-
The Journal of Biological Chemistry Jul 1987The pyruvate dehydrogenase kinase consists of a catalytic subunit (Kc) and a basic subunit (Kb) which appear to be anchored to the dihydrolipoyl transacetylase core...
The pyruvate dehydrogenase kinase consists of a catalytic subunit (Kc) and a basic subunit (Kb) which appear to be anchored to the dihydrolipoyl transacetylase core component (E2) by another subunit, referred to as protein X (Rahmatullah, M., Jilka, J. M., Radke, G. A., and Roche, T. E. (1986) J. Biol. Chem. 261, 6515-6523). We determined the catalytic requirements for reduction and acetylation of the lipoyl moiety in protein X and linked those changes in protein X to regulatory effects on kinase activity. Using fractions prepared by resolution and proteolytic treatments, we evaluated which subunits are required for regulatory effects on kinase activity. With X-KcKb fraction (treated to remove the mercurial agent used in its preparation), we found that the resolved pyruvate dehydrogenase component, the isolated inner domain of E2 (lacking the lipoyl-bearing region of E2), and the dihydrolipoyl dehydrogenase component directly utilize protein X as a substrate. The resulting reduction and acetylation of protein X occurs in association with enhancement of kinase activity. Following tryptic cleavage of E2 and protein X into subdomains, full acetylation of the lipoyl-bearing subdomains of these proteins is retained along with the capacity of acetylating substrates to stimulate kinase activity. All kinase-containing fractions, including those in which the Kb subunit was digested, were inhibited by pyruvate or ADP, alone, and synergistically by the combination suggesting that pyruvate and ADP bind to Kc. Our results suggest that the Kb subunit of the kinase does not contribute to the observed regulatory effects. A dynamic role of protein X in attenuating kinase activity based on changes in the mitochondrial redox and acetylating potentials is considered.
Topics: Acetylation; Adenosine Diphosphate; Alkylation; Animals; Kidney; Kinetics; Macromolecular Substances; NAD; Oxidation-Reduction; Phenylmercury Compounds; Protein Kinases; Protein Serine-Threonine Kinases; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Pyruvates; Pyruvic Acid
PubMed: 3611060
DOI: No ID Found -
Cellular and Molecular Life Sciences :... Apr 2010Lysine acetylation is a post-translational modification that critically regulates gene transcription by targeting histones as well as a variety of transcription factors... (Review)
Review
Lysine acetylation is a post-translational modification that critically regulates gene transcription by targeting histones as well as a variety of transcription factors in the nucleus. More recent reports have also demonstrated that numerous proteins located outside the nucleus are also acetylated and that this modification has profound consequences on their functions. This review describes the latest findings on the substrates acetylated outside the nucleus and on the acetylases and deacetylates that catalyse these modifications. Protein acetylation is emerging as a major mechanism by which key proteins are regulated in many physiological processes such as migration, metabolism and aging as well as in pathological circumstances such as cancer and neurodegenerative disorders.
Topics: Acetylation; Animals; Humans; Lysine; Protein Processing, Post-Translational; Proteins
PubMed: 20082207
DOI: 10.1007/s00018-009-0252-7 -
Proceedings of the National Academy of... Apr 2013Recent global proteomic and genomic studies have determined that lysine acetylation is a highly abundant posttranslational modification. The next challenge is connecting...
Recent global proteomic and genomic studies have determined that lysine acetylation is a highly abundant posttranslational modification. The next challenge is connecting lysine acetyltransferases (KATs) to their cellular targets. We hypothesize that proteins that physically interact with KATs may not only predict the cellular function of the KATs but may be acetylation targets. We have developed a mass spectrometry-based method that generates a KAT protein interaction network from which we simultaneously identify both in vivo acetylation sites and in vitro acetylation sites. This modified chromatin-immunopurification coupled to an in vitro KAT assay with mass spectrometry (mChIP-KAT-MS) was applied to the Saccharomyces cerevisiae KAT nucleosome acetyltransferase of histone H4 (NuA4). Using mChIP-KAT-MS, we define the NuA4 interactome and in vitro-enriched acetylome, identifying over 70 previously undescribed physical interaction partners for the complex and over 150 acetyl lysine residues, of which 108 are NuA4-specific in vitro sites. Through this method we determine NuA4 acetylation of its own subunit Epl1 is a means of self-regulation and identify a unique link between NuA4 and the spindle pole body. Our work demonstrates that this methodology may serve as a valuable tool in connecting KATs with their cellular targets.
Topics: Acetylation; Chromatin Immunoprecipitation; Histone Acetyltransferases; Lysine; Mass Spectrometry; Protein Interaction Mapping; Protein Processing, Post-Translational; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Substrate Specificity
PubMed: 23572591
DOI: 10.1073/pnas.1218515110 -
ACS Chemical Biology Sep 2015Acetylation is a post-translational modification that occurs on thousands of proteins located in many cellular organelles. This process mediates many protein functions...
Acetylation is a post-translational modification that occurs on thousands of proteins located in many cellular organelles. This process mediates many protein functions and modulates diverse biological processes. In mammalian cells, where acetyl-CoA is the primary acetyl donor, acetylation in the mitochondria is thought to occur by chemical means due to the relatively high concentration of acetyl-CoA located in this organelle. In contrast, acetylation outside of the mitochondria is thought to be mediated predominantly by acetyltransferase enzymes. Here, we address the possibility that nonenzymatic chemical acetylation outside of the mitochondria may be more common than previously appreciated. We employed the Nucleic Acid Programmable Protein Array platform to perform an unbiased screen for human proteins that undergo chemical acetylation, which resulted in the identification of a multitude of proteins with diverse functions and cellular localization. Mass spectrometry analysis revealed that basic residues typically precede the acetylated lysine in the -7 to -3 position, and we show by mutagenesis that these basic residues contribute to chemical acetylation capacity. We propose that these basic residues lower the pKa of the substrate lysine for efficient chemical acetylation. Many of the identified proteins reside outside of the mitochondria and have been previously demonstrated to be acetylated in vivo. As such, our studies demonstrate that chemical acetylation occurs more broadly throughout the eukaryotic cell than previously appreciated and suggests that this post-translational protein modification may have more diverse roles in protein function and pathway regulation.
Topics: Acetylation; Amino Acid Sequence; Humans; Lysine; Mass Spectrometry; Molecular Sequence Data; Protein Array Analysis; Proteins
PubMed: 26083674
DOI: 10.1021/acschembio.5b00342 -
Autophagy Jun 2016The Nϵ-lysine acetylation of cargo proteins in the lumen of the endoplasmic reticulum (ER) requires a membrane transporter (SLC33A1) and 2 acetyltransferases (NAT8B and...
The Nϵ-lysine acetylation of cargo proteins in the lumen of the endoplasmic reticulum (ER) requires a membrane transporter (SLC33A1) and 2 acetyltransferases (NAT8B and NAT8). The ER acetylation machinery regulates the homeostatic balance between quality control/efficiency of the secretory pathway and autophagy-mediated disposal of toxic protein aggregates. We recently reported that the autophagy pathway that acts downstream of the ER acetylation machinery specifically targets protein aggregates that form within the secretory pathway. Genetic and biochemical manipulation of ER acetylation in a mouse model of Alzheimer disease is able to restore normal proteostasis and rescue the disease phenotype. Here we summarize these findings and offer an overview of the ER-acetylation machinery.
Topics: Acetylation; Animals; Autophagy; Endoplasmic Reticulum; Homeostasis; Humans; Models, Biological; Proteins; Secretory Pathway
PubMed: 27124586
DOI: 10.1080/15548627.2016.1164369 -
ELife Oct 2022The NuA4 protein complex acetylates histones H4 and H2A to activate both transcription and DNA repair. We report the 3.1-Å resolution cryo-electron microscopy structure...
The NuA4 protein complex acetylates histones H4 and H2A to activate both transcription and DNA repair. We report the 3.1-Å resolution cryo-electron microscopy structure of the central hub of NuA4, which flexibly tethers the histone acetyltransferase (HAT) and Trimer Independent of NuA4 involved in Transcription Interactions with Nucleosomes (TINTIN) modules. The hub contains the large Tra1 subunit and a core that includes Swc4, Arp4, Act1, Eaf1, and the C-terminal region of Epl1. Eaf1 stands out as the primary scaffolding factor that interacts with the Tra1, Swc4, and Epl1 subunits and contributes the conserved HSA helix to the Arp module. Using nucleosome-binding assays, we find that the HAT module, which is anchored to the core through Epl1, recognizes H3K4me3 nucleosomes with hyperacetylated H3 tails, while the TINTIN module, anchored to the core via Eaf1, recognizes nucleosomes that have hyperacetylated H2A and H4 tails. Together with the known interaction of Tra1 with site-specific transcription factors, our data suggest a model in which Tra1 recruits NuA4 to specific genomic sites then allowing the flexible HAT and TINTIN modules to select nearby nucleosomes for acetylation.
Topics: Saccharomyces cerevisiae; Nucleosomes; Saccharomyces cerevisiae Proteins; Cryoelectron Microscopy; Histone Acetyltransferases; Acetylation
PubMed: 36263929
DOI: 10.7554/eLife.81400 -
Cell Metabolism Feb 2020The toxic effects of alcohol consumption are dependent upon its metabolism in the liver to downstream metabolites: acetaldehyde, acetate, and acetyl-CoA. Recently, in...
The toxic effects of alcohol consumption are dependent upon its metabolism in the liver to downstream metabolites: acetaldehyde, acetate, and acetyl-CoA. Recently, in Nature, Mews et al. (2019) have discovered that acetyl-CoA derived from alcohol plays an important epigenetic role in regulating ethanol's effects on the brain through histone acetylation.
Topics: Acetyl Coenzyme A; Acetylation; Brain; Epigenesis, Genetic; Ethanol; Histones
PubMed: 32023443
DOI: 10.1016/j.cmet.2020.01.008 -
Biochimica Et Biophysica Acta. General... Sep 2019The signal transducer and activator of transcription 3 (STAT3) protein is activated by phosphorylation of a specific tyrosine residue (Tyr705) in response to various...
The signal transducer and activator of transcription 3 (STAT3) protein is activated by phosphorylation of a specific tyrosine residue (Tyr705) in response to various extracellular signals. STAT3 activity was also found to be regulated by acetylation of Lys685. However, the molecular mechanism by which Lys685 acetylation affects the transcriptional activity of STAT3 remains elusive. By genetically encoding the co-translational incorporation of acetyl-lysine into position Lys685 and co-expression of STAT3 with the Elk receptor tyrosine kinase, we were able to characterize site-specifically acetylated, and simultaneously acetylated and phosphorylated STAT3. We measured the effect of acetylation on the crystal structure, and DNA binding affinity and specificity of Tyr705-phosphorylated and non-phosphorylated STAT3. In addition, we monitored the deacetylation of acetylated Lys685 by reconstituting the mammalian enzymatic deacetylation reaction in live bacteria. Surprisingly, we found that acetylation, per se, had no effect on the crystal structure, and DNA binding affinity or specificity of STAT3, implying that the previously observed acetylation-dependent transcriptional activity of STAT3 involves an additional cellular component. In addition, we discovered that Tyr705-phosphorylation protects Lys685 from deacetylation in bacteria, providing a new possible explanation for the observed correlation between STAT3 activity and Lys685 acetylation.
Topics: Acetylation; Betaine; Humans; Phosphorylation; Protein Processing, Post-Translational; STAT3 Transcription Factor; Signal Transduction
PubMed: 31170499
DOI: 10.1016/j.bbagen.2019.05.019 -
Molecular & Cellular Proteomics : MCP Dec 2018N-terminal acetylation (Nt-acetylation) is a highly abundant protein modification in eukaryotes and impacts a wide range of cellular processes, including protein quality...
N-terminal acetylation (Nt-acetylation) is a highly abundant protein modification in eukaryotes and impacts a wide range of cellular processes, including protein quality control and stress tolerance. Despite its prevalence, the mechanisms regulating Nt-acetylation are still nebulous. Here, we present the first global study of Nt-acetylation in yeast cells as they progress to stationary phase in response to nutrient starvation. Surprisingly, we found that yeast cells maintain their global Nt-acetylation levels upon nutrient depletion, despite a marked decrease in acetyl-CoA levels. We further observed two distinct sets of protein N termini that display differential and opposing Nt-acetylation behavior upon nutrient starvation, indicating a dynamic process. The first protein cluster was enriched for annotated N termini showing increased Nt-acetylation in stationary phase compared with exponential growth phase. The second protein cluster was conversely enriched for alternative nonannotated N termini ( N termini indicative of shorter N-terminal proteoforms) and, like histones, showed reduced acetylation levels in stationary phase when acetyl-CoA levels were low. Notably, the degree of Nt-acetylation of Pcl8, a negative regulator of glycogen biosynthesis and two components of the pre-ribosome complex (Rsa3 and Rpl7a) increased during starvation. Moreover, the steady-state levels of these proteins were regulated both by starvation and NatA activity. In summary, this study represents the first comprehensive analysis of metabolic regulation of Nt-acetylation and reveals that specific, rather than global, Nt-acetylation events are subject to metabolic regulation.
Topics: Acetyl Coenzyme A; Acetylation; Acetyltransferases; Analysis of Variance; Cells, Cultured; Chi-Square Distribution; Cyclins; Histones; N-Terminal Acetyltransferases; Proteome; Ribosomal Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Tandem Mass Spectrometry
PubMed: 30150368
DOI: 10.1074/mcp.RA118.000982 -
Methods in Molecular Biology (Clifton,... 2015Posttranslational modifications of NF-κB, including acetylation and methylation, have emerged as an important regulatory mechanism for determining the duration and...
Posttranslational modifications of NF-κB, including acetylation and methylation, have emerged as an important regulatory mechanism for determining the duration and strength of NF-κB nuclear activity as well as its transcriptional output. Within the seven NF-κB family proteins, the RelA subunit of NF-κB is the most studied for its regulation by lysine acetylation and methylation. Acetylation or methylation at different lysine residues modulates distinct functions of NF-κB, including DNA-binding and transcription activity, protein stability, and its interaction with NF-κB modulators. Here, we describe the experimental methods to monitor the in vitro and in vivo acetylated or methylated forms of NF-κB. These methods include radiolabeling the acetyl or methyl groups and immunoblotting with pan- or site-specific acetyl- or methyl-lysine antibodies. Radiolabeling is useful in the initial validation of the modifications. Immunoblotting with antibodies provides a rapid and powerful approach to detect and analyze the functions of these modifications in vitro and in vivo.
Topics: Acetylation; Blotting, Western; Cell Line; Chromatin Immunoprecipitation; Humans; Immunoprecipitation; In Vitro Techniques; Methylation; NF-kappa B; Protein Binding; Transcription Factor RelA
PubMed: 25736763
DOI: 10.1007/978-1-4939-2422-6_24