The protein acetylome and the regulation of metabolism

Acetyl-coenzyme A (CoA) is a central metabolite involved in numerous anabolic and catabolic pathways, as well as in protein acetylation. Beyond histones, a large number of metabolic enzymes are acetylated in both animal and bacteria, and the protein acetylome is now emerging in plants. Protein acetylation is influenced by the cellular level of both acetyl-CoA and NAD(+), and regulates the activity of several enzymes. Acetyl-CoA is thus ideally placed to act as a key molecule linking the energy balance of the cell to the regulation of gene expression and metabolic pathways via the control of protein acetylation. Better knowledge over how to influence acetyl-CoA levels and the acetylation process promises to be an invaluable tool to control metabolic pathways.     

Proteinase K improves quantitative acylation studies

Acetylation is a common PTM of proteins but is still challenging to analyze. Only few acetylome studies have been performed to tackle this issue. Yet, the detection of acetylated proteins in complex cell lysates remains to be improved. Here, we present a proteomic approach with proteinase K as a suitable protease to identify acetylated peptides quantitatively. We first optimized the digestion conditions using an artificial system of purified bovine histones to find the optimal protease. Subsequently, the capability of proteinase K was demonstrated in complex HEK293 cell lysates. Finally, SILAC in combination with MudPIT was used to show that quantification with proteinase K is possible. In this study, we identified a sheer number of 557 unique acetylated peptides originating from 633 acetylation sites.     

Proteome-wide lysine acetylation profiling of the human pathogen Mycobacterium tuberculosis

N^ɛ-Acetylation of lysine residues represents a pivotal post-translational modification used by both eukaryotes and prokaryotes to modulate diverse biological processes. Mycobacterium tuberculosis is the causative agent of tuberculosis, one of the most formidable public health threats. Many aspects of the biology of M. tuberculosis remain elusive, in particular the extent and function of N^ɛ-lysine acetylation. With a combination of anti-acetyllysine antibody-based immunoaffinity enrichment with high-resolution mass spectrometry, we identified 1128 acetylation sites on 658 acetylated M. tuberculosis proteins. GO analysis of the acetylome showed that acetylated proteins are involved in the regulation of diverse cellular processes including metabolism and protein synthesis. Six types of acetylated peptide sequence motif were revealed from the acetylome. Twenty lysine-acetylated proteins showed homology with acetylated proteins previously identified from Escherichia coli, Salmonella enterica, Bacillus subtilis and Streptomyces roseosporus, with several acetylation sites highly conserved among four or five bacteria, suggesting that acetylated proteins are more conserved. Notably, several proteins including isocitrate lyase involved in the persistence, virulence and antibiotic resistance are acetylated, and site-directed mutagenesis of isocitrate lyase acetylation site to glutamine led to a decrease of the enzyme activity, indicating major roles of KAc in these proteins engaged cellular processes. Our data firstly provides a global survey of M. tuberculosis acetylation, and implicates extensive regulatory role of acetylation in this pathogen. This may serve as an important basis to address the roles of lysine acetylation in M. tuberculosis metabolism, persistence and virulence.     

Histone deacetylases control lysine acetylation of ribosomal proteins in rice

Lysine acetylation (Kac) is well known to occur in histones for chromatin function and epigenetic regulation. In addition to histones, Kac is also detected in a large number of proteins with diverse biological functions. However, Kac function and regulatory mechanism for most proteins are unclear. In this work, we studied mutation effects of rice genes encoding cytoplasm-localized histone deacetylases (HDAC) on protein acetylome and found that the HDAC protein HDA714 was a major deacetylase of the rice non-histone proteins including many ribosomal proteins (r-proteins) and translation factors that were extensively acetylated. HDA714 loss-of-function mutations increased Kac levels but reduced abundance of r-proteins. In vitro and in vivo experiments showed that HDA714 interacted with r-proteins and reduced their Kac. Substitutions of lysine by arginine (depleting Kac) in several r-proteins enhance, while mutations of lysine to glutamine (mimicking Kac) decrease their stability in transient expression system. Ribo-seq analysis revealed that the hda714 mutations resulted in increased ribosome stalling frequency. Collectively, the results uncover Kac as a functional posttranslational modification of r-proteins which is controlled by histone deacetylases, extending the role of Kac in gene expression to protein translational regulation.     

The fasted/fed mouse metabolic acetylome: N6-acetylation differences suggest acetylation coordinates organ-specific fuel switching

The elucidation of extra-nuclear lysine acetylation has been of growing interest, as the cosubstrate for acetylation, acetyl CoA, is at a key metabolic intersection. Our hypothesis was that mitochondrial and cytoplasmic protein acetylation may be part of a fasted/re-fed feedback control system for the regulation of the metabolic network in fuel switching, where acetyl CoA would be provided by fatty acid oxidation, or glycolysis, respectively. To test this, we characterized the mitochondrial and cytoplasmic acetylome in various organs that have a high metabolic rate relative to their mass, and/or switch fuels, under fasted and re-fed conditions (brain, kidney, liver, skeletal muscle, heart muscle, white and brown adipose tissues). Using immunoprecipitation, coupled with LC-MS/MS label free quantification, we show there is a dramatic variation in global quantitative profiles of acetylated proteins from different organs. In total, 733 acetylated peptides from 337 proteins were identified and quantified, out of which 31 acetylated peptides from the metabolic proteins that may play organ-specific roles were analyzed in detail. Results suggest that fasted/re-fed acetylation changes coordinated by organ-specific (de)acetylases in insulin-sensitive versus -insensitive organs may underlie fuel use and switching. Characterization of the tissue-specific acetylome should increase understanding of metabolic conditions wherein normal fuel switching is disrupted, such as in Type II diabetes.     

Comparative acetylomic analysis reveals differentially acetylated proteins regulating fungal metabolism in hypovirus-infected chestnut blight fungus

Cryphonectria parasitica, the chestnut blight fungus, and hypoviruses are excellent models for examining fungal pathogenesis and virus–host interactions. Increasing evidence suggests that lysine acetylation plays a regulatory role in cell processes and signalling. To understand protein regulation in C. parasitica by hypoviruses at the level of posttranslational modification, a label-free comparative acetylome analysis was performed in the fungus with or without Cryphonectria hypovirus 1 (CHV1) infection. Using enrichment of acetyl-peptides with a specific anti-acetyl-lysine antibody, followed by high accuracy liquid chromatography–tandem mass spectrometry analysis, 638 lysine acetylation sites were identified on 616 peptides, corresponding to 325 unique proteins. Further analysis revealed that 80 of 325 proteins were differentially acetylated between C. parasitica strain EP155 and EP155/CHV1-EP713, with 43 and 37 characterized as up- and down-regulated, respectively. Moreover, 75 and 65 distinct acetylated proteins were found in EP155 and EP155/CHV1-EP713, respectively. Bioinformatics analysis revealed that the differentially acetylated proteins were involved in various biological processes and were particularly enriched in metabolic processes. Differences in acetylation in C. parasitica citrate synthase, a key enzyme in the tricarboxylic acid cycle, were further validated by immunoprecipitation and western blotting. Site-specific mutagenesis and biochemical studies demonstrated that the acetylation of lysine-55 plays a vital role in the regulation of the enzymatic activity of C. parasitica citrate synthase in vitro and in vivo. These findings provide a valuable resource for the functional analysis of lysine acetylation in C. parasitica, as well as improving our understanding of fungal protein regulation by hypoviruses from a protein acetylation perspective.     

Extensive lysine acetylation occurs in evolutionarily conserved metabolic pathways and parasite‐specific functions during Plasmodium falciparum intraerythrocytic development

Lysine acetylation has emerged as a major post‐translational modification involved in diverse cellular functions. Using a combination of immunoisolation and liquid chromatography coupled to accurate mass spectrometry, we determined the first acetylome of the human malaria parasite Plasmodium falciparum during its active proliferation in erythrocytes with 421 acetylation sites identified in 230 proteins. Lysine‐acetylated proteins are distributed in the nucleus, cytoplasm, mitochondrion and apicoplast. Whereas occurrence of lysine acetylation in a similarly wide range of cellular functions suggests conservation of lysine acetylation through evolution, the Plasmodium acetylome also revealed significant divergence from those of other eukaryotes and even the closely related parasite Toxoplasma. This divergence is reflected in the acetylation of a large number of Plasmodium‐specific proteins and different acetylation sites in evolutionarily conserved acetylated proteins. A prominent example is the abundant acetylation of proteins in the glycolysis pathway but relatively deficient acetylation of enzymes in the citrate cycle. Using specific transgenic lines and inhibitors, we determined that the acetyltransferase PfMYST and lysine deacetylases play important roles in regulating the dynamics of cytoplasmic protein acetylation. The Plasmodium acetylome provides an exciting start point for further exploration of functions of acetylation in the biology of malaria parasites.     

Recognition Imaging of Acetylated Chromatin Using a DNA Aptamer

Histone acetylation plays an important role in the regulation of gene expression. A DNA aptamer generated by in vitro selection to be highly specific for histone H4 protein acetylated at lysine 16 was used as a recognition element for atomic force microscopy-based recognition imaging of synthetic nucleosomal arrays with precisely controlled acetylation. The aptamer proved to be reasonably specific at recognizing acetylated histones, with recognition efficiencies of 60% on-target and 12% off-target. Though this selectivity is much poorer than the >2000:1 equilibrium specificity of the aptamer, it is a large improvement on the performance of a ChIP-quality antibody, which is not selective at all in this application, and it should permit high-fidelity recognition with repeated imaging. The ability to image the precise location of posttranslational modifications may permit nanometer-scale investigation of their effect on chromatin structure.     

Lysine acetylation contributes to development,aflatoxin biosynthesis and pathogenicity in Aspergillus flavus

Aspergillus flavus is a pathogenic fungus that produces carcinogenic aflatoxins, posing a great threat to crops, animals and humans. Lysine acetylation is one of the most important reversible post-translational modifications and plays a vital regulatory role in various cellular processes. However, current information on the extent and function of lysine acetylation and aflatoxin biosynthesis in A. flavus is limited. Here, a global acetylome analysis of A. flavus was performed by peptide pre-fractionation, pan-acetylation antibody enrichment and liquid chromatography–mass spectrometry. A total of 1313 high-confidence acetylation sites in 727 acetylated proteins were identified in A. flavus. These acetylation proteins are widely involved in glycolysis/gluconeogenesis, pentose phosphate pathway, citric acid cycle and aflatoxin biosynthesis. AflO (O-methyltransferase), a key enzyme in aflatoxin biosynthesis, was found to be acetylated at K241 and K384. Deletion of aflO not only impaired conidial and sclerotial developments, but also dramatically suppressed aflatoxin production and pathogenicity of A. flavus. Further site-specific mutations showed that lysine acetylation of AflO could also result in defects in development, aflatoxin production and pathogenicity, suggesting that acetylation plays a vital role in the regulation of the enzymatic activity of AflO in A. flavus. Our findings provide evidence for the involvement of lysine acetylation in various biological processes in A. flavus and facilitating in the elucidation of metabolic networks.     

Generation of an antibody recognizing a set of acetylated proteins,including subunits of BAF complexes

The aim of this study was to generate an antibody specific to Ki-67 acetylated at lysine 3180, whose existence was reported in an acetylome study (Scholz, C., B.T. Weinert, S.A. Wagner, P. Beli, Y. Miyake, J. Qi, L.J. Jensen, W. Streicher, A.R. McCarthy, N.J. Westwood, S. Lain, J. Cox, P. Matthias, M. Mann, J.E. Bradner, and C. Choudhary. 2015). Rabbits were immunized with a synthetic acetylated peptide corresponding to acetylated lysine 3180 of Ki-67 and the residues flanking it. The obtained antibody, referred to as Ab3180 in this study, was affinity purified with the antigen peptide and characterized. Immunoblot analysis of cell extracts using Ab3180 revealed that this antibody unexpectedly recognized a set of acetylated proteins unrelated to Ki-67. Ab3180-recognizable proteins were immunoprecipitated from cell extracts in a stringent condition and identified by mass-spec analysis as subunits of BAF (mammalian SWI/SNF) chromatin remodeling complexes. The unique specificity of Ab3180 will allow this antibody to be a useful tool for analyzing the acetylation of BAF complexes and its significance to the formation/function of BAF complexes.     

Comprehensive Profiling of Lysine Acetylome in Baculovirus Infected Silkworm (Bombyx mori) Cells

Bombyx mori is one of the key lepidopteran model species, and is economically important for silk production and proteinaceous drug expression. Baculovirus and insect host are important natural biological models for studying host–pathogen interactions. The impact of Bombyx mori nucleopolyhedrovirus (BmNPV) infection on the proteome and acetylome of Bombyx mori ovarian (BmN) cells are explored to facilitate a better understanding of infection‐driven interactions between BmNPV and host in vitro. The proteome and acetylome are profiled through six‐plex Tandem mass tag (TMT) labeling‐based quantitative proteomics. A total of 4194 host proteins are quantified, of which 33 are upregulated and 47 are downregulated in BmN cells at 36 h post‐infection. Based on the proteome, quantifiable differential Kac proteins are identified and functionally annotated to gene expression regulation, energy metabolism, substance synthesis, and metabolism after BmNPV infection. Altogether, 644 Kac sites in 431 host proteins and 39 Kac sites in 22 viral proteins are identified and quantified in infected BmN cells. Our study demonstrates that BmNPV infection globally impacts the proteome and acetylome of BmN cells. The viral proteins are also acetylated by the host acetyltransferase. Protein acetylation is essential for cellular self‐regulation and response to virus infection. This study provides new insights for understanding the host–virus interaction mechanisms, and the role of acetylation in BmN cellular response to viral infection.     

Proteome-wide lysine acetylation identification in developing rice (Oryza sativa) seeds and protein co-modification by acetylation,succinylation, ubiquitination,and phosphorylation

Protein lysine acetylation is a highly conserved post-translational modification with various biological functions. However, only a limited number of acetylation sites have been reported in plants, especially in cereals, and the function of non-histone protein acetylation is still largely unknown. In this report, we identified 1003 lysine acetylation sites in 692 proteins of developing rice seeds, which greatly extended the number of known acetylated sites in plants. Seven distinguished motifs were detected flanking acetylated lysines. Functional annotation analyses indicated diverse biological processes and pathways engaged in lysine acetylation. Remarkably, we found that several key enzymes in storage starch synthesis pathway and the main storage proteins were heavily acetylated. A comprehensive comparison of the rice acetylome, succinylome, ubiquitome and phosphorylome with available published data was conducted. A large number of proteins carrying multiple kinds of modifications were identified and many of these proteins are known to be key enzymes of vital metabolic pathways. Our study provides extending knowledge of protein acetylation. It will have critical reference value for understanding the mechanisms underlying PTM mediated multiple signal integration in the regulation of metabolism and development in plants.