their N-terminal signature is conserved in Saccharomycotina yeasts. Finally, systematic mutagenesis of the position 2 in a prototypal yeast mitochondrial protein confirmed its critical role in mitochondrial protein import. Our work highlights the hydrophobicity of MTS N-terminal residues and their targeting by NatC as important features for the definition of the mitochondrial proteome, providing a molecular explanation for mitochondrial defects observed in yeast or human NatC-depleted cells. Functional mapping of N-terminal residues thus has the potential to support the discovery of novel mechanisms of protein regulation or targeting.

Abstract P1037: Dysfunction Of N-terminal Acetylation Causes Multiple Electrical Abnormalities Leading To Long QT Syndrome And Dilated Cardiomyopathy

Introduction: NAA10-related syndrome is characterized by long QT syndrome (LQTS), cardiomyopathy, hypotonia, and neurodevelopmental delay. NAA10 complexes with the regulatory subunit NAA15 to make the post-translationally modifying N-terminal acetyltransferase, NatA. Despite the association of LQTS and NAA10 mutations, the mechanism is unknown. Hypothesis: NAA10 dysfunction affects the function of cardiac ion channels or related proteins causing LQTS, and sarcomere abnormalities causing cardiomyopathy. Methods: Target sequencing of large kindred with multiple family members who had LQTS and sudden death revealed a segregating mutation in NAA10 (c.10C>A; p.R4S). Somatic cell reprogramming was used to create an induced pluripotent stem cell (iPSC) line from a gene-positive male patient with LQTS and dilated cardiomyopathy (pNAA10 R4S ). Genome-editing was used to create the control iPSC line (eNAA10 R4S ). iPSC lines were differentiated into cardiomyocytes (iPSC-CMs) for further analysis. In addition, we performed biochemical assays to investigate the enzymatic activities of the NAA10-R4S mutation. Results: The NAA10-R4S mutation had reduced enzymatic activity, decreased expression levels of NAA10/NAA15 and destabilized the NatA complex. Action potential duration was prolonged in pNAA10 R4S - and eNAA10 R4S -iPSC-CMs as compared to WT iPSC-CMs (498.9 ± 38.8 ms and 514.1 ± 52.9 ms, vs 316.7 ± 48.2 ms; p<0.05). Investigation of the underlying ion channel currents revealed increased late I Na (Late I Na / Peak I Na : pNAA10 R4S : 0.062 ± 0.010 % and eNAA10 R4S : 0.074 ± 0.020 % vs. WT: 0.026 ± 0.005 %; p<0.05) and reduced I Ks (pNAA10 R4S : 0.18 ± 0.05 pA/pF and eNAA10 R4S : 0.26 ± 0.04 pA/pF vs. WT: 0.61 ± 0.15 pA/pF; p<0.05). There were no significant differences in I Kr and I CaL . Diastolic Ca 2+ levels were increased in mutant iPSC-CMs (F340/F380: 0.18 ± 0.02 vs 0.04 ± 0.01; p<0.05) consistent with impaired Ca 2+ handling. Plating iPSC-CMs on micro-contact printed islands revealed alterations in sarcomeric structure. Conclusion: We demonstrate that the NAA10 mutation cause abnormalities in ion channels and sarcomere development. Our data indicated a novel role for N-terminal acetylation in cardiac ion channel regulation leading to cardiovascular disorders.

Loss of N-terminal acetyltransferase A activity induces thermally unstable ribosomal proteins and increases their turnover in Saccharomyces cerevisiae

Protein N-terminal (Nt) acetylation is one of the most abundant modifications in eukaryotes, covering ~50-80 % of the proteome, depending on species. Cells with defective Nt-acetylation display a wide array of phenotypes such as impaired growth, mating defects and increased stress sensitivity. However, the pleiotropic nature of these effects has hampered our understanding of the functional impact of protein Nt-acetylation. The main enzyme responsible for Nt-acetylation throughout the eukaryotic kingdom is the N-terminal acetyltransferase NatA. Here we employ a multi-dimensional proteomics approach to analyze Saccharomyces cerevisiae lacking NatA activity, which causes global proteome remodeling. Pulsed-SILAC experiments reveals that NatA-deficient strains consistently increase degradation of ribosomal proteins compared to wild type. Explaining this phenomenon, thermal proteome profiling uncovers decreased thermostability of ribosomes in NatA-knockouts. Our data are in agreement with a role for Nt-acetylation in promoting stability for parts of the proteome by enhancing the avidity of protein-protein interactions and folding.

Chemoproteomics Yields a Selective Molecular Host for Acetyl-CoA.

Chemoproteomic profiling is a powerful approach to define the selectivity of small molecules and endogenous metabolites with the human proteome. In addition to mechanistic studies, proteome specificity profiling also has the potential to identify new scaffolds for biomolecular sensing. Here, we report a chemoproteomics-inspired strategy for selective sensing of acetyl-CoA. First, we use chemoproteomic capture experiments to validate the N-terminal acetyltransferase NAA50 as a protein capable of differentiating acetyl-CoA and CoA. A Nanoluc-NAA50 fusion protein retains this specificity and can be used to generate a bioluminescence resonance energy transfer (BRET) signal in the presence of a CoA-linked fluorophore. This enables the development of a ligand displacement assay in which CoA metabolites are detected via their ability to bind the Nanoluc-NAA50 protein "host" and compete binding of the CoA-linked fluorophore "guest". We demonstrate that the specificity of ligand displacement reflects the molecular recognition of the NAA50 host, while the window of dynamic sensing can be controlled by tuning the binding affinity of the CoA-linked fluorophore guest. Finally, we show that the method's specificity for acetyl-CoA can be harnessed for gain-of-signal optical detection of enzyme activity and quantification of acetyl-CoA from cellular samples. Overall, our studies demonstrate the potential of harnessing insights from chemoproteomics for molecular sensing and provide a foundation for future applications in target engagement and selective metabolite detection.