Introduction to Protein Acylation
Protein acylation constitutes a critical class of post-translational modifications (PTMs) involving the covalent attachment of acyl groups to specific amino acid residues, most commonly lysine and cysteine. These modifications regulate a wide array of cellular processes, including gene expression, protein stability, subcellular localization, and signal transduction. Unlike phosphorylation or ubiquitination, which primarily act through charge alterations or proteasomal targeting, acylation confers hydrophobic properties that influence both structural and functional dynamics of proteins.
In recent years, advancements in mass spectrometry-based proteomics and chemical biology have uncovered a diverse spectrum of acylation types. These discoveries underscore the necessity of a systematic understanding of protein acylation, not only as a fundamental biological phenomenon but also as a driver of disease pathogenesis and a targetable axis for therapeutic intervention.
Types of Protein Acylation
Acetylation
Acetylation involves the transfer of an acetyl moiety to lysine ε-amino groups. It neutralizes positive charges. This change modulates protein-DNA interactions. Histone acetylation loosens chromatin structure. It promotes transcriptional activation. Non-histone acetylation influences protein stability, localization, and interactions.
Lactylation
Lactylation refers to the addition of a lactyl group. It derives from lactate, a glycolytic metabolite. Histone lactylation emerges under high glycolytic flux. This mark promotes the expression of genes involved in wound healing and immune responses. Non-histone lactylation remains under investigation but shows potential regulatory roles.
Crotonylation
Crotonylation installs a crotonyl moiety on lysine residues. The group features a conjugated double bond. Histone crotonylation associates with active promoters and enhancers. It confers unique steric and electrostatic properties. Non-histone crotonylation modulates metabolic enzymes and signal mediators.
Succinylation
Succinylation transfers a succinyl group from succinyl-CoA. The modification adds two negative charges. This profound change alters protein conformation. Mitochondrial proteins exhibit abundant succinylation. It influences energy metabolism, redox state, and metabolic flux.
Propionylation
Propionylation attaches a propionyl moiety. This acyl group is one carbon longer than acetyl. Histone propionylation correlates with gene activation. Non-histone propionylation modulates enzymes of fatty-acid oxidation and amino-acid metabolism.
Butyrylation
Butyrylation adds a butyryl group to lysine. It introduces a hydrophobic character. Histone butyrylation links to gene transcription under ketogenic conditions. This mark reflects fatty-acid metabolism. Emerging studies reveal roles in stress responses.
Figure 1. Timeline of the historical milestone for the discovery of protein acylation, and the chemical structures of acyl groups (Shang S, et al., 2022).
Enzymology of Protein Acylation
Acetyltransferases (KATs and NATs)
Lysine acetyltransferases (KATs), often called histone acetyltransferases (HATs), add acetyl groups to lysine residues using acetyl-CoA. Key members of this enzyme family include p300/CBP, GCN5, and PCAF. By modifying both histone and non-histone proteins, KATs help control how tightly DNA is packed, making it easier or harder for genes to be turned on. This process shapes transcription, alters protein behavior, and influences how proteins interact inside cells.
N-terminal acetyltransferases (NATs) modify the α-amino group of protein N-termini, often co-translationally. NATs influence protein stability, subcellular targeting, and degradation. They display substrate specificity based on the N-terminal amino acid sequence.
Deacetylases (HDACs and Sirtuins)
Histone deacetylases (HDACs) remove acetyl groups from lysine residues, restoring positive charge and promoting chromatin condensation.
Sirtuins exhibit broad deacylase activity beyond deacetylation. They can remove succinyl, malonyl, crotonyl, and long-chain fatty acyl groups, linking cellular energy status to post-translational regulation. SIRT3 and SIRT5 are notable for regulating mitochondrial acylation states and oxidative metabolism.
Palmitoyltransferases (DHHC Family) and Thioesterases
Protein S-palmitoylation adds a 16-carbon palmitoyl chain to cysteine side chains through a thioester link. This lipid tag helps proteins stick to membranes and find the right spot inside the cell. A family of over 20 DHHC enzymes—named for their Asp-His-His-Cys active motif—carries out this reaction. Each DHHC isoform has its own set of targets and is found in different tissues. Well-studied examples include DHHC2, DHHC5, and DHHC17.
Acyl-protein thioesterases (APTs) and palmitoyl-protein thioesterases (PPTs) remove palmitate groups from proteins by breaking thioester bonds. This action controls how certain signaling proteins—like Ras and G-protein subunits—move between the cell membrane and the cytoplasm, helping fine-tune their activity.
Cellular Pathways Regulated by Acylation
Protein acylation serves as a key regulatory mechanism across multiple cellular pathways. By altering charge, hydrophobicity, and structural conformation, acyl modifications fine-tune protein interactions, localization, and activity. Below are the principal pathways modulated by acylation.
Epigenetic Control
Histone acylation plays a key role in regulating how tightly DNA is packed in the nucleus. When lysine residues on histones H3 and H4 are acetylated, their positive charges are neutralized. This loosens the interaction between histones and DNA, making the chromatin more open and allowing genes to be actively transcribed. Other types of acylation—such as crotonylation, butyrylation, and propionylation—also affect gene activity by creating unique chromatin states. These marks are often found near gene promoters and enhancers, where they help turn genes on.
Histone acylation recruits chromatin readers, such as bromodomains (acetyl-lysine) and YEATS domains (crotonyl-lysine), which facilitate the assembly of transcriptional coactivator complexes. This regulation is critical during cell differentiation, stress response, and metabolic adaptation.
Signal Transduction
Dynamic lipid acylation, such as palmitoylation and myristoylation, directs proteins to specific membrane compartments. These lipid anchors localize signaling proteins—like Ras, Src-family kinases, and G-protein subunits—to lipid rafts and plasma membranes, where signal transduction is initiated.
Palmitoylation enhances protein–membrane affinity and promotes complex formation at signalosomes. Reversible depalmitoylation allows proteins to recycle between the membrane and cytosol, supporting spatial and temporal control of signaling events.
Metabolic Enzyme Modulation
Acylation of metabolic enzymes regulates their catalytic function, stability, and subcellular localization. For example:
- Succinylation of mitochondrial enzymes, such as succinate dehydrogenase and isocitrate dehydrogenase, modulates flux through the tricarboxylic acid (TCA) cycle.
- Crotonylation and lactylation of glycolytic enzymes, including enolase and pyruvate kinase, alter glucose metabolism in response to nutrient availability.
- Acetylation of enzymes like acetyl-CoA synthetase controls substrate utilization by switching enzymes between active and inactive states.
Membrane Dynamics
Lipidation, particularly palmitoylation, is essential for protein sorting within membrane microdomains. Acylated proteins partition into lipid rafts, which serve as platforms for receptor clustering and intracellular signaling.
Acylation also regulates vesicular transport in both endocytic and exocytic pathways. Proteins such as SNAREs and Rab GTPases require palmitoylation for proper localization and trafficking. Moreover, reversible acylation ensures precise delivery and turnover of membrane proteins, supporting cellular polarity, immune signaling, and neurotransmission.
Analytical Techniques for Protein Acylation Detection & Quantification
Accurate detection and quantification of protein acylation are critical for understanding its biological functions. A range of complementary techniques has been developed, each tailored to detect specific acyl modifications, assess stoichiometry, and define cellular context.
Mass Spectrometry-Based Profiling
Mass spectrometry (MS) is the gold standard for global acylation analysis. It offers site-specific identification and quantitative profiling of acylated peptides.
- Enrichment: Due to the low abundance of acylated peptides, enrichment strategies are required. These include acyl-specific antibody immunoprecipitation, resin-based affinity capture, or chemical derivatization (e.g., alkynyl-CoA labeling for palmitoylation).
- Data Acquisition and Analysis: High-resolution instruments (e.g., Orbitrap, Q-TOF) detect mass shifts associated with specific acyl groups. Software tools (e.g., MaxQuant, Proteome Discoverer) support modification site assignment and quantification.
Chemical Probes & Bioorthogonal Labeling
Metabolic labeling with bioorthogonal acyl-CoA analogs allows selective incorporation of acyl groups into proteins in living cells. Alkyne- or azide-tagged acyl donors (e.g., alkynyl-palmitate or alkynyl-acetate) are incorporated into proteins. Subsequent click chemistry reactions conjugate biotin or fluorescent reporters for detection.
Antibody-Based Methods
Immunochemical techniques remain essential for validating acylation at specific sites or within specific proteins.
- Western Blotting: Pan-acylation or site-specific antibodies detect modified proteins. Signal intensity provides semi-quantitative information.
- Immunoprecipitation (IP): Acyl-specific antibodies enrich modified proteins before MS or blotting, enhancing detection sensitivity.
- Immunofluorescence: Visualizes spatial distribution of acylated proteins in fixed cells, supporting co-localization and functional studies.
Quantitative Proteomics: Label-Free vs. Isotope Labeling
MS-based quantification strategies compare acylation levels across different biological states.
- Label-Free Quantification (LFQ): Measures peptide ion intensities or spectral counts. Simpler and broadly applicable but less precise.
- Isotope Labeling: Includes SILAC (Stable Isotope Labeling by Amino acids in Cell culture), TMT (Tandem Mass Tags), and iTRAQ. These methods enable multiplexed, high-accuracy comparisons of acylation levels across conditions or timepoints.
Emerging Platforms: Single-Cell and Live-Cell Imaging
Recent advances allow acylation to be studied at high spatial and temporal resolution.
- Single-Cell Proteomics: Uses miniaturized MS workflows and nanoLC-MS to quantify acylation in individual cells, revealing heterogeneity in acylation landscapes.
- Live-Cell Imaging: Genetically encoded biosensors and fluorophore-conjugated chemical probes enable real-time tracking of dynamic acylation events. Techniques such as FRET and BiFC have been adapted to monitor acylation-dependent protein interactions.
Select Service
Related Article
Dysregulated Acylation in Pathology
Cancer
Acetylation and Myc regulation: The cancer-related protein c-Myc is modified by the acetyltransferase enzyme p300 (also known as CBP). This acetylation speeds up the breakdown of c-Myc while also boosting its ability to activate genes linked to cell growth, including hTERT, which supports continuous cell division ( Faiola F, et al., 2005).
KRAS‑mutant lung adenocarcinoma: Blocking EGFR palmitoylation by DHHC20 knockdown or mutation of palmitoylated cysteines suppresses PI3K signaling and reduces Myc levels. Consequently, tumor proliferation declines and sensitivity to PI3K inhibitors increases in KRAS‑mutant lung tumor models (Bollu L R, et al., 2015).
Neurological Disorders
In an Alzheimer's disease (AD) mouse model, total S-palmitoylation in the hippocampus increased by approximately 138%. Palmitoylation levels of key synaptic proteins—such as BACE1, SNAP-25, CAMKIIα, GRIK2, and GluA2—also rose significantly. These shifts are associated with abnormal amyloid processing and impaired synaptic function. Together, they suggest that disrupted palmitoylation contributes to early disease development. Notably, blocking zDHHC7, an enzyme that adds palmitate to proteins, prevented memory loss in this model, linking faulty palmitoylation to cognitive decline. (Natale F, et al., 2024).
Metabolic Syndromes
Mitochondrial succinylation and ischemia/reperfusion injury: In models of liver ischemia–reperfusion (I/R), SIRT5 deficiency led to accumulation of succinylated mitochondrial proteins, including PRDX3 at lysine 84. SIRT5 overexpression or desuccinylation of PRDX3 alleviated oxidative stress, apoptosis, and inflammation, highlighting SIRT5's protective role via lysine desuccinylation (Hu Y, et al., 2024).
Figure 2. Protein succinylation and malonylation on metabolic enzymes or kinases in tumor, inflammatory, cardiovascular and metabolic diseases (Shang S, et al., 2022).
People Also Ask
What distinguishes enzymatic from non‑enzymatic protein acylation?
Enzymatic acylation involves writer enzymes such as lysine acetyltransferases (KATs) or DHHC palmitoyltransferases. These enzymes transfer acyl groups from acyl‑CoA donors to specific amino‑acid residues with substrate sequence specificity. In contrast, non‑enzymatic acylation occurs spontaneously, primarily in the mitochondrial matrix, where high concentrations of acyl‑CoA and alkaline pH enable direct lysine modification without an enzyme.
Why are some lysine residues susceptible to multiple acyl modifications?
Certain lysines are chemically predisposed to modification due to local protein structure and reactivity. Proteomic analyses show significant overlap between acetylation and succinylation sites. This suggests that lysines with appropriate accessibility and charge environment are modified enzymatically or spontaneously by multiple acyl‑CoAs.
How does cellular metabolism influence the levels and types of protein acylation?
Cellular metabolism controls the intracellular availability of acyl-CoA donors, directly impacting protein acylation. For example, increased glycolysis elevates acetyl-CoA and lactyl-CoA, promoting acetylation and lactylation. Fatty acid oxidation generates butyryl-CoA, influencing butyrylation. Metabolic shifts in cancer or hypoxia alter the acylation landscape, linking nutrient status to epigenetic and signaling regulation.
References
- Shang S, Liu J, Hua F. Protein acylation: mechanisms, biological functions and therapeutic targets. Signal transduction and targeted therapy, 2022, 7(1): 396.
- Xu J Y, et al. Protein acylation is a general regulatory mechanism in biosynthetic pathway of acyl-CoA-derived natural products. Cell Chemical Biology, 2018, 25(8): 984-995. e6.
- Lanyon-Hogg T, et al. Dynamic protein acylation: new substrates, mechanisms, and drug targets. Trends in biochemical sciences, 2017, 42(7): 566-581.
Our products and services are for research use only.
