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Cell Reports Jan 2023Advanced mass spectrometry methods have detected thousands of post-translational phosphorylation and acetylation sites in bacteria, but their functional role and the...
Advanced mass spectrometry methods have detected thousands of post-translational phosphorylation and acetylation sites in bacteria, but their functional role and the enzymes catalyzing these modifications remain largely unknown. In addition to enzymatic acetylation, lysine residues can also be chemically acetylated by the metabolite acetyl phosphate. In Escherichia coli, acetylation at over 3,000 sites has been linked to acetyl phosphate, but the functionality of this widespread non-enzymatic acetylation is even less clear than the enzyme-catalyzed one. Here, we investigate the role of acetyl-phosphate-mediated acetylation in E. coli central metabolism. Out of 19 enzymes investigated, only GapA and GpmA are acetylated at high stoichiometry, which inhibits their activity by interfering with substrate binding, effectively reducing glycolysis when flux to or from acetate is high. Extrapolating our results to the whole proteome, maximally 10% of the reported non-enzymatically acetylated proteins are expected to reach a stoichiometry that could inhibit their activity.
Topics: Escherichia coli; Acetylation; Organophosphates; Protein Processing, Post-Translational; Glycolysis
PubMed: 36640332
DOI: 10.1016/j.celrep.2022.111950 -
MBio Jun 2022ε-lysine acetylation is a common posttranslational modification observed in diverse species of bacteria. Aside from a few central metabolic enzymes and transcription...
ε-lysine acetylation is a common posttranslational modification observed in diverse species of bacteria. Aside from a few central metabolic enzymes and transcription factors, little is known about how this posttranslational modification regulates protein activity. In this work, we investigated how lysine acetylation affects translation in Escherichia coli. In multiple species of bacteria, ribosomal proteins are highly acetylated at conserved lysine residues, suggesting that this modification may regulate translation. In support of this hypothesis, we found that the addition of either of the acetyl donors acetyl phosphate and acetyl-coenzyme A inhibits translation but not transcription using an E. coli cell-free system. Further investigations using assays revealed that acetylation does not appear to alter the rate of translation elongation but, rather, increases the proportions of dissociated 30S and 50S ribosomes, based on polysome profiles of mutants or growth conditions known to promote lysine acetylation. Furthermore, ribosomal proteins are more acetylated in the disassociated 30S and 50S ribosomal subunits than in the fully assembled 70S complex. The effect of acetylation is also growth rate dependent, with disassociation of the subunits being most pronounced during late-exponential and early-stationary-phase growth-the same growth phase where protein acetylation is greatest. Collectively, our data demonstrate that lysine acetylation inhibits translation, most likely by interfering with subunit association. These results have also uncovered a new mechanism for coupling translation to the metabolic state of the cell. Numerous cellular processes are regulated in response to the metabolic state of the cell. One such regulatory mechanism involves lysine acetylation, a covalent modification involving the transfer of an acetyl group from central metabolite acetyl-coenzyme A or acetyl phosphate to a lysine residue in a protein. This posttranslational modification is known to regulate some central metabolic enzymes and transcription factors in bacteria, though a comprehensive understanding of its effect on cellular physiology is still lacking. In the present study, lysine acetylation was also found to inhibit translation in Escherichia coli by impeding ribosome association, most likely by disrupting salt bridges along the binding interface of the 30S and 50S ribosomal subunits. These results further our understanding of lysine acetylation by uncovering protein synthesis as a new target of regulation and aid in the design of bacteria for biotechnology applications where the growth conditions are known to promote lysine acetylation.
Topics: Acetyl Coenzyme A; Acetylation; Escherichia coli; Lysine; Protein Processing, Post-Translational; Ribosomal Proteins; Transcription Factors
PubMed: 35604121
DOI: 10.1128/mbio.01224-22 -
International Journal of Molecular... Jul 2021Histone deacetylase (HDAC) and histone acetyltransferase (HAT) regulate transcription and the most important functions of cells by acetylating/deacetylating histones and... (Review)
Review
Histone deacetylase (HDAC) and histone acetyltransferase (HAT) regulate transcription and the most important functions of cells by acetylating/deacetylating histones and non-histone proteins. These proteins are involved in cell survival and death, replication, DNA repair, the cell cycle, and cell responses to stress and aging. HDAC/HAT balance in cells affects gene expression and cell signaling. There are very few studies on the effects of stroke on non-histone protein acetylation/deacetylation in brain cells. HDAC inhibitors have been shown to be effective in protecting the brain from ischemic damage. However, the role of different HDAC isoforms in the survival and death of brain cells after stroke is still controversial. HAT/HDAC activity depends on the acetylation site and the acetylation/deacetylation of the main proteins (c-Myc, E2F1, p53, ERK1/2, Akt) considered in this review, that are involved in the regulation of cell fate decisions. Our review aims to analyze the possible role of the acetylation/deacetylation of transcription factors and signaling proteins involved in the regulation of survival and death in cerebral ischemia.
Topics: Acetylation; Animals; Brain Ischemia; Histone Acetyltransferases; Histone Deacetylases; Humans; Models, Neurological; Protein Processing, Post-Translational; Signal Transduction; Transcription Factors
PubMed: 34360712
DOI: 10.3390/ijms22157947 -
Molecular Endocrinology (Baltimore, Md.) Mar 2008It has been known since the early 1970s that nuclear receptor complexes bind DNA in association with coregulatory proteins. Characterization of these nuclear receptor... (Review)
Review
It has been known since the early 1970s that nuclear receptor complexes bind DNA in association with coregulatory proteins. Characterization of these nuclear receptor coregulators has revealed diverse enzymatic activities that temporally and spatially coordinate nuclear receptor activity within the context of local chromatin in response to diverse hormone signals. Chromatin-modifying proteins, which dictate the higher-order chromatin structure in which DNA is packaged, in turn orchestrate orderly recruitment of nuclear receptor complexes. Modifications of histones include acetylation, methylation, phosphorylation, ubiquitylation, sumoylation, ADP ribosylation, deimination, and proline isomerization. At this time, we understand how a subset of these modifications regulates nuclear receptor signaling. However, the effects, particularly of acetylation and demethylation, are profound. The finding that nuclear receptors are directly acetylated and that acetylation in turn directly regulates contact-independent growth has broad therapeutic implications. Studies over the past 7 yr have led to the understanding that nuclear receptor acetylation is a conserved function, regulating diverse nuclear receptor activity. Furthermore, we now know that acetylation of multiple and distinct substrates within nuclear receptor signaling pathways, form an acetylation signaling network from the cell surface to the nucleus. The finding that nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylases, the sirtuins, are capable of deacetylating nuclear receptors provides a new level of complexity in the control of nuclear receptor activity in which local intracellular concentrations of NAD may regulate nuclear receptor physiology.
Topics: Acetylation; Histones; Protein Processing, Post-Translational; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Sirtuins
PubMed: 18165438
DOI: 10.1210/me.2007-0379 -
Journal of Proteome Research Apr 2020Lysine acetylation has emerged as one of the most important post-translational modifications, regulating different biological processes. However, its regulation by...
Lysine acetylation has emerged as one of the most important post-translational modifications, regulating different biological processes. However, its regulation by lysine acetyltransferases is still unclear in most cases. Hat1 is a lysine acetyltransferase originally identified based on its ability to acetylate histones. Using an unbiased proteomics approach, we have determined how loss of Hat1 affects the mammalian acetylome. Hat1 and Hat1 mouse embryonic fibroblast cell lines were grown in both glucose- and galactose-containing media, as Hat1 is required for growth on galactose, and Hat1 cells exhibit defects in mitochondrial function. Following trypsin digestion of whole cell extracts, acetylated peptides were enriched by acetyllysine affinity purification, and acetylated peptides were identified and analyzed by label-free quantitation. Comparison of the acetylome from Hat1 cells grown on galactose and glucose demonstrated that there are large carbon source-dependent changes in the mammalian acetylome where the acetylation of enzymes involved in glycolysis were the most affected. Comparisons of the acetylomes from Hat1 and Hat1 cells identified 65 proteins whose acetylation decreased by at least 2.5-fold in cells lacking Hat1. In Hat1 cells, acetylation of the autoregulatory loop of CBP (CREB-binding protein) was the most highly affected, decreasing by up to 20-fold. In addition to the proteins involved in chromatin structure, Hat1-dependent acetylation was also found in a number of transcriptional regulators, including p53 and mitochondrial proteins. Hat1 mitochondrial localization suggests that it may be directly involved in the acetylation of mitochondrial proteins. Data are available via ProteomeXchange with identifier PXD017362.
Topics: Acetylation; Animals; Fibroblasts; Histones; Lysine; Mice; Protein Processing, Post-Translational
PubMed: 32081014
DOI: 10.1021/acs.jproteome.9b00843 -
Cell Reports Aug 2017Replication protein A (RPA) is a multifunctional, single-stranded DNA-binding protein complex and plays a critical role in DNA replication and damage response. Herein,...
Replication protein A (RPA) is a multifunctional, single-stranded DNA-binding protein complex and plays a critical role in DNA replication and damage response. Herein, we show that the 70-kDa subunit of RPA (RPA1) is acetylated on lysine 163 by the acetyltransferases GCN5 and PCAF and that such acetylation is reversed principally via the action of the deacetylase HDAC6. UV irradiation promotes cytoplasmic translocation of HDAC6, thereby disrupting the interaction of HDAC6 with RPA1 and increasing RPA1 acetylation. Mutation of the acetylation site of RPA1 specifically impairs the ability of the protein to interact with the key nucleotide excision repair (NER) protein XPA, reduces XPA retention at sites of DNA damage caused by UV, compromises NER, and renders the cell hypersensitive to UV irradiation. Our data suggest that the acetylation status of RPA1 played a crucial role in repair of DNA damage via NER.
Topics: Acetylation; Cell Cycle Checkpoints; DNA Damage; DNA Repair; HEK293 Cells; HeLa Cells; Histone Deacetylase 6; Humans; Lysine; Protein Binding; Replication Protein A; Sirtuin 1; Ultraviolet Rays
PubMed: 28854355
DOI: 10.1016/j.celrep.2017.08.016 -
Methods in Enzymology 2019Lysine acetylation is a prevalent posttranslational modification that acts as a regulator of protein function, subcellular localization, and interactions. A growing body...
Lysine acetylation is a prevalent posttranslational modification that acts as a regulator of protein function, subcellular localization, and interactions. A growing body of work has highlighted the importance of temporal alterations in protein acetylation during infection with a range of human viruses. It has become clear that both cellular and viral proteins are decorated by lysine acetylations, and that these modifications contribute to core host defense and virus replication processes. Further defining the extent and dynamics of protein acetylation events during the progression of an infection can provide an important new perspective on the intricate mechanisms underlying the biology and pathogenesis of virus infections. Here, we provide protocols for identifying, quantifying, and probing the regulation of lysine acetylations during viral infection. We describe the use of acetyl-lysine immunoaffinity purification and quantitative mass spectrometry for assessing the cellular acetylome at different stages of an infection. As an alternative to traditional antibody-mediated western blotting, we discuss the benefits of targeted mass spectrometry approaches for detecting and quantifying site-specific acetylations on proteins of interest. Specifically, we provide a protocol using parallel reaction monitoring (PRM). We further discuss experimental considerations that are specific to studying viral infections. Finally, we provide a brief overview of the types of assays that can be employed to characterize the function of an acetylation event in the context of infection. As a method to interrogate the regulation of acetylation, we describe the Fluor de Lys assay for monitoring the enzymatic activities of deacetylases.
Topics: Acetylation; Host-Pathogen Interactions; Humans; Lysine; Mass Spectrometry; Protein Processing, Post-Translational; Proteins; Proteome; Proteomics; Viral Proteins; Virus Diseases; Virus Physiological Phenomena
PubMed: 31606092
DOI: 10.1016/bs.mie.2019.06.030 -
International Journal of Molecular... Feb 2021Histone deacetylases (HDACs) are conserved enzymes that remove acetyl groups from lysine side chains in histones and other proteins and play a crucial role in epigenetic...
Histone deacetylases (HDACs) are conserved enzymes that remove acetyl groups from lysine side chains in histones and other proteins and play a crucial role in epigenetic regulation. Previously, we showed that histone acetylation is implicated in ultraviolet (UV)-induced inflammation and matrix impairment. To elucidate the histone acetylation status and specific HDACs involved in skin aging, we examined the changes in histone acetylation, global HDAC activity, and the expression of HDACs and sirtuins (SIRTs) in intrinsically aged and photoaged human skin as well as in UV-irradiated human skin in vivo. Following acute UV irradiation, the acetylated histone H3 (AcH3) level was increased, but HDAC activity and the expression levels of HDAC4, HDAC11, and SIRT4 were significantly decreased. In intrinsically aged skin, AcH3 levels were increased, but HDAC activity and the expression levels of HDAC4, HDAC5, HDAC10, HDAC11, SIRT6, and SIRT7 were significantly decreased. However, histone acetylation and HDAC expression in photoaged skin were not significantly different from those in intrinsically aged skin. Collectively, HDAC4 and HDAC11 were decreased in both UV-irradiated and intrinsically aged skin, suggesting that they may play a universal role in increased histone acetylation associated with skin aging.
Topics: Acetylation; Histone Deacetylases; Histones; Humans; Mitochondrial Proteins; Sirtuins; Skin; Skin Aging; Ultraviolet Rays
PubMed: 33670779
DOI: 10.3390/ijms22042032 -
Position-specific analysis and prediction for protein lysine acetylation based on multiple features.PloS One 2012Protein lysine acetylation is a type of reversible post-translational modification that plays a vital role in many cellular processes, such as transcriptional...
Protein lysine acetylation is a type of reversible post-translational modification that plays a vital role in many cellular processes, such as transcriptional regulation, apoptosis and cytokine signaling. To fully decipher the molecular mechanisms of acetylation-related biological processes, an initial but crucial step is the recognition of acetylated substrates and the corresponding acetylation sites. In this study, we developed a position-specific method named PSKAcePred for lysine acetylation prediction based on support vector machines. The residues around the acetylation sites were selected or excluded based on their entropy values. We incorporated features of amino acid composition information, evolutionary similarity and physicochemical properties to predict lysine acetylation sites. The prediction model achieved an accuracy of 79.84% and a Matthews correlation coefficient of 59.72% using the 10-fold cross-validation on balanced positive and negative samples. A feature analysis showed that all features applied in this method contributed to the acetylation process. A position-specific analysis showed that the features derived from the critical neighboring residues contributed profoundly to the acetylation site determination. The detailed analysis in this paper can help us to understand more of the acetylation mechanism and can provide guidance for the related experimental validation.
Topics: Acetylation; Amino Acid Sequence; Binding Sites; Chemical Phenomena; Computational Biology; Conserved Sequence; Evolution, Molecular; Lysine; Protein Processing, Post-Translational; Proteins; User-Computer Interface
PubMed: 23173045
DOI: 10.1371/journal.pone.0049108 -
Laboratory Investigation; a Journal of... Jan 2022Ameloblastoma (AB) is the most common benign epithelial odontogenic tumor occurring in the jawbone. AB is a slowly growing tumor but sometimes shows a locally invasive...
Ameloblastoma (AB) is the most common benign epithelial odontogenic tumor occurring in the jawbone. AB is a slowly growing tumor but sometimes shows a locally invasive and an aggressive growth pattern with a marked bone resorption. In addition, the local recurrence and distant metastasis of AB also sometimes occurs, which resembles one of the typical malignant potentials. From these points of view, to understand better the mechanisms of AB cell migration or invasion is necessary for the better clinical therapy and improvements of the patients' quality of life. Microtubules in eukaryotic cells reveal the shape of hollow cylinders made up of polymerized alpha (α)- and beta (β)-tubulin dimers and form the cytoskeleton together with microfilaments and intermediate filaments. Microtubules play important roles in cell migration by undergoing assembly and disassembly with post-translational modifications. Stability of microtubules caused by their acetylation is involved in cell migration. In this study, we investigated the expression and distribution of acetylated α-tubulin and alpha-tubulin N-acetyltransferase 1 (αTAT1), an enzyme which acetylates Lys-40 in α-tubulin, in AB specimens, and analyzed how tubulin was acetylated by αTAT1 activation in a human AB cell line, AM-1. Finally, we clarified that TGF-β-activated kinase1 (TAK1) was phosphorylated by TGF-β stimulation, then, induced tubulin acetylation via αTAT1 activation, which subsequently activated the migration and invasion of AB cells.
Topics: Acetylation; Acetyltransferases; Adolescent; Adult; Aged; Ameloblastoma; Cell Line, Tumor; Cell Movement; Female; Humans; Immunohistochemistry; Jaw Neoplasms; MAP Kinase Kinase Kinases; Male; Microtubule Proteins; Middle Aged; Neoplasm Invasiveness; RNA Interference; Transforming Growth Factor beta; Tubulin; Young Adult
PubMed: 34508164
DOI: 10.1038/s41374-021-00671-w