Protein acylation is a reversible post-translational modifications (PTMs) that controls how proteins work, where they go in the cell, how stable they are, and how they interact with other molecules. It plays a key role in normal cell function and is now known to contribute to diseases like cancer, neurodegeneration, and infections.
What is Protein Acylation
Protein acylation is the process of attaching acyl groups—like acetyl, myristoyl, palmitoyl, or crotonyl—to certain amino acids, usually lysine or cysteine. This chemical change can influence how the protein behaves, such as where it goes in the cell, how it attaches to membranes, and how it interacts with other proteins. Unlike more uniform modifications like phosphorylation, acylation includes a wide range of types—such as N-myristoylation, S-palmitoylation, and lysine crotonylation—each controlled by specific enzymes that add or remove the acyl groups.
Types of Protein Acylation and Their Functions
Protein acylation encompasses a range of modification types with distinct biochemical characteristics and biological outcomes:
Select Service
Related Article
Experimental Techniques for Protein Acylation Analysis
Accurate profiling of protein acylation relies on complementary techniques that enrich, detect, and quantify modified residues with high specificity and sensitivity. The most widely adopted approaches are:
Affinity Enrichment
Antibody-Based Capture: Pan- or site-specific anti-acyl-lysine antibodies are used to capture modified proteins or peptides from complex samples. To ensure accurate results, it is crucial to validate antibody specificity and check for consistency between batches, reducing unwanted binding and background noise.
Chemical Probes: Cells can incorporate bioorthogonal acyl-donor analogs, like alkynyl- or azido-acyl CoA, into proteins during metabolism. These chemical tags allow selective labeling and enrichment of modified proteins using click chemistry, making it possible to detect even low-abundance acylation events.
Mass Spectrometry (MS)-Based Detection
LC-MS/MS Analysis
The enriched peptides are analyzed using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). High-resolution instruments such as Orbitrap or Q-TOF systems provide accurate mass measurements and deep proteome coverage. Parameters are optimized for labile acyl groups to prevent modification loss during fragmentation.
Fragmentation methods commonly used include:
- Higher-energy collisional dissociation (HCD) for reliable PTM site localization
- Electron-transfer dissociation (ETD) for preserving fragile acyl modifications
Quantitative Approaches
- Label-Free Quantification: Measures peptide ion intensities or spectral counts without chemical labels. This method is cost-effective and flexible but can be less precise for low-abundance modifications.
- Isobaric Tagging (iTRAQ/TMT): Peptides from multiple samples are chemically labeled with tags of identical mass but different reporter ions. Multiplexing enables simultaneous quantification across conditions with high precision, though ratio distortion can occur due to co-isolation interference.
- Metabolic Labeling (SILAC): Cells are cultured in media containing heavy isotope-labeled amino acids, enabling incorporation into nascent proteins. SILAC provides accurate relative quantification between experimental conditions, especially in cell culture systems.
- Data-Independent Acquisition (DIA): Offers reproducible and deep quantification without the need for labeling.
Figure 1. A schematic workflow for the large-scale lysine modification proteome analysis (Kozuka-Hata H, et al., 2020).
Metabolic and Chemical Labeling Approaches
Stable Isotope Tracing: Incorporation of ^13C or ^15N-labeled acyl donors tracks dynamic acylation flux. This allows differentiation of newly formed acyl modifications from pre-existing ones, revealing insights into metabolic regulation of acylation.
Photo-Crosslinking Probes: Photoactivatable acyl-CoA analogs enable covalent capture of transient enzyme-substrate interactions upon UV activation. This approach facilitates identification of acyltransferases and their substrates, which is difficult to achieve with conventional methods.
Orthogonal Validation and Functional Studies
Western Blotting and Immunoprecipitation: These methods provide targeted validation of candidate acylation sites or proteins. Immunoprecipitation using specific antibodies enriches the protein of interest, which is then detected by acylation-specific antibodies via Western blot. This confirms the presence and regulation of modifications under physiological conditions.
Site-Directed Mutagenesis: Substituting modified residues (e.g., lysine to arginine or cysteine to serine) enables functional analysis of acylation. Loss of modification sites can affect protein stability, subcellular localization, or interaction networks, providing insights into the biological impact of acylation.
Enzyme Inhibition Studies: Treatment with specific inhibitors of acyltransferases (e.g., HAT inhibitors) or deacylases (e.g., HDAC inhibitors) modulates acylation levels. Monitoring changes using MS or immunodetection validates enzyme involvement and therapeutic potential.
Global vs Site-Specific Acylation Profiling
Global profiling aims to catalog the total repertoire of acylated proteins (the "acylome"), whereas site-specific profiling identifies the precise amino acid residues modified. Combined approaches are often needed for comprehensive analysis—starting with global screens, followed by site-specific validation.
| Aspect |
Global Acylation Profiling |
Site-Specific Acylation Profiling |
| Definition |
Measures overall acylation levels across the proteome |
Identifies exact amino acid residues modified by acyl groups |
| Objective |
Determine the presence and relative abundance of acylated proteins |
Define modification sites for mechanistic and functional analysis |
| Techniques Used |
Label-free LC-MS/MS, enrichment by chemical probes or antibodies |
LC-MS/MS with high-resolution fragmentation, MS/MS site localization algorithms |
| Enrichment Strategy |
Broad, often targets protein-level enrichment (e.g., metabolic labeling, ABE) |
Peptide-level enrichment and high-resolution tandem MS |
| Resolution |
Low – provides protein-level information only |
High – provides site-level and positional data |
| Throughput |
High – suitable for global acylome profiling |
Moderate – requires deeper analysis and data validation |
| Advantages |
Fast, scalable, suitable for screening |
Enables functional interpretation and downstream biological studies |
| Limitations |
Cannot determine specific sites; prone to false positives |
Technically demanding; requires more sample and data processing |
Bioinformatics Tools for Acylation Site Identification
Accurately identifying protein acylation sites depends on advanced bioinformatics tools that can analyze complex mass spectrometry data and uncover meaningful biological information. After data collection, these platforms match peptides to spectra, pinpoint modification sites, and assign confidence scores to ensure reliable results. Commonly used tools and databases include:
MaxQuant and Proteome Discoverer: Widely used platforms that support identification and quantification of acylated peptides from raw MS data. They implement algorithms for PTM site localization scoring and control false discovery rates (FDR), ensuring high-confidence site assignments.
Motif Analysis Tools (e.g., Motif-X, pLogo): These analyze sequence context around acylation sites to discover conserved motifs or amino acid preferences. Understanding motif patterns helps predict novel acylation targets and reveals potential regulatory mechanisms.
PTM Databases (e.g., UniProt, PhosphoSitePlus, iPTMnet): These repositories catalog experimentally validated acylation sites and related enzyme information. They assist in functional annotation, cross-referencing with known PTMs, and integrating acylation data into broader signaling networks.
Challenges in Acylation Analysis
Despite technological advances, several analytical hurdles persist:
- Low Stoichiometry: Many acylation events are substoichiometric, requiring enrichment for detection.
- Dynamic and Reversible Nature: Acylation is often transient, making snapshot analysis potentially misleading.
- Antibody Specificity: Cross-reactivity and inconsistent performance can lead to false positives or negatives.
- Site Ambiguity: Overlapping PTMs at the same site may confound assignment.
Applications of Acylation Profiling in Research
Role of Acylation in Human Diseases
- Cancer: Changes in protein acylation are closely linked to cancer development. For instance, excess palmitoylation of proteins like H-Ras boosts their attachment to cell membranes, increasing cancer-related signaling. Acetylation of transcription factors such as p53 affects how they bind DNA and control tumor growth. Targeting the enzymes that add or remove these acyl groups is becoming a promising approach in cancer therapy.
- Neurological Disorders: Palmitoylation of synaptic proteins regulates neuronal signaling, plasticity, and membrane trafficking. Dysregulated acylation of proteins such as PSD-95 and huntingtin is associated with neurodevelopmental and neurodegenerative conditions, including autism and Huntington's disease.
- Immunology: Acylation modulates immune receptor signaling and cytokine production. S-acylation of immune proteins like STING and IFITM3 influences innate immune responses. Aberrant acylation can impair pathogen clearance or contribute to chronic inflammation.
Acylation Signatures in Drug Discovery and Target Validation
Analyzing protein acylation patterns can uncover cancer-specific changes and identify targets for treatment. Drugs that block acylation enzymes, like palmitoyltransferases, are being tested for cancer and viral infections. Quantitative acyl-proteomics helps track how well these drugs work, detect resistance, and confirm treatment response.
- Target Engagement: Measuring acylation changes upon compound treatment confirms on-target activity.
- Mechanism of Action: Drug-induced acylation patterns help elucidate downstream signaling effects.
- Biomarker Discovery: Acylated proteins differentially expressed in disease vs. control samples may serve as diagnostic or prognostic indicators.
Host-Pathogen Interactions Mediated by Acylated Proteins
Pathogens often exploit or mimic host acylation processes to support infection and immune evasion.
- Viruses: Viral envelope proteins (e.g., influenza HA, HIV-1 gp41) are S-palmitoylated to promote membrane fusion and viral assembly.
- Bacteria: Some bacterial effectors hijack host acylation machinery to modify signaling pathways.
- Host Defense: Acylation of antimicrobial proteins enhances membrane targeting and pathogen clearance.
Case study
Ldha desuccinylase Sirtuin 5 as a novel cancer metastatic stimulator in aggressive prostate cancer.
Journal: Genomics, Proteomics & Bioinformatics
Published: 2023
DOI: 10.1016/j.gpb.2022.02.004
Background
Lysine succinylation is an emerging post-translational acylation that profoundly alters enzyme function and cellular metabolism. Sirtuin 5 (SIRT5) is a mitochondrial NAD⁺-dependent desuccinylase, yet its role in cancer progression—especially in castration-resistant prostate cancer (CRPC)—remained poorly defined prior to this study.
Purpose
To elucidate how SIRT5 and succinylation dynamics contribute to prostate cancer aggressiveness, and to identify specific succinylated substrates that drive malignant phenotypes.
Methods
- Protein Expression Analysis: Western blotting measured SIRT5 levels across PCa cell lines of varying metastatic potential.
- Global Succinylome Profiling: Quantitative mass-spectrometry–based succinylome analysis compared lysine succinylation patterns between high- and low-metastatic PCa cells.
- Site-Specific Validation: Immunoblotting and mutagenesis confirmed succinylation at LDHA-K118.
- Functional Assays: Cell migration, invasion, and LDH enzymatic activity assays assessed the biological impact of LDHA-K118 succinylation.
Results
- SIRT5 Downregulation: Aggressive PCa cells exhibited significantly lower SIRT5 protein levels than non-metastatic counterparts.
- Elevated Succinylation: Global analysis revealed a marked increase in succinylation at LDHA lysine 118 (LDHA-K118su) in high-metastatic cells.
- Functional Consequences: LDHA-K118su enhanced LDH enzymatic activity and promoted PCa cell migration and invasion.
- Clinical Correlation: Lower SIRT5 expression and higher LDHA-K118su levels correlated with poorer patient survival.
Figure 2. Increased Ksu in the cytoplasm in PCa.
Conclusion
The study uncovers a novel SIRT5–LDHA axis in prostate cancer: loss of SIRT5 leads to hypersuccinylation of LDHA at K118, boosting LDH activity and metastatic behavior. Targeting this desuccinylation pathway may offer new strategies to impede CRPC progression.
People Also Ask
Which experimental techniques yield the most comprehensive acylation profiles?
Mass spectrometry (MS)–based proteomics combined with affinity enrichment (using acyl-specific antibodies or chemical probes) currently offers the deepest, site-specific coverage. Label-free or isotope-labeling strategies (SILAC, TMT, DIA) provide quantitative readouts, while metabolic labeling with bioorthogonal acyl analogs can capture dynamic changes.
Can multiple types of acylation occur on the same protein?
Yes, proteins can carry different types of acylation on separate sites—or even on the same lysine, but not at the same time. For instance, one lysine might be acetylated, crotonylated, or succinylated depending on cell conditions. This flexibility adds complexity to how proteins are regulated and function.
How does acylation influence chromatin accessibility?
Acylation removes positive charges from lysine residues on histone tails, loosening the bond between DNA and histones. This opens up the chromatin, making it easier for genes to be read and activated. Different acyl groups—like acetyl, crotonyl, or butyryl—have distinct sizes and shapes, which fine-tune how genes are turned on or off.
References
- Kozuka-Hata H, et al. System-wide analysis of protein acetylation and ubiquitination reveals a diversified regulation in human cancer cells. Biomolecules, 2020, 10(3): 411.
- Diallo I, et al. Current trends in protein acetylation analysis. Expert review of proteomics, 2019, 16(2): 139-159.
- Lanyon-Hogg T, Faronato M, Serwa R A, 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.
