The combined effect of acetylation and glycation on the chaperone and anti-apoptotic functions of human α-crystallin

N^ε-acetylation occurs on select lysine residues in α-crystallin of the human lens and alters its chaperone function. In this study, we investigated the effect of N^ε-acetylation on advanced glycation end product (AGE) formation and consequences of the combined N^ε-acetylation and AGE formation on the function of α-crystallin. Immunoprecipitation experiments revealed that N^ε-acetylation of lysine residues and AGE formation co-occurs in both αA- and αB-crystallin of the human lens. Prior acetylation of αA- and αB-crystallin with acetic anhydride (Ac₂O) before glycation with methylglyoxal (MGO) resulted in significant inhibition of the synthesis of two AGEs, hydroimidazolone (HI) and argpyrimidine. Similarly, synthesis of ascorbate-derived AGEs, pentosidine and N^ε-carboxymethyl lysine (CML), was inhibited in both proteins by prior acetylation. In all cases, inhibition of AGE synthesis was positively related to the degree of acetylation. While prior acetylation further increased the chaperone activity of MGO-glycated αA-crystallin, it inhibited the loss of chaperone activity by ascorbate-glycation in both proteins. BioPORTER-mediated transfer of αA- and αB-crystallin into CHO cells resulted in significant protection against hyperthermia-induced apoptosis. This effect was enhanced in acetylated and MGO-modified αA- and αB-crystallin. Caspase-3 activity was reduced in α-crystallin transferred cells. Glycation of acetylated proteins with either MGO or ascorbate produced no significant change in the anti-apoptotic function. Collectively, these data demonstrate that lysine acetylation and AGE formation can occur concurrently in α-crystallin of human lens, and that lysine acetylation improves anti-apoptotic function of α-crystallin and prevents ascorbate-mediated loss of chaperone function.     

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.     

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.     

Lysine acetylome profiling uncovers novel histone deacetylase substrate proteins in Arabidopsis

Histone deacetylases have central functions in regulating stress defenses and development in plants. However, the knowledge about the deacetylase functions is largely limited to histones, although these enzymes were found in diverse subcellular compartments. In this study, we determined the proteome‐wide signatures of the RPD3/HDA1 class of histone deacetylases in Arabidopsis. Relative quantification of the changes in the lysine acetylation levels was determined on a proteome‐wide scale after treatment of Arabidopsis leaves with deacetylase inhibitors apicidin and trichostatin A. We identified 91 new acetylated candidate proteins other than histones, which are potential substrates of the RPD3/HDA1‐like histone deacetylases in Arabidopsis, of which at least 30 of these proteins function in nucleic acid binding. Furthermore, our analysis revealed that histone deacetylase 14 (HDA14) is the first organellar‐localized RPD3/HDA1 class protein found to reside in the chloroplasts and that the majority of its protein targets have functions in photosynthesis. Finally, the analysis of HDA14 loss‐of‐function mutants revealed that the activation state of RuBisCO is controlled by lysine acetylation of RuBisCO activase under low‐light conditions.     

Digestibility of Acetylated and Succinylated Proteins by Pepsin-Pancreatin and Some Intracellular Peptidases

The molecular sizes of hydrolysates of acetylated and succinylated caseins by pepsin-pancreatin were examined by gel filtration on Sephadex G-15. There were more large peptides (average residual number > 15) in the hydrolysates of the acylated caseins than there were in the hydrolysate of unmodified casein. In these large peptides from the acylated caseins the contents of N^ε-acyl-lysines were high. The digestibility of N^ε-acetyl and N^ε-succinyl lysine bonds in peptides by aminopeptidases [(EC 3.4.11.1) and (EC 3.4.11.2)] and watermelon carboxypeptidase [model enzyme of cathepsin A (EC 3.4.16.1)] was examined using digest of acylated caseins by pepsin, trypsin and α-chymotrypsin and some synthetic peptides. All peptidases released either N^ε-acetyl or N^ε-succinyl-lysine from peptides.

The hydrolytic processes of acetylated and succinylated proteins before and after intestinal absorption are discussed.     

Bromodomains shake the hegemony of pan‐acetyl antibodies

Acetylation signaling pathways are involved in numerous cellular processes and are used as therapeutic targets in several disease contexts. However, acetylated proteins only represent a minor fraction of the full proteome, and the identification and quantification of acetylated sites remain a technological challenge. Currently, pan‐acetyl antibodies are used to increase the abundance of acetylated peptides through affinity purification before MS analysis. These antibodies are powerful reagents, but they are hampered by a lack of specificity, affinity, and batch‐to‐batch reproducibility. In this issue, Bryson et al. (Proteomics 2015 15, 1470–1475) present an interesting alternative to these antibodies, in the form of bromodomains. These domains specifically recognize acetylated lysines, and were successfully used in this study to enrich for acetylated peptides before MS analysis. Future development of this pioneering approach could help overcome this limiting step in the characterization of acetylproteomes.     

Proteomic profiling of lysine acetylation in Pseudomonas aeruginosa reveals the diversity of acetylated proteins

Protein lysine acetylation is a reversible and highly regulated post‐translational modification with the well demonstrated physiological relevance in eukaryotes. Recently, its important role in the regulation of metabolic processes in bacteria was highlighted. Here, we reported the lysine acetylproteome of Pseudomonas aeruginosa using a proteomic approach. We identified 430 unique peptides corresponding to 320 acetylated proteins. In addition to the proteins involved in various metabolic pathways, several enzymes contributing to the lipopolysaccharides biosynthesis were characterized as acetylated. This data set illustrated the abundance and the diversity of acetylated lysine proteins in P. aeruginosa and opens opportunities to explore the role of the acetylation in the bacterial physiology.     

Acetylation of αA-crystallin in the human lens: Effects on structure and chaperone function

α-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a N^ε-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it affects the chaperone function of the protein.     

Quantitative proteome‐based systematic identification of SIRT7 substrates

SIRT7 is a class III histone deacetylase that is involved in numerous cellular processes. Only six substrates of SIRT7 have been reported thus far, so we aimed to systematically identify SIRT7 substrates using stable‐isotope labeling with amino acids in cell culture (SILAC) coupled with quantitative mass spectrometry (MS). Using SIRT7^+/+ and SIRT7 ^?/? mouse embryonic fibroblasts as our model system, we identified and quantified 1493 acetylation sites in 789 proteins, of which 261 acetylation sites in 176 proteins showed ≥2‐fold change in acetylation state between SIRT7^?/? and SIRT7^+/+ cells. These proteins were considered putative SIRT7 substrates and were carried forward for further analysis. We then validated the predictive efficiency of the SILAC–MS experiment by assessing substrate acetylation status in vitro in six predicted proteins. We also performed a bioinformatic analysis of the MS data, which indicated that many of the putative protein substrates were involved in metabolic processes. Finally, we expanded our list of candidate substrates by performing a bioinformatics‐based prediction analysis of putative SIRT7 substrates, using our list of putative substrates as a positive training set, and again validated a subset of the proteins in vitro. In summary, we have generated a comprehensive list of SIRT7 candidate substrates.     

In Streptomyces lividans,acetyl‐CoA synthetase activity is controlled by O‐serine and Nɛ‐lysine acetylation

Protein acetylation is a rapid mechanism for control of protein function. Acetyl‐CoA synthetase (AMP‐forming, Acs) is the paradigm for the control of metabolic enzymes by lysine acetylation. In many bacteria, type I or II protein acetyltransferases acetylate Acs, however, in actinomycetes type III protein acetyltransferases control the activity of Acs. We measured changes in the activity of the Streptomyces lividans Acs (SlAcs) enzyme upon acetylation by PatB using in vitro and in vivo analyses. In addition to the acetylation of residue K610, residue S608 within the acetylation motif of SlAcs was also acetylated (PKTRSGK⁶¹⁰). S608 acetylation rendered SlAcs inactive and non‐acetylatable by PatB. It is unclear whether acetylation of S608 is enzymatic, but it was clear that this modification occurred in vivo in Streptomyces. In S. lividans, an NAD⁺‐dependent sirtuin deacetylase from Streptomyces, SrtA (a homologue of the human SIRT4 protein) was needed to maintain SlAcs function in vivo. We have characterized a sirtuin‐dependent reversible lysine acetylation system in Streptomyces lividans that targets and controls the Acs enzyme of this bacterium. These studies raise questions about acetyltransferase specificity, and describe the first Acs enzyme in any organism whose activity is modulated by O‐Ser and N^?‐Lys acetylation.     

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.     

KDAC8 substrate specificity quantified by a biologically relevant,label‐free deacetylation assay

Analysis of the human proteome has identified thousands of unique protein sequences that contain acetylated lysine residues in vivo. These modifications regulate a variety of biological processes and are reversed by the lysine deacetylase (KDAC) family of enzymes. Despite the known prevalence and importance of acetylation, the details of KDAC substrate recognition are not well understood. While several methods have been developed to monitor protein deacetylation, none are particularly suited for identifying enzyme‐substrate pairs of label‐free substrates across the entire family of lysine deacetylases. Here, we present a fluorescamine‐based assay which is more biologically relevant than existing methods and amenable to probing substrate specificity. Using this assay, we evaluated the activity of KDAC8 and other lysine deacetylases, including a sirtuin, for several peptides derived from known acetylated proteins. KDAC8 showed clear preferences for some peptides over others, indicating that the residues immediately surrounding the acetylated lysine play an important role in substrate specificity. Steady‐state kinetics suggest that the sequence surrounding the acetylated lysine affects binding affinity and catalytic rate independently. Our results provide direct evidence that potential KDAC8 substrates previously identified through cell based experiments can be directly deacetylated by KDAC8. Conversely, the data from this assay did not correlate well with predictions from previous screens for KDAC8 substrates using less biologically relevant substrates and assay conditions. Combining results from our assay with mass spectrometry‐based experiments and cell‐based experiments will allow the identification of specific KDAC‐substrate pairs and lead to a better understanding of the biological consequences of these interactions.     

Side‐chain thioamides as fluorescence quenching probes

Thioamides, single atom oxygen‐to‐sulfur substitutions of canonical amide bonds, can be valuable probes for protein folding and protease studies. Here, we investigate the fluorescence quenching properties of thioamides incorporated into the side‐chains of amino acids. We synthesize and incorporate Fmoc‐protected, solid‐phase peptide synthesis building blocks for introducing N^ε‐thioacetyl‐lysine and γ‐thioasparagine. Using rigid model peptides, we demonstrate the distance‐dependent fluorescence quenching of these thioamides. Furthermore, we describe attempts to incorporate of N^ε‐thioacetyl‐lysine into proteins expressed in Escherichia coli using amber codon suppression.     

Proteomic profiling of the mitochondrial inner membrane of rat renal proximal convoluted tubules

The proximal convoluted tubule is the primary site of renal fluid, electrolyte, and nutrient reabsorption, processes that consume large amounts of adenosine‐5′‐triphosphate. Previous proteomic studies have profiled the adaptions that occur in this segment of the nephron in response to the onset of metabolic acidosis. To extend this analysis, a proteomic workflow was developed to characterize the proteome of the mitochondrial inner membrane of the rat renal proximal convoluted tubule. Separation by LC coupled with analysis by MS/MS (LC‐MS/MS) confidently identified 206 proteins in the combined samples. Further proteomic analysis identified 14 peptides that contain an N‐?‐acetyl‐lysine, seven of which are novel sites. This study provides the first proteomic profile of the mitochondrial inner membrane proteome of this segment of the rat renal nephron. The MS data have been deposited in the ProteomeXchange with the identifier PXD000121.     

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.     

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.