What is Acetylation?
Protein acetylation is an important post-translational modification (PTM). It refers to the transfer of an acetyl group to the amino acid residues of a protein, thereby altering the protein's structure and function. This modification can be classified into two categories: N-terminal acetylation and lysine acetylation.
N-terminal acetylation: N-terminal acetylation usually occurs in the early stage of protein translation. The enzyme N-terminal acetyltransferase is responsible for adding an acetyl group to the N-terminus of the protein. This modification is common in eukaryotes. Its main function is to stabilize the protein structure, prevent its degradation, and promote protein-protein interactions.
Lysine acetylation: Lysine acetylation is usually catalyzed by acetyltransferases (often referred to as HATs), which transfer an acetyl group to lysine residues. Conversely, deacetylases (often referred to as HDACs) are responsible for removing the acetyl group. Lysine acetylation mainly affects histones (DNA-packaging proteins) and non-histone proteins. Histone acetylation can regulate gene expression: high levels of histone acetylation are usually associated with active gene expression. Non-histone acetylation can affect various cellular processes, including DNA repair, cell division, and metabolic regulation.
Molecular Mechanisms and Classification of Acetylation
N-Terminal Acetylation
N-terminal acetylation occurs co-translationally on a vast majority of eukaryotic proteins, permanently modifying their α-amino groups. This process is catalyzed by N-terminal acetyltransferases (NATs), which transfer an acetyl group from acetyl-CoA to the exposed N-terminus of newly synthesized proteins.
Catalytic Enzymes and Substrate Specificity:
NATs, such as NatA, NatB, and NatC, exhibit distinct substrate preferences based on the first few amino acids of the nascent protein. For example, NatA primarily acetylates small N-terminal residues like Ser, Ala, and Thr.
Impact on Protein Stability and Interactions:
N-terminal acetylation can enhance or reduce protein stability by influencing degradation pathways. It also acts as a molecular tag for protein interactions, mediating key cellular processes such as signal transduction and complex formation. This modification is crucial in the assembly of multiprotein complexes such as ribosomes and actin filaments.
Lysine Acetylation
Unlike N-terminal acetylation, lysine acetylation is reversible, dynamically regulated by two opposing enzyme classes:
Histone Acetyltransferases (HATs):
Enzymes like p300/CBP, GCN5, and PCAF catalyze the transfer of an acetyl group to lysine residues, neutralizing their positive charge. This leads to changes in protein-protein interactions, chromatin accessibility, and enzymatic activity.
Histone Deacetylases (HDACs):
HDACs, including classical HDACs and sirtuins (SIRT1-7), remove acetyl groups, restoring lysine's positive charge and reversing acetylation-dependent effects. Sirtuins, which are NAD⁺-dependent, link acetylation to cellular metabolism.
Functional and Pathological Relevance
Gene Expression Regulation
Histone acetylation (e.g., H3K27ac) marks active enhancers and promoters, facilitating chromatin relaxation and transcriptional activation. Dysregulation of these marks is linked to oncogenic gene expression in cancers like leukemia.
Metabolism and Disease
Cancer: Hyperacetylation of non-histone proteins, such as tubulin, promotes metastasis by altering cell motility. Conversely, hypoacetylation of tumor suppressors like p53 impairs their anti-cancer functions.
Neurodegeneration: Reduced acetylation of mitochondrial proteins (e.g., SOD2) exacerbates oxidative stress in Alzheimer's disease. HDAC inhibitors (e.g., sodium butyrate) show promise in restoring acetylation and cognitive function.
How to Study Protein Acetylation?
Antibody-Based Methods
- Western Blotting: Uses acetylation-specific antibodies to detect acetylated proteins in a sample.
- Immunofluorescence (IF): Visualizes acetylation within cells by staining with specific antibodies, revealing subcellular localization patterns.
Chromatin Immunoprecipitation (ChIP-seq)
A powerful technique for mapping histone acetylation across the genome. Helps identify enhancer regions and transcriptionally active chromatin states (e.g., H3K27ac as a marker of active enhancers).
Mass Spectrometry-Based Acetyl-proteomics Analysis
Mass spectrometry (MS) has revolutionized acetylation research by enabling high-throughput and site-specific identification of acetylated proteins.
Experimental Workflow
- Sample Preparation: Enrichment of acetylated peptides using anti-acetyllysine antibodies or chemical affinity methods (e.g., TiO₂ purification).
- Liquid chromatography-tandem mass spectrometry (LC-MS/MS) Analysis: LC-MS/MS separates and identifies acetylated peptides with high precision.
- Data Processing and Bioinformatics: Software tools like MaxQuant and Proteome Discoverer facilitate site-specific acetylation mapping and quantitative analysis.
Quantitative Strategies
- Stable Isotope Labeling (SILAC, TMT): Uses isotopic labeling for comparative acetylation profiling between experimental conditions (e.g., cancer vs. normal tissue).
- Label-Free Quantification: Enables large-scale profiling of acetylation dynamics under different stimuli (e.g., response to HDAC inhibitors).
Applications of Mass Spectrometry in Acetylation Research
Discovery of novel acetylation sites on metabolic enzymes, transcription factors, and signaling proteins.
System-wide correlation of acetylation changes with cellular pathways (e.g., mitochondrial metabolism, DNA damage response).
Challenges
- Low Stoichiometry: Acetylation occurs at low abundance, requiring high-sensitivity techniques for detection.
- Discrimination from Other PTMs: Acetylation must be distinguished from modifications like ubiquitination and methylation that also target lysine residues.
Chemical Proteomics Approaches
Pan-Antibody Enrichment: Anti-acetyllysine antibodies coupled with MS enable proteome-wide profiling. For example, a 2022 study in Cell Reports identified over 3,000 acetylation sites in human liver tissue, linking acetylation to lipid metabolism.
Metabolic Labeling: Heavy isotope-labeled acetyl-CoA analogs (e.g., ¹³C-acetyl-CoA) track acetylation turnover rates in vivo, revealing dynamics in mitochondrial proteins.
Structural Mass Spectrometry
Hydrogen-Deuterium Exchange (HDX-MS): Detects acetylation-induced conformational changes. For instance, acetylation of histone H4 at K16 reduces DNA-histone binding, as shown by altered deuterium uptake patterns.
Crosslinking-MS: Identifies acetylation-dependent protein interactions. A 2023 Nature Structural & Molecular Biology study mapped acetylation-mediated interactions between p300 and chromatin remodelers.
Dynamic and Functional Analysis
Pulse-Chase Experiments: Combining SILAC with timed HDAC inhibition quantifies acetylation half-lives (e.g., p53 acetylation turnover in response to DNA damage).
Functional Validation: siRNA knockdown of specific acetyltransferases (e.g., p300) followed by phenotypic assays (e.g., RNA-seq) links acetylation to transcriptional outcomes.
How to Interpret Acetylation Assay Results?
Case 1 MSL1 Promotes Liver Regeneration by Driving Phase Separation of STAT3 and Histone H4 and Enhancing Their Acetylation
This image presents the results of a neuroscience experiment involving brain tissue slices from the CA1 region of the hippocampus in mice. The study compares the acetylation status of a specific protein between wild-type (WT) and familial Alzheimer's disease (FAD) model mice.
The different components of the image include:
- ac-H4K12: This refers to the acetylation of histone H4 at lysine 12. In the image, this acetylation marker is shown in green fluorescence.
- NeuN: A neuronal-specific nuclear protein labeled with red fluorescence, primarily marking neurons.
- DAPI: A fluorescent dye used to stain cell nuclei, appearing blue in the image.
- Merge: A composite image combining the three stains, showing blue-stained nuclei, red-labeled neurons, and the green fluorescence of the histone acetylation marker overlapping with them.
On the right side of the image, a bar graph illustrates the relative intensity of the ac-H4K12 signal in CA1 neurons of both WT and FAD model mice. Statistical analysis indicates a significant reduction in ac-H4K12 levels in FAD model mice compared to WT, as denoted by the double asterisks (**), indicating statistical significance.
Case 2 MSL1 Promotes Liver Regeneration by Driving Phase Separation of STAT3 and Histone H4 and Enhancing Their Acetylation
This image consists of two parts: a gel electrophoresis image (c) and a bar graph (d), presenting experimental results on the acetylation of histone H4 at lysine 16 (H4K16ac). The experiments were conducted under different conditions, including wild-type (WT) and MSL1 knockout (LKO) mice, as well as different treatments (Sham control vs. PH 36 hours post-treatment). Below is a detailed breakdown of each part:
Gel Electrophoresis Image (c)
- Marker: A DNA marker used to determine fragment sizes.
- Input: Represents the initial sample used in the experiment.
- IgG: A negative control, where immunoprecipitation was performed using immunoglobulin G (IgG) instead of a specific antibody.
- H4K16ac: Immunoprecipitation using a specific antibody targeting the acetylation state of H4K16.
- Cyclin A2, B1, D1: These bands represent PCR amplification results for promoter regions of specific cyclin genes under different conditions, used to assess H4K16 acetylation levels.
Bar Graph (d)
- Illustrates the enrichment of H4K16 acetylation under different conditions, expressed as the fold increase relative to IgG.
- Cyclin A2, B1, D1: Each bar represents the H4K16 acetylation level at the promoter region of the corresponding cyclin gene.
Analysis
- Changes in Acetylation Levels: A noticeable reduction in H4K16 acetylation at cyclin gene promoter regions is observed in MSL1 knockout (LKO) mice compared to wild-type.
- This suggests that the loss of MSL1 may impact the regulation of these genes, potentially affecting their transcriptional activity.
References
- Lin, Yingbin, et al. "ACSS2-dependent histone acetylation improves cognition in mouse model of Alzheimer's disease." Molecular neurodegeneration 18.1 (2023): 47. https://doi.org/10.1186/s13024-023-00625-4
- He, Yucheng, et al. "MSL1 promotes liver regeneration by driving phase separation of STAT3 and histone h4 and enhancing their acetylation." Advanced Science 10.23 (2023): 2301094. https://doi.org/10.1002/advs.202301094
