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Scientific Reports Aug 2017Protein acetylation plays a critical role in biological processes by regulating the functions and properties of proteins. Thus, the study of protein acetylation dynamics...
Protein acetylation plays a critical role in biological processes by regulating the functions and properties of proteins. Thus, the study of protein acetylation dynamics is critical for understanding of how this modification influences protein stability, localization, and function. Here we performed a comprehensive characterization of protein acetylation dynamics using mass spectrometry (MS) based proteomics through utilization of C-glucose or D-acetate, which are metabolized into acetyl-coA, labeling acetyl groups through subsequent incorporation into proteins. Samples were collected at eight time points to monitor rates and trends of heavy acetyl incorporation. Through this platform, we characterized around 1,000 sites with significantly increasing acetylation trends, which we clustered based on their rates of acetylation. Faster rates were enriched on proteins associated with chromatin and RNA metabolism, while slower rates were more typical on proteins involved with lipid metabolism. Among others, we identified sites catalyzed at faster rates with potential critical roles in protein activation, including the histone acetyltransferase p300 acetylated in its activation loop, which could explain self-acetylation as an important feedback mechanism to regulate acetyltransferases. Overall, our studies highlight the dynamic nature of protein acetylation, and how metabolism plays a central role in this regulation.
Topics: Acetates; Acetyl Coenzyme A; Acetylation; Computational Biology; Glucose; HeLa Cells; Humans; Peptides; Protein Conformation; Protein Processing, Post-Translational; Proteome; Proteomics; Structure-Activity Relationship
PubMed: 28860605
DOI: 10.1038/s41598-017-09918-3 -
Methods in Molecular Biology (Clifton,... 2019Lysine acetylation is an important posttranslational modification (PTM) that regulates the function of proteins by affecting their localization, stability, binding, and...
Lysine acetylation is an important posttranslational modification (PTM) that regulates the function of proteins by affecting their localization, stability, binding, and enzymatic activity. Aberrant acetylation patterns have been observed in numerous diseases, most notably cancer, which has spurred the development of potential therapeutics that target acetylation pathways. Mass spectrometry (MS) has become the most adopted tool not only for the qualitative identification of acetylation sites but also for their large-scale quantification. By using heavy isotope labeling in cell culture combined with MS, it is now possible to accurately quantify newly synthesized acetyl groups and other PTMs, allowing differentiation between dynamically regulated and steady-state modifications. Here, we describe MS-based protocols to identify acetylation sites and quantify acetylation rates on both proteins in general and in the special case of histones. In the experimental approach for the former, C-glucose and D-acetate are used to metabolically label protein acetylation in cells with stable isotopes, thus allowing isotope incorporation to be tracked over time. After protein extraction and digestion, acetylated peptides are enriched via immunoprecipitation and then analyzed by MS. For histones, a similar metabolic labeling approach is performed, followed by acid extraction, derivatization with propionic anhydride, and trypsin digestion prior to MS analysis. The procedures presented may be adapted to investigate acetylation dynamics in a broad range of experimental contexts, including different cell types and stimulation conditions.
Topics: Acetylation; Cell Culture Techniques; Chromatography, Liquid; Data Interpretation, Statistical; Databases, Protein; Histones; Humans; Isotope Labeling; Lysine; Protein Processing, Post-Translational; Proteomics; Tandem Mass Spectrometry
PubMed: 30980322
DOI: 10.1007/978-1-4939-9232-4_5 -
Advances in Experimental Medicine and... 2019Mitochondria have a central role in cellular metabolism and reversible post-translational modifications regulate activity of mitochondrial proteins. Thanks to advances...
Mitochondria have a central role in cellular metabolism and reversible post-translational modifications regulate activity of mitochondrial proteins. Thanks to advances in proteomics, lysine acetylation has arisen as an important post-translational modification in the mitochondrion. During acetylation an acetyl group is covalently attached to the epsilon amino group in the side chain of lysine residues using acetyl-CoA as the substrate donor. Therefore the positive charge is neutralized, and this can affect the function of proteins thereby regulating enzyme activity, protein interactions, and protein stability. The major deacetylase in mitochondria is SIRT3 whose activity regulates many mitochondrial enzymes. The method of choice for the analysis of acetylated proteins foresees the combination of mass spectrometry-based proteomics with affinity enrichment techniques. Beyond the identification of lysine-acetylated proteins, many studies are moving towards the characterization of acetylated patterns in different diseases. Indeed, modifications in lysine acetylation status can directly alter mitochondrial function and, therefore, be linked to human diseases such as metabolic diseases, cancer, myocardial injury and neurodegenerative diseases. Despite the progress in the characterization of different lysine acetylation sites, additional studies are needed to differentiate the specific changes with a significant biological relevance.
Topics: Acetylation; Humans; Lysine; Mitochondria; Mitochondrial Proteins; Phenotype; Protein Processing, Post-Translational
PubMed: 31452135
DOI: 10.1007/978-981-13-8367-0_4 -
Scientific Reports Apr 2023The majority of proteins in mammalian cells are modified by covalent attachment of an acetyl-group to the N-terminus (Nt-acetylation). Paradoxically, Nt-acetylation has...
The majority of proteins in mammalian cells are modified by covalent attachment of an acetyl-group to the N-terminus (Nt-acetylation). Paradoxically, Nt-acetylation has been suggested to inhibit as well as to promote substrate degradation. Contrasting these findings, proteome-wide stability measurements failed to detect any correlation between Nt-acetylation status and protein stability. Accordingly, by analysis of protein stability datasets, we found that predicted Nt-acetylation positively correlates with protein stability in case of GFP, but this correlation does not hold for the entire proteome. To further resolve this conundrum, we systematically changed the Nt-acetylation and ubiquitination status of model substrates and assessed their stability. For wild-type Bcl-B, which is heavily modified by proteasome-targeting lysine ubiquitination, Nt-acetylation did not correlate with protein stability. For a lysine-less Bcl-B mutant, however, Nt-acetylation correlated with increased protein stability, likely due to prohibition of ubiquitin conjugation to the acetylated N-terminus. In case of GFP, Nt-acetylation correlated with increased protein stability, as predicted, but our data suggest that Nt-acetylation does not affect GFP ubiquitination. Similarly, in case of the naturally lysine-less protein p16, Nt-acetylation correlated with protein stability, regardless of ubiquitination on its N-terminus or on an introduced lysine residue. A direct effect of Nt-acetylation on p16 stability was supported by studies in NatB-deficient cells. Together, our studies argue that Nt-acetylation can stabilize proteins in human cells in a substrate-specific manner, by competition with N-terminal ubiquitination, but also by other mechanisms that are independent of protein ubiquitination status.
Topics: Animals; Humans; Lysine; Proteome; Acetylation; Protein Processing, Post-Translational; Ubiquitination; Mammals
PubMed: 37005459
DOI: 10.1038/s41598-023-32380-3 -
Journal of Proteome Research Jan 2021Acetylation was initially discovered as a post-translational modification (PTM) on the unstructured, highly basic N-terminal tails of eukaryotic histones in the 1960s.... (Review)
Review
Acetylation was initially discovered as a post-translational modification (PTM) on the unstructured, highly basic N-terminal tails of eukaryotic histones in the 1960s. Histone acetylation constitutes part of the "histone code", which regulates chromosome compaction and various DNA processes such as gene expression, recombination, and DNA replication. In bacteria, nucleoid-associated proteins (NAPs) are responsible these functions in that they organize and compact the chromosome and regulate some DNA processes. The highly conserved DNABII family of proteins are considered functional homologues of eukaryotic histones despite having no sequence or structural conservation. Within the past decade, a growing interest in N-lysine acetylation led to the discovery that hundreds of bacterial proteins are acetylated with diverse cellular functions, in direct contrast to the original thought that this was a rare phenomenon. Similarly, other previously undiscovered bacterial PTMs, like serine, threonine, and tyrosine phosphorylation, have also been characterized. In this review, the various PTMs that were discovered among DNABII family proteins, specifically histone-like protein (HU) orthologues, from large-scale proteomic studies are discussed. The functional significance of these modifications and the enzymes involved are also addressed. The discovery of novel PTMs on these proteins begs this question: is there a histone-like code in bacteria?
Topics: Acetylation; Bacteria; Histone Code; Histones; Protein Processing, Post-Translational; Proteomics
PubMed: 32962352
DOI: 10.1021/acs.jproteome.0c00442 -
MBio Aug 2023The protozoan pathogen relies on tight regulation of gene expression to invade and establish infection in its host. The divergent gene regulatory mechanisms of and...
The protozoan pathogen relies on tight regulation of gene expression to invade and establish infection in its host. The divergent gene regulatory mechanisms of and related apicomplexan pathogens rely heavily on regulators of chromatin structure and histone modifications. The important contribution of histone acetylation for in both acute and chronic infection has been demonstrated, where histone acetylation increases at active gene loci. However, the direct consequences of specific histone acetylation marks and the chromatin pathway that influences transcriptional regulation in response to the modification are unclear. As a reader of lysine acetylation, the bromodomain serves as a mediator between the acetylated histone and transcriptional regulators. Here we show that the bromodomain protein, TgBDP1, which is conserved among Apicomplexa and within the Alveolata superphylum, is essential for asexual proliferation. Using cleavage under targets and tagmentation, we demonstrate that TgBDP1 is recruited to transcriptional start sites of a large proportion of parasite genes. Transcriptional profiling during TgBDP1 knockdown revealed that loss of TgBDP1 leads to major dysregulation of gene expression, implying multiple roles for TgBDP1 in both gene activation and repression. This is supported by interactome analysis of TgBDP1 demonstrating that TgBDP1 forms a core complex with two other bromodomain proteins and an ApiAP2 factor. This core complex appears to interact with other epigenetic factors such as nucleosome remodeling complexes. We conclude that TgBDP1 interacts with diverse epigenetic regulators to exert opposing influences on gene expression in the tachyzoite. IMPORTANCE Histone acetylation is critical for proper regulation of gene expression in the single-celled eukaryotic pathogen . Bromodomain proteins are "readers" of histone acetylation and may link the modified chromatin to transcription factors. Here, we show that the bromodomain protein TgBDP1 is essential for parasite survival and that loss of TgBDP1 results in global dysregulation of gene expression. TgBDP1 is recruited to the promoter region of a large proportion of parasite genes, forms a core complex with two other bromodomain proteins, and interacts with different transcriptional regulatory complexes. We conclude that TgBDP1 is a key factor for sensing specific histone modifications to influence multiple facets of transcriptional regulation in .
Topics: Animals; Toxoplasma; Histones; Chromatin; Gene Expression Regulation; Parasites; Epigenesis, Genetic; Acetylation; Protozoan Proteins
PubMed: 37350586
DOI: 10.1128/mbio.03573-22 -
Molecules (Basel, Switzerland) Jul 2018Nowadays advanced mass spectrometry techniques make the identification of protein posttranslational modifications (PTMs) much easier than ever before. A series of... (Review)
Review
Nowadays advanced mass spectrometry techniques make the identification of protein posttranslational modifications (PTMs) much easier than ever before. A series of proteomic studies have demonstrated that large numbers of proteins in cells are modified by phosphorylation, acetylation and many other types of PTMs. However, only limited studies have been performed to validate or characterize those identified modification targets, mostly because PTMs are very dynamic, undergoing large changes in different growth stages or conditions. To overcome this issue, the genetic code expansion strategy has been introduced into PTM studies to genetically incorporate modified amino acids directly into desired positions of target proteins. Without using modifying enzymes, the genetic code expansion strategy could generate homogeneously modified proteins, thus providing powerful tools for PTM studies. In this review, we summarized recent development of genetic code expansion in PTM studies for research groups in this field.
Topics: Acetylation; Animals; Genetic Code; Humans; Mass Spectrometry; Phosphorylation; Protein Processing, Post-Translational; Proteins; Proteomics
PubMed: 29986538
DOI: 10.3390/molecules23071662 -
Acetylation of histones and non-histone proteins is not a mere consequence of ongoing transcription.Nature Communications Jun 2024In all eukaryotes, acetylation of histone lysine residues correlates with transcription activation. Whether histone acetylation is a cause or consequence of...
In all eukaryotes, acetylation of histone lysine residues correlates with transcription activation. Whether histone acetylation is a cause or consequence of transcription is debated. One model suggests that transcription promotes the recruitment and/or activation of acetyltransferases, and histone acetylation occurs as a consequence of ongoing transcription. However, the extent to which transcription shapes the global protein acetylation landscapes is not known. Here, we show that global protein acetylation remains virtually unaltered after acute transcription inhibition. Transcription inhibition ablates the co-transcriptionally occurring ubiquitylation of H2BK120 but does not reduce histone acetylation. The combined inhibition of transcription and CBP/p300 further demonstrates that acetyltransferases remain active and continue to acetylate histones independently of transcription. Together, these results show that histone acetylation is not a mere consequence of transcription; acetyltransferase recruitment and activation are uncoupled from the act of transcription, and histone and non-histone protein acetylation are sustained in the absence of ongoing transcription.
Topics: Acetylation; Histones; Transcription, Genetic; Humans; Ubiquitination; p300-CBP Transcription Factors; Protein Processing, Post-Translational; Histone Acetyltransferases; Lysine
PubMed: 38862536
DOI: 10.1038/s41467-024-49370-2 -
The Journal of Biological Chemistry Aug 2021Sialic acids are nine-carbon sugars that frequently cap glycans at the cell surface in cells of vertebrates as well as cells of certain types of invertebrates and... (Review)
Review
Sialic acids are nine-carbon sugars that frequently cap glycans at the cell surface in cells of vertebrates as well as cells of certain types of invertebrates and bacteria. The nine-carbon backbone of sialic acids can undergo extensive enzymatic modification in nature and O-acetylation at the C-4/7/8/9 position in particular is widely observed. In recent years, the detection and analysis of O-acetylated sialic acids have advanced, and sialic acid-specific O-acetyltransferases (SOATs) and O-acetylesterases (SIAEs) that add and remove O-acetyl groups, respectively, have been identified and characterized in mammalian cells, invertebrates, bacteria, and viruses. These advances now allow us to draw a more complete picture of the biosynthetic pathway of the diverse O-acetylated sialic acids to drive the generation of genetically and biochemically engineered model cell lines and organisms with altered expression of O-acetylated sialic acids for dissection of their roles in glycoprotein stability, development, and immune recognition, as well as discovery of novel functions. Furthermore, a growing number of studies associate sialic acid O-acetylation with cancer, autoimmunity, and infection, providing rationale for the development of selective probes and inhibitors of SOATs and SIAEs. Here, we discuss the current insights into the biosynthesis and biological functions of O-acetylated sialic acids and review the evidence linking this modification to disease. Furthermore, we discuss emerging strategies for the design, synthesis, and potential application of unnatural O-acetylated sialic acids and inhibitors of SOATs and SIAEs that may enable therapeutic targeting of this versatile sialic acid modification.
Topics: Acetylation; Acetyltransferases; Animals; Biosynthetic Pathways; Carboxylic Ester Hydrolases; Disease; Glycoproteins; Humans; N-Acetylneuraminic Acid; Polysaccharides
PubMed: 34157283
DOI: 10.1016/j.jbc.2021.100906 -
Acetylation of Lactate Dehydrogenase Negatively Regulates the Acidogenicity of Streptococcus mutans.MBio Oct 2022Lysine acetylation, a ubiquitous and dynamic regulatory posttranslational modification (PTM), affects hundreds of proteins across all domains of life. In bacteria,...
Lysine acetylation, a ubiquitous and dynamic regulatory posttranslational modification (PTM), affects hundreds of proteins across all domains of life. In bacteria, lysine acetylation can be found in many essential pathways, and it is also crucial for bacterial virulence. However, the biological significance of lysine acetylation events to bacterial virulence factors remains poorly characterized. In Streptococcus mutans, the acetylome profiles help identify several lysine acetylation sites of lactate dehydrogenase (LDH), which catalyzes the conversion of pyruvate to lactic acid, causing the deterioration of teeth. We investigated the regulatory mechanism of LDH acetylation and characterized the effect of LDH acetylation on its function. We overexpressed the 15 Gcn5 -acetyltransferases (GNAT) family members in S. mutans and showed that the acetyltransferase ActA impaired its acidogenicity by acetylating LDH. Additionally, enzymatic acetyltransferase reactions demonstrated that purified ActA could acetylate LDH , and 10 potential lysine acetylation sites of LDH were identified by mass spectrometry, 70% of which were also detected . We further demonstrated that the lysine acetylation of LDH inhibited its enzymatic activity, and a subsequent rat caries model showed that ActA impaired the cariogenicity of S. mutans. Collectively, we demonstrated that ActA, the first identified and characterized acetyltransferase in S. mutans, acetylated the LDH enzymatically and inhibited its enzymatic activity, thereby providing a starting point for the further analysis of the biological significance of lysine acetylation in the virulence of S. mutans. Lysine acetylation, a dynamic regulatory posttranslational modification, remains poorly characterized in bacteria. Hundreds of proteins have been identified to be acetylated in bacteria, with advances made in acetylome analyses. However, the regulatory mechanisms and functional significance of the majority of these acetylated proteins remain unclear. We analyzed the acetylome profiles of Streptococcus mutans and found that lactate dehydrogenase (LDH) contains several lysine acetylation sites. We also demonstrated that the acetyltransferase ActA, a member of the Gcn5 -acetyltransferases (GNAT) family in S. mutans, acetylated LDH, inhibited its enzymatic ability to catalyze the conversion of pyruvate to lactic acid, and impaired its cariogenicity in a rat caries model. Therefore, LDH acetylation might be a potential target that can be exploited in the design of novel therapeutics to prevent dental caries.
Topics: Rats; Animals; Acetylation; Streptococcus mutans; Lysine; L-Lactate Dehydrogenase; Dental Caries; Protein Processing, Post-Translational; Virulence Factors; Acetyltransferases; Lactic Acid; Pyruvates
PubMed: 36043788
DOI: 10.1128/mbio.02013-22