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Structure (London, England : 1993) Sep 2023Assembly of tau into beta-sheet-rich amyloids dictates the pathology of a diversity of diseases. Lysine acetylation has been proposed to drive tau amyloid assembly, but...
Assembly of tau into beta-sheet-rich amyloids dictates the pathology of a diversity of diseases. Lysine acetylation has been proposed to drive tau amyloid assembly, but no direct mechanism has emerged. Using tau fragments, we identify patterns of acetylation that flank amyloidogenic motifs on the tau fragments that promote rapid fibril assembly. We determined a 3.9 Å cryo-EM amyloid fibril structure assembled from an acetylated tau fragment uncovering how lysine acetylation can mediate gain-of-function interactions. Comparison of the structure to an ex vivo tauopathy fibril reveals regions of structural similarity. Finally, we show that fibrils encoding disease-associated patterns of acetylation are active in cell-based tau aggregation assays. Our data uncover the dual role of lysine residues in limiting tau aggregation while their acetylation leads to stabilizing pro-aggregation interactions. Design of tau sequence with specific acetylation patterns may lead to controllable tau aggregation to direct folding of tau into distinct amyloid folds.
Topics: Acetylation; Amyloid; Lysine; Protein Processing, Post-Translational; tau Proteins; Humans; Animals; Mice; Tauopathies
PubMed: 37348495
DOI: 10.1016/j.str.2023.05.020 -
Methods in Molecular Biology (Clifton,... 2023Acetylation is one of the most abundant post-translational protein modifications that regulates all cellular compartments ranging from chromatin to cytoskeleton and...
Acetylation is one of the most abundant post-translational protein modifications that regulates all cellular compartments ranging from chromatin to cytoskeleton and Golgi. The dynamic acetylation of the Golgi stacking protein GRASP55 was shown to regulate Golgi reassembly after mitosis. Here we provide a detailed protocol for the analysis of Golgi acetylation including in vitro assays to detect protein acetylation and mass spectrometry analysis to identify specific acetylation sites and their relative abundance.
Topics: Golgi Matrix Proteins; Acetylation; Golgi Apparatus; Protein Processing, Post-Translational; Mass Spectrometry
PubMed: 36512247
DOI: 10.1007/978-1-0716-2639-9_43 -
Nature Communications Jun 2023Acetyl-CoA utilized by histone acetyltransferases (HAT) for chromatin modification is mainly generated by ATP-citrate lyase (ACL) from glucose sources. How ACL locally...
Acetyl-CoA utilized by histone acetyltransferases (HAT) for chromatin modification is mainly generated by ATP-citrate lyase (ACL) from glucose sources. How ACL locally establishes acetyl-CoA production for histone acetylation remains unclear. Here we show that ACL subunit A2 (ACLA2) is present in nuclear condensates, is required for nuclear acetyl-CoA accumulation and acetylation of specific histone lysine residues, and interacts with Histone AcetylTransferase1 (HAT1) in rice. The rice HAT1 acetylates histone H4K5 and H4K16 and its activity on H4K5 requires ACLA2. Mutations of rice ACLA2 and HAT1 (HAG704) genes impair cell division in developing endosperm, result in decreases of H4K5 acetylation at largely the same genomic regions, affect the expression of similar sets of genes, and lead to cell cycle S phase stagnation in the endosperm dividing nuclei. These results indicate that the HAT1-ACLA2 module selectively promotes histone lysine acetylation in specific genomic regions and unravel a mechanism of local acetyl-CoA production which couples energy metabolism with cell division.
Topics: Histones; ATP Citrate (pro-S)-Lyase; Acetyl Coenzyme A; Lysine; Histone Acetyltransferases; Cell Proliferation; Acetylation
PubMed: 37277331
DOI: 10.1038/s41467-023-39101-4 -
Nature Communications Nov 2018Emerging evidence highlights protein acetylation, a prevalent lysine posttranslational modification, as a regulatory mechanism and promising therapeutic target in human...
Emerging evidence highlights protein acetylation, a prevalent lysine posttranslational modification, as a regulatory mechanism and promising therapeutic target in human viral infections. However, how infections dynamically alter global cellular acetylation or whether viral proteins are acetylated remains virtually unexplored. Here, we establish acetylation as a highly-regulated molecular toggle of protein function integral to the herpesvirus human cytomegalovirus (HCMV) replication. We offer temporal resolution of cellular and viral acetylations. By interrogating dynamic protein acetylation with both protein abundance and subcellular localization, we discover finely tuned spatial acetylations across infection time. We determine that lamin acetylation at the nuclear periphery protects against virus production by inhibiting capsid nuclear egress. Further studies within infectious viral particles identify numerous acetylations, including on the viral transcriptional activator pUL26, which we show represses virus production. Altogether, this study provides specific insights into functions of cellular and viral protein acetylations and a valuable resource of dynamic acetylation events.
Topics: Acetylation; Cell Nucleus; Cytomegalovirus; Cytomegalovirus Infections; Host-Pathogen Interactions; Humans; Lamins; Proteins; Viral Proteins; Virus Replication
PubMed: 30470744
DOI: 10.1038/s41467-018-07179-w -
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 -
The FEBS Journal Jan 2024Lysine acetylation is a common reversible post-translational modification of proteins that plays a key role in regulating gene expression. Nuclear receptors (NRs)... (Review)
Review
Lysine acetylation is a common reversible post-translational modification of proteins that plays a key role in regulating gene expression. Nuclear receptors (NRs) include ligand-inducible transcription factors and orphan receptors for which the ligand is undetermined, which together regulate the expression of genes involved in development, metabolism, homeostasis, reproduction and human diseases including cancer. Since the original finding that the ERα, AR and HNF4 are acetylated, we now understand that the vast majority of NRs are acetylated and that this modification has profound effects on NR function. Acetylation sites are often conserved and involve both ordered and disordered regions of NRs. The acetylated residues function as part of an intramolecular signalling platform intersecting phosphorylation, methylation and other modifications. Acetylation of NR has been shown to impact recruitment into chromatin, co-repressor and coactivator complex formation, sensitivity and specificity of regulation by ligand and ligand antagonists, DNA binding, subcellular distribution and transcriptional activity. A growing body of evidence in mice indicates a vital role for NR acetylation in metabolism. Additionally, mutations of the NR acetylation site occur in human disease. This review focuses on the role of NR acetylation in coordinating signalling in normal physiology and disease.
Topics: Humans; Animals; Mice; Acetylation; Ligands; Receptors, Cytoplasmic and Nuclear; Chromatin; Protein Processing, Post-Translational
PubMed: 36471658
DOI: 10.1111/febs.16695 -
PloS One 2017Neisseria gonorrhoeae, the causative agent of gonorrhea, has a number of factors known to contribute to pathogenesis; however, a full understanding of these processes...
Neisseria gonorrhoeae, the causative agent of gonorrhea, has a number of factors known to contribute to pathogenesis; however, a full understanding of these processes and their regulation has proven to be elusive. Post-translational modifications (PTMs) of bacterial proteins are now recognized as one mechanism of protein regulation. In the present study, Western blot analyses, with an anti-acetyl-lysine antibody, indicated that a large number of gonococcal proteins are post-translationally modified. Previous work has shown that Nε-lysine acetylation can occur non-enzymatically with acetyl-phosphate (AcP) as the acetyl donor. In the current study, an acetate kinase mutant (1291ackA), which accumulates AcP, was generated in N. gonorrhoeae. Broth cultures of N. gonorrhoeae 1291wt and 1291ackA were grown, proteins extracted and digested, and peptides containing acetylated-lysines (K-acetyl) were affinity-enriched from both strains. Mass spectrometric analyses of these samples identified a total of 2686 unique acetylation sites. Label-free relative quantitation of the K-acetyl peptides derived from the ackA and wild-type (wt) strains demonstrated that 109 acetylation sites had an ackA/wt ratio>2 and p-values <0.05 in at least 2/3 of the biological replicates and were designated as "AckA-dependent". Regulated K-acetyl sites were found in ribosomal proteins, central metabolism proteins, iron acquisition and regulation proteins, pilus assembly and regulation proteins, and a two-component response regulator. Since AckA is part of a metabolic pathway, comparative growth studies of the ackA mutant and wt strains were performed. The mutant showed a growth defect under aerobic conditions, an inability to grow anaerobically, and a defect in biofilm maturation. In conclusion, the current study identified AckA-dependent acetylation sites in N. gonorrhoeae and determined that these sites are found in a diverse group of proteins. This work lays the foundation for future studies focusing on specific acetylation sites that may have relevance in gonococcal pathogenesis and metabolism.
Topics: Acetate Kinase; Acetylation; Bacterial Proteins; Gene Expression Regulation, Bacterial; Mass Spectrometry; Metabolic Networks and Pathways; Neisseria gonorrhoeae; Phosphorylation; Protein Processing, Post-Translational
PubMed: 28654654
DOI: 10.1371/journal.pone.0179621 -
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 -
Journal of Proteome Research Sep 2021Linker histone H1 plays a key role in chromatin organization and maintenance, yet our knowledge of the regulation of H1 functions by post-translational modifications is...
Linker histone H1 plays a key role in chromatin organization and maintenance, yet our knowledge of the regulation of H1 functions by post-translational modifications is rather limited. In this study, we report on the generation of site-specifically mono- and di-acetylated linker histone H1.2 by genetic code expansion. We used these modified histones to identify and characterize the acetylation-dependent cellular interactome of H1.2 by affinity purification mass spectrometry and show that site-specific acetylation results in overlapping but distinct groups of interacting partners. Among these, we find multiple translational initiation factors and transcriptional regulators such as the NAD-dependent deacetylase SIRT1, which we demonstrate to act on acetylated H1.2. Taken together, our data suggest that site-specific acetylation of H1.2 plays a role in modulating protein-protein interactions.
Topics: Acetylation; Chromatin; Histones; Mass Spectrometry; Protein Processing, Post-Translational
PubMed: 34351766
DOI: 10.1021/acs.jproteome.1c00396 -
Nature Structural & Molecular Biology Sep 2023Histone acetylation regulates most DNA transactions and is dynamically controlled by highly conserved enzymes. The only essential histone acetyltransferase (HAT) in...
Histone acetylation regulates most DNA transactions and is dynamically controlled by highly conserved enzymes. The only essential histone acetyltransferase (HAT) in yeast, Esa1, is part of the 1-MDa NuA4 complex, which plays pivotal roles in both transcription and DNA-damage repair. NuA4 has the unique capacity to acetylate histone targets located several nucleosomes away from its recruitment site. Neither the molecular mechanism of this activity nor its physiological importance are known. Here we report the structure of the Pichia pastoris NuA4 complex, with its core resolved at 3.4-Å resolution. Three subunits, Epl1, Eaf1 and Swc4, intertwine to form a stable platform that coordinates all other modules. The HAT module is firmly anchored into the core while retaining the ability to stretch out over a long distance. We provide structural, biochemical and genetic evidence that an unfolded linker region of the Epl1 subunit is critical for this long-range activity. Specifically, shortening the Epl1 linker causes severe growth defects and reduced H4 acetylation levels over broad chromatin regions in fission yeast. Our work lays the foundations for a mechanistic understanding of NuA4's regulatory role and elucidates how its essential long-range activity is attained.
Topics: Histones; Saccharomyces cerevisiae Proteins; Chromatin; Nucleosomes; Saccharomyces cerevisiae; Histone Acetyltransferases; DNA; Acetylation
PubMed: 37550452
DOI: 10.1038/s41594-023-01056-x