HYPK controls stability and catalytic activity of the N-terminal acetyltransferase A in Arabidopsis thaliana.

The ribosome-tethered N-terminal acetyltransferase A (NatA) acetylates 52% of soluble proteins in Arabidopsis thaliana. This co-translational modification of the N terminus stabilizes diverse cytosolic plant proteins. The evolutionary conserved Huntingtin yeast partner K (HYPK) facilitates NatA activity in planta, but in vitro, its N-terminal helix α1 inhibits human NatA activity. To dissect the regulatory function of HYPK protein domains in vivo, we genetically engineer CRISPR-Cas9 mutants expressing a HYPK fragment lacking all functional domains (hypk-cr1) or an internally deleted HYPK variant truncating helix α1 but retaining the C-terminal ubiquitin-associated (UBA) domain (hypk-cr2). We find that the UBA domain of HYPK is vital for stabilizing the NatA complex in an organ-specific manner. The N terminus of HYPK, including helix α1, is critical for promoting NatA activity on substrates starting with various amino acids. Consequently, deleting only 42 amino acids inside the HYPK N terminus causes substantial destabilization of the plant proteome and higher tolerance toward drought stress.

The Global Acetylation Profiling Pipeline for Quick Assessment of Protein N-Acetyltransferase Specificity In Cellulo.

Global acetylation profiling (GAP) consists of heterologous expression of a given N-acetyltransferase (NAT) in Escherichia coli to assess its specificity. The remarkable sensitivity and robustness of the GAP pipeline relies on the very low frequency of known N-terminal acetylated proteins in E. coli, including their degree of N-terminal acetylation. Using the SILProNAQ mass spectrometry strategy on bacterial protein extracts, GAP permits easy acquisition of both qualitative and quantitative data to decipher the impact of any putative NAT of interest on the N-termini of newly acetylated proteins. This strategy allows rapid determination of the substrate specificity of any NAT.

Nt-acetylation-independent turnover of SQUALENE EPOXIDASE 1 by Arabidopsis DOA10-like E3 ligases.

The acetylation-dependent (Ac/)N-degron pathway degrades proteins through recognition of their acetylated N-termini (Nt) by E3 ligases called Ac/N-recognins. To date, specific Ac/N-recognins have not been defined in plants. Here we used molecular, genetic, and multiomics approaches to characterize potential roles for Arabidopsis (Arabidopsis thaliana) DEGRADATION OF ALPHA2 10 (DOA10)-like E3 ligases in the Nt-acetylation-(NTA)-dependent turnover of proteins at global- and protein-specific scales. Arabidopsis has two endoplasmic reticulum (ER)-localized DOA10-like proteins. AtDOA10A, but not the Brassicaceae-specific AtDOA10B, can compensate for loss of yeast (Saccharomyces cerevisiae) ScDOA10 function. Transcriptome and Nt-acetylome profiling of an Atdoa10a/b RNAi mutant revealed no obvious differences in the global NTA profile compared to wild type, suggesting that AtDOA10s do not regulate the bulk turnover of NTA substrates. Using protein steady-state and cycloheximide-chase degradation assays in yeast and Arabidopsis, we showed that turnover of ER-localized SQUALENE EPOXIDASE 1 (AtSQE1), a critical sterol biosynthesis enzyme, is mediated by AtDOA10s. Degradation of AtSQE1 in planta did not depend on NTA, but Nt-acetyltransferases indirectly impacted its turnover in yeast, indicating kingdom-specific differences in NTA and cellular proteostasis. Our work suggests that, in contrast to yeast and mammals, targeting of Nt-acetylated proteins is not a major function of DOA10-like E3 ligases in Arabidopsis and provides further insight into plant ERAD and the conservation of regulatory mechanisms controlling sterol biosynthesis in eukaryotes.

HYPK promotes the activity of the Nα-acetyltransferase A complex to determine proteostasis of nonAc-X2/N-degron-containing proteins.

In humans, the Huntingtin yeast partner K (HYPK) binds to the ribosome-associated Nα-acetyltransferase A (NatA) complex that acetylates ~40% of the proteome in humans and Arabidopsis thaliana. However, the relevance of HsHYPK for determining the human N-acetylome is unclear. Here, we identify the AtHYPK protein as the first in vivo regulator of NatA activity in plants. AtHYPK physically interacts with the ribosome-anchoring subunit of NatA and promotes Nα-terminal acetylation of diverse NatA substrates. Loss-of-AtHYPK mutants are remarkably resistant to drought stress and strongly resemble the phenotype of NatA-depleted plants. The ectopic expression of HsHYPK rescues this phenotype. Combined transcriptomics, proteomics, and N-terminomics unravel that HYPK impairs plant metabolism and development, predominantly by regulating NatA activity. We demonstrate that HYPK is a critical regulator of global proteostasis by facilitating masking of the recently identified nonAc-X2/N-degron. This N-degron targets many nonacetylated NatA substrates for degradation by the ubiquitin-proteasome system.

N-acetylation of secreted proteins in Apicomplexa is widespread and is independent of the ER acetyl-CoA transporter AT1.

Acetyl-CoA participates in post-translational modification of proteins and in central carbon and lipid metabolism in several cell compartments. In mammals, acetyl-CoA transporter 1 (AT1, also known as SLC33A1) facilitates the flux of cytosolic acetyl-CoA into the endoplasmic reticulum (ER), enabling the acetylation of proteins of the secretory pathway, in concert with the activity of dedicated acetyltransferases such as NAT8. However, the involvement of the ER acetyl-CoA pool in acetylation of ER-transiting proteins in Apicomplexa is unknown. Here, we identified homologs of AT1 and NAT8 in Toxoplasma gondii and Plasmodium berghei parasites. Proteome-wide analyses revealed widespread N-terminal acetylation of secreted proteins in both species. Such extensive acetylation of N-terminally processed proteins has not been observed previously in any other organism. Deletion of AT1 homologs in both T. gondii and P. berghei resulted in considerable reductions in parasite fitness. In P. berghei, AT1 was found to be important for growth of asexual blood stages, production of female gametocytes and male gametocytogenesis, implying its requirement for parasite transmission. In the absence of AT1, lysine acetylation and N-terminal acetylation in T. gondii remained globally unaltered, suggesting an uncoupling between the role of AT1 in development and active acetylation occurring along the secretory pathway.

A Continuous Assay Set to Screen and Characterize Novel Protein N-Acetyltransferases Unveils Rice General Control Non-repressible 5-Related N-Acetyltransferase2 Activity.

Protein N-acetyltransferases (NATs) belong to the general control non-repressible 5 (Gcn5)-related N-acetyltransferases (GNATs) superfamily. GNATs catalyze the transfer of acetyl from acetyl-CoA to the reactive amine moiety of a wide range of acceptors. NAT sequences are difficult to distinguish from other members of the GNAT superfamily and there are many uncharacterized GNATs. To facilitate the discovery and characterization of new GNATs, we have developed a new continuous, non-radioactive assay. This assay is virtually independent of the substrate and can be used to get substrate specificity hints. We validated first the assay with the well-characterized Schizosaccharomyces pombe NatA (SpNatA). The SpNatA kinetic parameters were determined with various peptides confirming the robustness of the new assay. We reveal that the longer the peptide substrate the more efficient the enzyme. As a proof of concept of the relevance of the new assay, we characterized a NAA90 member from rice (Oryza sativa), OsGNAT2. We took advantage of an in vivo medium-scale characterization of OsGNAT2 specificity to identify and then validate in vitro several specific peptide substrates. With this assay, we reveal long-range synergic effects of basic residues on OsGNAT2 activity. Overall, this new, high-throughput assay allows better understanding of the substrate specificity and activity of any GNAT.

Lysine-specific acetylated proteome from the archaeon Thermococcus gammatolerans reveals the presence of acetylated histones.

Thermococcus gammatolerans EJ3 is an extremophile archaeon which was revealed as one of the most radioresistant organisms known on Earth, withstanding up to 30 kGy gamma-ray radiations. While its theoretical proteome is rather small, T. gammatolerans may enhance its toolbox by post-translational modification of its proteins. Here, we explored its extent of Nε-acetylation of lysines. For this, we immunopurified with two acetylated-lysine antibodies the acetylated peptides resulting from a proteolysis of soluble proteins with trypsin. The comparison of acetylated proteomes of two archaea highlights some common acetylation patterns but only 4 out of 26 orthologous proteins found to be acetylated in both species, are acetylated on the same lysine site. We evidenced that histone B is acetylated in T. gammatolerans at least at two different sites (K27 and K36), and a peptide common at the C-terminus of histones A and B is also acetylated. We verified that acetylation of histones is a common trait among Thermococcales after recording data on Thermococcus kodakaraensis histones and identifying three acetylated sites. This discovery reinforces the strong evolutionary link between Archaea and Eukaryotes and should be an incentive for further investigation on the extent and role of acetylation of histones in Archaea. SIGNIFICANCE: Acetylation is an important post-translational modification of proteins that has been extensively described in Eukaryotes, and more recently in Bacteria. Here, we report for the first time ever that histones in Archaea are also modified by acetylation after a systematic survey of acetylated peptides in Thermococcus gammatolerans. Structural models of histones A and B indicates that acetylation of the identified modified residues may play an important role in histone assembly and/or interaction with DNA. The in-depth protein acetylome landscape in T. gammatolerans includes at least 181 unique protein sequences, some of them being modified on numerous residues. Proteins involved in metabolic processes, information storage and processing mechanisms are over-represented categories in this dataset, highlighting the ancient role of this protein post-translational modification in primitive cells.

Dual lysine and N-terminal acetyltransferases reveal the complexity underpinning protein acetylation.

Protein acetylation is a highly frequent protein modification. However, comparatively little is known about its enzymatic machinery. N-α-acetylation (NTA) and ε-lysine acetylation (KA) are known to be catalyzed by distinct families of enzymes (NATs and KATs, respectively), although the possibility that the same GCN5-related N-acetyltransferase (GNAT) can perform both functions has been debated. Here, we discovered a new family of plastid-localized GNATs, which possess a dual specificity. All characterized GNAT family members display a number of unique features. Quantitative mass spectrometry analyses revealed that these enzymes exhibit both distinct KA and relaxed NTA specificities. Furthermore, inactivation of GNAT2 leads to significant NTA or KA decreases of several plastid proteins, while proteins of other compartments were unaffected. The data indicate that these enzymes have specific protein targets and likely display partly redundant selectivity, increasing the robustness of the acetylation process in vivo. In summary, this study revealed a new layer of complexity in the machinery controlling this prevalent modification and suggests that other eukaryotic GNATs may also possess these previously underappreciated broader enzymatic activities.

NAA50 Is an Enzymatically Active N α-Acetyltransferase That Is Crucial for Development and Regulation of Stress Responses.

Nα-terminal acetylation (NTA) is a prevalent protein modification in eukaryotes. In plants, the biological function of NTA remains enigmatic. The dominant N-acetyltransferase (Nat) in Arabidopsis (Arabidopsis thaliana) is NatA, which cotranslationally catalyzes acetylation of ∼40% of the proteome. The core NatA complex consists of the catalytic subunit NAA10 and the ribosome-anchoring subunit NAA15. In human (Homo sapiens), fruit fly (Drosophila melanogaster), and yeast (Saccharomyces cerevisiae), this core NatA complex interacts with NAA50 to form the NatE complex. While in metazoa, NAA50 has N-acetyltransferase activity, yeast NAA50 is catalytically inactive and positions NatA at the ribosome tunnel exit. Here, we report the identification and characterization of Arabidopsis NAA50 (AT5G11340). Consistent with its putative function as a cotranslationally acting Nat, AtNAA50-EYFP localized to the cytosol and the endoplasmic reticulum but also to the nuclei. We demonstrate that purified AtNAA50 displays Nα-terminal acetyltransferase and lysine-ε-autoacetyltransferase activity in vitro. Global N-acetylome profiling of Escherichia coli cells expressing AtNAA50 revealed conservation of NatE substrate specificity between plants and humans. Unlike the embryo-lethal phenotype caused by the absence of AtNAA10 and AtNAA15, loss of NAA50 expression resulted in severe growth retardation and infertility in two Arabidopsis transfer DNA insertion lines (naa50-1 and naa50-2). The phenotype of naa50-2 was rescued by the expression of HsNAA50 or AtNAA50. In contrast, the inactive ScNAA50 failed to complement naa50-2 Remarkably, loss of NAA50 expression did not affect NTA of known NatA substrates and caused the accumulation of proteins involved in stress responses. Overall, our results emphasize a relevant role of AtNAA50 in plant defense and development, which is independent of the essential NatA activity.

Integrative proteomic and phosphoproteomic profiling of prostate cell lines.

BACKGROUND: Prostate cancer is a major public health issue, mainly because patients relapse after androgen deprivation therapy. Proteomic strategies, aiming to reflect the functional activity of cells, are nowadays among the leading approaches to tackle the challenges not only of better diagnosis, but also of unraveling mechanistic details related to disease etiology and progression. METHODS: We conducted here a large SILAC-based Mass Spectrometry experiment to map the proteomes and phosphoproteomes of four widely used prostate cell lines, namely PNT1A, LNCaP, DU145 and PC3, representative of different cancerous and hormonal status. RESULTS: We identified more than 3000 proteins and phosphosites, from which we quantified more than 1000 proteins and 500 phosphosites after stringent filtering. Extensive exploration of this proteomics and phosphoproteomics dataset allowed characterizing housekeeping as well as cell-line specific proteins, phosphosites and functional features of each cell line. In addition, by comparing the sensitive and resistant cell lines, we identified protein and phosphosites differentially expressed in the resistance context. Further data integration in a molecular network highlighted the differentially expressed pathways, in particular migration and invasion, RNA splicing, DNA damage repair response and transcription regulation. CONCLUSIONS: Overall, this study proposes a valuable resource toward the characterization of proteome and phosphoproteome of four widely used prostate cell lines and reveals candidates to be involved in prostate cancer progression for further experimental validation.

O-GlcNAcylation site mapping by (azide-alkyne) click chemistry and mass spectrometry following intensive fractionation of skeletal muscle cells proteins.

The O-linked-N-acetyl-d-glucosaminylation (O-GlcNAcylation) modulates numerous aspects of cellular processes. Akin to phosphorylation, O-GlcNAcylation is highly dynamic, reversible, and responds rapidly to extracellular demand. Despite the absolute necessity to determine post-translational sites to fully understand the role of O-GlcNAcylation, it remains a high challenge for the major reason that unmodified proteins are in excess comparing to the O-GlcNAcylated ones. Based on a click chemistry approach, O-GlcNAcylated proteins were labelled with azido-GalNAc and coupled to agarose beads. The proteome extracted from C2C12 myotubes was submitted to an intensive fractionation prior to azide-alkyne click chemistry. This combination of fractionation and click chemistry is a powerful methodology to map O-GlcNAc sites; indeed, 342 proteins were identified through the identification of 620 peptides containing one or more O-GlcNAc sites. We localized O-GlcNAc sites on proteins involved in signalling pathways or in protein modification, as well as structural proteins. Considering the recent role of O-GlcNAcylation in the modulation of sarcomere morphometry and interaction between key structural protein, we focused on proteins involved in the cytoarchitecture of skeletal muscle cells. In particular, several O-GlcNAc sites were located into protein-protein interaction domains, suggesting that O-GlcNAcylation could be strongly involved in the organization and reorganization of sarcomere and myofibrils. SIGNIFICANCE: O-GlcNAcylation is an atypical glycosylation involved in the regulation of almost all if not all cellular processes, but its precise role remains sometimes obscure because of the ignorance of the O-GlcNAc site localization; thus, it remains indispensable to precisely map the O-GlcNAcylated sites to fully understand the role of O-GlcNAcylation on a given protein. For this purpose, we combined extensive fractionation of skeletal muscle cells proteome with click chemistry to map O-GlcNAc sites without an a priori consideration. A total of 620 peptides containing one or more O-GlcNAc sites were identified; interestingly, several of them belong to low expressed proteins, in particular proteins involved in signalling pathways. We also focused on structural proteins in view of recent data supporting the role of O-GlcNAcylation in the modulation of sarcomere cytoarchitecture; importantly, some of the O-GlcNAc sites were mapped into protein-protein interaction domains, reinforcing the involvement of O-GlcNAcylation in the organization and reorganization of sarcomere, and in larger extent, of myofibrils.

How may targeted proteomics complement genomic data in breast cancer?

INTRODUCTION: Breast cancer (BC) is the most common female cancer in the world and was recently deconstructed in different molecular entities. Although most of the recent assays to characterize tumors at the molecular level are genomic-based, proteins are the actual executors of cellular functions and represent the vast majority of targets for anticancer drugs. Accumulated data has demonstrated an important level of quantitative and qualitative discrepancies between genomic/transcriptomic alterations and their protein counterparts, mostly related to the large number of post-translational modifications. Areas covered: This review will present novel proteomics technologies such as Reverse Phase Protein Array (RPPA) or mass-spectrometry (MS) based approaches that have emerged and that could progressively replace old-fashioned methods (e.g. immunohistochemistry, ELISA, etc.) to validate proteins as diagnostic, prognostic or predictive biomarkers, and eventually monitor them in the routine practice. Expert commentary: These different targeted proteomic approaches, able to complement genomic data in BC and characterize tumors more precisely, will permit to go through a more personalized treatment for each patient and tumor.

Prioritizing targets for structural biology through the lens of proteomics: the archaeal protein TGAM_1934 from Thermococcus gammatolerans.

ORFans are hypothetical proteins lacking any significant sequence similarity with other proteins. Here, we highlighted by quantitative proteomics the TGAM_1934 ORFan from the hyperradioresistant Thermococcus gammatolerans archaeon as one of the most abundant hypothetical proteins. This protein has been selected as a priority target for structure determination on the basis of its abundance in three cellular conditions. Its solution structure has been determined using multidimensional heteronuclear NMR spectroscopy. TGAM_1934 displays an original fold, although sharing some similarities with the 3D structure of the bacterial ortholog of frataxin, CyaY, a protein conserved in bacteria and eukaryotes and involved in iron-sulfur cluster biogenesis. These results highlight the potential of structural proteomics in prioritizing ORFan targets for structure determination based on quantitative proteomics data. The proteomic data and structure coordinates have been deposited to the ProteomeXchange with identifier PXD000402 (http://proteomecentral.proteomexchange.org/dataset/PXD000402) and Protein Data Bank under the accession number 2mcf, respectively.

N- and O-acetylation of threonine residues in the context of proteomics.

The detection of post-translational modifications (PTMs) of proteins is a matter of intensive research. Among all possible pitfalls that may lead to misidentifications, the chemical stability of modified peptides is scarcely questioned. Global proteomic studies devoted to protein acetylation are becoming popular. Thus, we were concerned about the intrinsic stability of O-acetylated peptides because of the O-N acyl transfer reactivity occurring when an amino moiety is present in the vicinity of the acylated hydroxyl group. Here, the behavior of isomeric O- and N-acetylated, N-terminal threonine-containing peptides was explored in a standard proteomic workflow. We demonstrated a strong chemical instability of O-acetylation, which prevents its detection.