GO:0090433 palmitoyl-CoA ligase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0090433 (palmitoyl-CoA ligase activity) catalyzes the ATP-dependent ligation of palmitic acid with coenzyme A to form palmitoyl-CoA, AMP, and diphosphate.
• ACSL4 is the most extensively studied enzyme with this activity; it preferentially activates long-chain polyunsaturated fatty acids and is a key driver of ferroptosis.
• ACSL4-mediated phospholipid remodeling promotes cancer metastasis and is associated with poor prognosis in triple-negative breast cancer.
• Ferroptosis-dependent inflammation driven by ACSL4 contributes to pulmonary hypertension, highlighting its role beyond cancer.
• Other ACSL family members (ACSL1, ACSL3, ACSL5, ACSL6) exhibit distinct substrate preferences and tissue distributions, influencing diverse metabolic and signaling pathways.
• CRISPR knockout, point mutation, and overexpression models are essential for dissecting the causal roles of ACSL enzymes in disease.
Description
Palmitoyl-CoA ligase activity (GO:0090433) is a molecular function that catalyzes the ATP-dependent formation of palmitoyl-CoA from palmitic acid and coenzyme A. This reaction is the first step in the activation of long-chain fatty acids for subsequent metabolic processes, including phospholipid synthesis, beta-oxidation, and protein acylation. The enzyme activity is critical for maintaining lipid homeostasis and is carried out by members of the acyl-CoA synthetase long-chain (ACSL) family. Dysregulation of palmitoyl-CoA ligase activity has been implicated in a wide range of pathological conditions, from cancer to cardiovascular disease. Recent studies have highlighted the role of ACSL4, a major enzyme with this activity, in ferroptosis, a form of regulated cell death driven by lipid peroxidation. Understanding the molecular mechanisms and regulatory networks of palmitoyl-CoA ligase activity is therefore of paramount importance for both basic biology and therapeutic development. This article provides a comprehensive overview of the GO term, its associated genes, disease relevance, and cutting-edge research methods, with a focus on CRISPR-based models for functional interrogation.
palmitoyl-CoA ligase activity At A Glance
| GO ID | GO:0090433 |
|---|---|
| GO term | palmitoyl-CoA ligase activity |
| Ontology | molecular_function |
| Synonym | palmitoyl-CoA synthetase activity |
| Major function | Catalyzes ATP-dependent ligation of palmitic acid with CoA to form palmitoyl-CoA, AMP, and diphosphate |
| EC number | 6.2.1.3 (long-chain fatty acid-CoA ligase) |
| Substrates | Palmitic acid, CoA, ATP |
| Products | Palmitoyl-CoA, AMP, diphosphate |
| Cofactors | Mg2+ (required for ATP binding) |
| Representative genes | ACSL1, ACSL3, ACSL4, ACSL5, ACSL6 |
What Is GO:0090433?
Palmitoyl-CoA ligase activity (GO:0090433) is defined as the catalysis of the reaction: ATP + palmitic acid + CoA = AMP + diphosphate + palmitoyl-CoA. In other words, it is the enzyme activity that activates palmitic acid by attaching it to coenzyme A, consuming ATP in the process. This thioesterification reaction is essential for channeling palmitate into various metabolic pathways, such as phospholipid biosynthesis and fatty acid oxidation.
Why Is palmitoyl-CoA ligase activity Important in Cell Biology?
Palmitoyl-CoA ligase activity is a central node in lipid metabolism, controlling the activation of palmitic acid for anabolic and catabolic pathways. Its dysregulation is linked to cancer progression, ferroptosis, and inflammatory diseases, making it a promising therapeutic target. Moreover, the enzyme activity influences membrane phospholipid composition, which in turn affects cell signaling and survival.
• Provides activated palmitoyl-CoA for phospholipid synthesis and membrane remodeling.
• Drives ferroptosis by incorporating polyunsaturated fatty acids into phospholipids, a process dependent on ACSL4.
• Promotes cancer metastasis through integrin β1 activation in triple-negative breast cancer.
• Contributes to pulmonary hypertension via ferroptosis-mediated inflammation.
• Supports tumor immune evasion by activating IL-18R1-NF-κB signaling (ACSL6).
• Regulates lipid droplet formation and energy storage.
• Modulates protein palmitoylation and subcellular localization.
• Influences intercellular interactions and cancer cell sensitivity to ferroptosis.
• Is a potential biomarker for intervertebral disc degeneration.
• Offers targets for CRISPR-based functional screens in metabolic and cancer research.
Molecular Mechanism of palmitoyl-CoA ligase activity
Substrate Binding and ATP Utilization
In simple terms: The enzyme grabs palmitic acid and ATP, then uses ATP energy to activate the fatty acid.
Palmitoyl-CoA ligase activity begins with the binding of palmitic acid, ATP, and CoA to the enzyme's active site. ATP is hydrolyzed to AMP and diphosphate, forming an acyl-AMP intermediate. This step requires Mg2+ as a cofactor.
Thioesterification and Product Release
In simple terms: The activated fatty acid is linked to CoA, forming palmitoyl-CoA, which is then released.
The acyl-AMP intermediate reacts with CoA to form palmitoyl-CoA, releasing AMP and diphosphate. The product, palmitoyl-CoA, is a key metabolite that can enter various pathways, including phospholipid synthesis and beta-oxidation.
Enzyme Isoforms and Substrate Specificity
In simple terms: Different versions of the enzyme prefer different fatty acids, affecting what they do in the cell.
The ACSL family comprises five isoforms (ACSL1, ACSL3, ACSL4, ACSL5, ACSL6) with distinct substrate preferences and tissue distributions. ACSL4 preferentially activates arachidonic acid and other polyunsaturated fatty acids, linking it to ferroptosis. ACSL6 is involved in IL-18-mediated immune evasion.
Regulation by Phosphorylation and Signaling
In simple terms: The enzyme's activity can be turned up or down by chemical modifications and signaling pathways.
ACSL4 is phosphorylated by PKCβII at Thr328, which amplifies lipid peroxidation and promotes ferroptosis. Additionally, glycolysis-derived lactate induces ACSL4 expression and lactylation, activating ferroptosis in intervertebral disc degeneration. These modifications fine-tune enzyme activity in response to cellular stress.
Role in Phospholipid Remodeling
In simple terms: The enzyme helps build and reshape cell membranes by incorporating specific fatty acids.
ACSL4-mediated activation of polyunsaturated fatty acids leads to their incorporation into membrane phospholipids, a process that can alter membrane fluidity and signaling. This remodeling is critical for cancer cell metastasis and ferroptosis sensitivity.
Key Genes Involved in GO:0090433 palmitoyl-CoA ligase activity
The following genes encode enzymes with palmitoyl-CoA ligase activity or are directly involved in its regulation and downstream effects.
| Gene | Major Role | Research Relevance |
|---|---|---|
| ACSL1 | Activates long-chain fatty acids for beta-oxidation and lipid synthesis | Metabolic disorders, insulin resistance |
| ACSL3 | Activates fatty acids for phospholipid synthesis | Lipid droplet formation, cancer |
| ACSL4 | Preferentially activates arachidonic acid and other PUFAs | Ferroptosis, cancer metastasis, pulmonary hypertension |
| ACSL5 | Activates fatty acids in the intestine and liver | Lipid absorption, metabolic syndrome |
| ACSL6 | Activates long-chain fatty acids in the brain | Tumor immune evasion, neurological disorders |
| PKCβII | Phosphorylates ACSL4 at Thr328 | Amplifies ferroptosis |
| NF2 | Regulates YAP signaling and ACSL4 expression | Cancer cell ferroptosis |
| YAP | Transcriptionally regulates ACSL4 | Ferroptosis resistance |
| PCK2 | Supports phospholipid remodeling and ferroptosis evasion | Tumor-repopulating cells |
| IL-18R1 | Receptor activated by ACSL6-mediated IL-18 signaling | Tumor immune evasion |
| NF-κB | Transcription factor downstream of IL-18R1 | Inflammation and cancer |
| Integrin β1 | Activated by ACSL4-mediated membrane remodeling | Triple-negative breast cancer metastasis |
| Lactate | Induces ACSL4 expression and lactylation | Intervertebral disc degeneration |
| GPX4 | Glutathione peroxidase that counteracts lipid peroxidation | Ferroptosis regulation |
| SLC7A11 | Cystine/glutamate antiporter, affects ferroptosis | Cancer therapy |
| ACSL4 (phospho-Thr328) | Phosphorylated form with enhanced activity | Ferroptosis induction |
| ACSL4 (lactylated) | Lactylation enhances activity | Intervertebral disc degeneration |
How Is palmitoyl-CoA ligase activity Regulated?
Palmitoyl-CoA ligase activity is regulated at multiple levels. Post-translational modifications, such as phosphorylation of ACSL4 by PKCβII at Thr328, enhance its enzymatic activity and promote lipid peroxidation. Lactylation of ACSL4, induced by glycolysis-derived lactate, also increases its activity and triggers ferroptosis in intervertebral disc degeneration. Transcriptional regulation by NF2-YAP signaling modulates ACSL4 expression, affecting cancer cell sensitivity to ferroptosis. Additionally, metabolic cues, such as PCK2-dependent phospholipid remodeling, can bypass the need for ACSL4 in tumor-repopulating cells. These regulatory mechanisms fine-tune palmitoyl-CoA ligase activity in response to cellular stress and metabolic demands.
palmitoyl-CoA ligase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| ACSL4 | Triple-negative breast cancer metastasis | Knockout and overexpression in MDA-MB-231 cells |
| ACSL4 | Pulmonary hypertension | Endothelial cell-specific knockout in mice |
| ACSL4 | Intervertebral disc degeneration | Lactate-treated nucleus pulposus cells with ACSL4 knockdown |
| ACSL6 | Tumor immune evasion | Knockout in melanoma cells and syngeneic mouse models |
| ACSL4 | Ferroptosis in cancer | CRISPR knockout in cancer cell lines followed by ferroptosis inducers |
Cancer and Metastasis
ACSL4-mediated membrane phospholipid remodeling activates integrin β1, facilitating triple-negative breast cancer metastasis. ACSL6-activated IL-18R1-NF-κB signaling promotes tumor immune evasion and progression. Tumor-repopulating cells evade ferroptosis via PCK2-dependent phospholipid remodeling, highlighting metabolic plasticity. NF2-YAP signaling dictates cancer cell ferroptosis by regulating ACSL4.
Ferroptosis and Inflammatory Diseases
PKCβII phosphorylation of ACSL4 amplifies lipid peroxidation to induce ferroptosis. Ferroptosis-mediated inflammation promotes pulmonary hypertension, with ACSL4 playing a central role. Glycolysis-derived lactate induces ACSL4 expression and lactylation, activating ferroptosis during intervertebral disc degeneration.
Metabolic and Neurological Disorders
ACSL family members are implicated in metabolic syndrome, insulin resistance, and neurological disorders due to their roles in lipid metabolism. ACSL6 is particularly important in brain lipid metabolism and has been linked to tumor immune evasion.
From palmitoyl-CoA ligase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does ACSL4 loss prevent ferroptosis? | ACSL4 knockout cell lines (e.g., HT-1080, MDA-MB-231) |
| Does ACSL4 phosphorylation at Thr328 enhance ferroptosis? | Point mutation (T328A) knock-in cells |
| Does ACSL4 lactylation affect intervertebral disc degeneration? | Knock-in of lactylation-deficient ACSL4 mutant |
| Can ACSL6 overexpression promote tumor immune evasion? | ACSL6 overexpression in melanoma cells |
| Does ACSL4-mediated phospholipid remodeling drive metastasis? | ACSL4 knockout in triple-negative breast cancer cells |
| What is the role of ACSL4 in pulmonary hypertension? | Endothelial-specific ACSL4 knockout mice |
How to Study the palmitoyl-CoA ligase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| CRISPR knockout screen | Gene essentiality for ferroptosis | Identify ACSL4 as key regulator |
| Phosphoproteomics | Phosphorylation sites on ACSL4 | Detect PKCβII-mediated Thr328 phosphorylation |
| Lactylome analysis | Lactylation sites on ACSL4 | Study lactate-induced ACSL4 activation |
| Lipidomics | Phospholipid composition and oxidation | Assess ACSL4-mediated membrane remodeling |
| Metabolomics | Palmitoyl-CoA and fatty acid levels | Measure enzyme activity |
| C11-BODIPY staining | Lipid peroxidation | Monitor ferroptosis |
| Western blot | Protein expression and phosphorylation | Validate ACSL4 levels and modifications |
| Immunofluorescence | Subcellular localization | Visualize ACSL4 at membranes |
CRISPR-Cas9 Knockout Screens
Genome-wide CRISPR knockout screens can identify genes required for palmitoyl-CoA ligase activity and ferroptosis. For example, ACSL4 was identified as a key ferroptosis promoter in a CRISPR screen. These screens are powerful for uncovering novel regulators of lipid metabolism.
Phosphoproteomics and Lactylome Analysis
Mass spectrometry-based phosphoproteomics can detect phosphorylation of ACSL4 at Thr328, while lactylome analysis can identify lactylation sites induced by lactate. These methods reveal post-translational modifications that regulate enzyme activity.
Lipidomics and Metabolomics
Lipidomic profiling quantifies phospholipid species and oxidized lipids, providing insights into ACSL4-mediated membrane remodeling. Metabolomics can measure palmitoyl-CoA levels and other intermediates.
Imaging and Flow Cytometry
Fluorescent probes for lipid peroxidation (e.g., C11-BODIPY) and ferroptosis markers (e.g., GPX4) allow visualization of ACSL4 activity in live cells. Flow cytometry can assess integrin β1 activation.
How CRISPR Can Be Used to Study GO:0090433 palmitoyl-CoA ligase activity
Knockout
CRISPR-Cas9 knockout of ACSL4 or other ACSL genes is used to abolish palmitoyl-CoA ligase activity, revealing its role in ferroptosis, cancer metastasis, and lipid metabolism. Knockout cell lines are valuable for drug sensitivity studies and pathway analysis.
Point Mutation
Point mutations, such as ACSL4 T328A, can be introduced via CRISPR to prevent phosphorylation, thereby dissecting the contribution of specific post-translational modifications to enzyme activity and ferroptosis. Similarly, lactylation-deficient mutants can test the role of lactate-induced ACSL4 activation.
Knock-in
Knock-in of tagged ACSL4 (e.g., FLAG or GFP) allows for affinity purification, imaging, and interaction studies. Knock-in of disease-associated variants can model human conditions and test therapeutic interventions.
Overexpression
CRISPR activation (CRISPRa) or lentiviral overexpression of ACSL4 or ACSL6 can enhance palmitoyl-CoA ligase activity, promoting ferroptosis or immune evasion. Overexpression models are useful for gain-of-function studies and identifying downstream effectors.
How EDITGENE Supports palmitoyl-CoA ligase activity Research
Researchers studying palmitoyl-CoA ligase activity-related genes often need to determine whether a candidate gene is causally involved in lipid metabolism, ferroptosis, or cancer progression. EDITGENE provides comprehensive CRISPR-based services to generate precisely engineered cell models, enabling rigorous functional validation.
Contact EDITGENE today to design your custom CRISPR model for palmitoyl-CoA ligase activity research.
Frequently Asked Questions About palmitoyl-CoA ligase activity
What is palmitoyl-CoA ligase activity?
Palmitoyl-CoA ligase activity (GO:0090433) is the enzyme activity that catalyzes the ATP-dependent formation of palmitoyl-CoA from palmitic acid and coenzyme A, producing AMP and diphosphate.
What genes are involved in palmitoyl-CoA ligase activity?
The main genes are ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6, which encode acyl-CoA synthetase long-chain family members.
Which enzyme is most important for ferroptosis?
ACSL4 is the key enzyme whose palmitoyl-CoA ligase activity preferentially activates polyunsaturated fatty acids, driving ferroptosis.
How is ACSL4 regulated?
ACSL4 is regulated by phosphorylation at Thr328 by PKCβII and by lactylation induced by glycolysis-derived lactate.
What diseases are linked to palmitoyl-CoA ligase activity?
It is linked to cancer metastasis, pulmonary hypertension, intervertebral disc degeneration, and metabolic disorders.
Can CRISPR knockout of ACSL4 prevent ferroptosis?
Yes, CRISPR knockout of ACSL4 abolishes its activity and confers resistance to ferroptosis inducers.
What is the role of ACSL6 in cancer?
ACSL6 activates IL-18R1-NF-κB signaling to promote tumor immune evasion and progression.
How can I study palmitoyl-CoA ligase activity in the lab?
Use CRISPR knockout, overexpression, lipidomics, and ferroptosis assays to measure enzyme function and downstream effects.
What are the substrates of palmitoyl-CoA ligase?
The substrates are palmitic acid, coenzyme A, and ATP; the products are palmitoyl-CoA, AMP, and diphosphate.
Is palmitoyl-CoA ligase activity a drug target?
Yes, inhibitors of ACSL4 are being explored for cancer therapy and ferroptosis modulation.
Conclusion
Palmitoyl-CoA ligase activity (GO:0090433) is a fundamental molecular function that governs lipid metabolism and cell fate. Its dysregulation is implicated in cancer, ferroptosis, and inflammatory diseases, making it a high-priority target for therapeutic development. CRISPR-based models are indispensable for dissecting the causal roles of ACSL enzymes and for identifying novel regulators. EDITGENE's comprehensive services empower researchers to generate precise knockout, knock-in, and overexpression models, accelerating discoveries in this dynamic field.
References
- 1. Sun K et al.. 2025. Glycolysis-Derived Lactate Induces ACSL4 Expression and Lactylation to Activate Ferroptosis during Intervertebral Disc Degeneration.. Adv Sci (Weinh) 12(21):e2416149 PMID: 40171826
- 2. Zhang HL et al.. 2022. PKCβII phosphorylates ACSL4 to amplify lipid peroxidation to induce ferroptosis.. Nat Cell Biol 24(1):88-98 PMID: 35027735
- 3. Qiu Y et al.. 2024. ACSL4-Mediated Membrane Phospholipid Remodeling Induces Integrin β1 Activation to Facilitate Triple-Negative Breast Cancer Metastasis.. Cancer Res 84(11):1856-1871 PMID: 38471082
- 4. Kazmirczak F et al.. 2024. Ferroptosis-Mediated Inflammation Promotes Pulmonary Hypertension.. Circ Res 135(11):1067-1083 PMID: 39421926
- 5. Wu J et al.. 2019. Intercellular interaction dictates cancer cell ferroptosis via NF2-YAP signalling.. Nature 572(7769):402-406 PMID: 31341276
- 6. Li Z et al.. 2024. Tumor-repopulating cells evade ferroptosis via PCK2-dependent phospholipid remodeling.. Nat Chem Biol 20(10):1341-1352 PMID: 38720107
- 7. Di Y et al.. 2024. ACSL6-activated IL-18R1-NF-κB promotes IL-18-mediated tumor immune evasion and tumor progression.. Sci Adv 10(38):eadp0719 PMID: 39292786
- 8. Quan J et al.. 2021. ACSL family: The regulatory mechanisms and therapeutic implications in cancer.. Eur J Pharmacol 909:174397 PMID: 34332918
