GO:0031957 very long-chain fatty acid-CoA ligase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0031957 (very long-chain fatty acid-CoA ligase activity) catalyzes the ATP-dependent ligation of a very long-chain fatty acid (more than 22 carbons) with coenzyme A to form a very long-chain fatty acyl-CoA plus AMP and diphosphate.
• This activity is a molecular_function that activates very long-chain fatty acids for downstream metabolism, including phospholipid remodeling and ferroptosis-related lipid peroxidation.
• ACSL4 is the best-characterized enzyme with very long-chain fatty acid-CoA ligase activity and is a central regulator of ferroptosis sensitivity in cancer and degenerative disease.
• ACSL6 also exhibits activity toward long-chain and very long-chain fatty acids and drives tumor immune evasion through IL-18R1-NF-kB signaling.
• The long-chain acyl-CoA synthetase family (ACSL1, ACSL3, ACSL4, ACSL5, ACSL6) shows overlapping but distinct substrate preferences, subcellular localization, and disease associations.
• CRISPR knockout, point-mutation, knock-in, and overexpression models are essential to dissect how very long-chain fatty acid-CoA ligase activity contributes to ferroptosis, cancer metastasis, and inflammation.
Description
Very long-chain fatty acid-CoA ligase activity (GO:0031957) is a molecular_function that catalyzes the ATP-dependent activation of very long-chain fatty acids, defined as fatty acids with an aliphatic tail containing more than 22 carbons, by ligating them to coenzyme A to produce very long-chain fatty acyl-CoA, AMP, and diphosphate. This reaction is the obligatory first step for very long-chain fatty acids to enter most downstream metabolic pathways, because acyl-CoA thioesters are the biologically active forms used by acyltransferases, oxidases, and other enzymes. The activity is therefore a gatekeeper for the incorporation of very long-chain fatty acids into membrane phospholipids, lipid droplets, and oxidative reactions. Among the long-chain acyl-CoA synthetase (ACSL) family, ACSL4 is the most extensively studied enzyme with very long-chain fatty acid-CoA ligase activity and preferentially activates arachidonic acid and other polyunsaturated very long-chain fatty acids. ACSL4-mediated membrane phospholipid remodeling is required for integrin beta1 activation and triple-negative breast cancer metastasis, and ACSL4 is also a key driver of ferroptosis, an iron-dependent form of regulated cell death characterized by lipid peroxidation. Glycolysis-derived lactate induces ACSL4 expression and lactylation to activate ferroptosis during intervertebral disc degeneration, and PKCbetaII phosphorylates ACSL4 to amplify lipid peroxidation during ferroptosis. These findings place GO:0031957 at the center of cancer biology, degenerative disease, and inflammation research. ACSL6 is another enzyme with activity toward long-chain and very long-chain fatty acids; ACSL6-activated IL-18R1-NF-kB signaling promotes IL-18-mediated tumor immune evasion and tumor progression. The broader ACSL family, including ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6, displays distinct but overlapping substrate specificities, tissue distributions, and regulatory mechanisms, making GO:0031957 a rich area for functional genomics and therapeutic target discovery. Understanding this activity requires integrating enzymology, lipidomics, cell biology, and CRISPR-based perturbation, which is the focus of this article.
very long-chain fatty acid-CoA ligase activity At A Glance
| GO ID | GO:0031957 |
|---|---|
| GO term | very long-chain fatty acid-CoA ligase activity |
| Ontology | molecular_function |
| Synonym | very-long-chain fatty acid activation; very-long-chain fatty acid-CoA ligase activity; very-long-chain-fatty-acid-CoA ligase activity |
| Major function | ATP-dependent ligation of very long-chain fatty acids (>22 carbons) to coenzyme A, forming very long-chain fatty acyl-CoA, AMP, and diphosphate |
| Substrate specificity | Very long-chain fatty acids with aliphatic tails longer than 22 carbons; ACSL4 preferentially activates arachidonic acid and other polyunsaturated fatty acids |
| Representative enzymes | ACSL4, ACSL6, and other long-chain acyl-CoA synthetase family members |
| Biological context | Membrane phospholipid remodeling, ferroptosis, cancer metastasis, inflammation, and intervertebral disc degeneration |
What Is GO:0031957?
GO:0031957 very long-chain fatty acid-CoA ligase activity is defined as catalysis of the reaction: a very long-chain fatty acid + ATP + CoA = a very long-chain fatty acyl-CoA + AMP + diphosphate, where a very long-chain fatty acid has an aliphatic tail containing more than 22 carbons. In other words, the enzyme uses ATP to activate a very long-chain fatty acid by attaching it to coenzyme A, releasing AMP and diphosphate, and generating a very long-chain fatty acyl-CoA thioester that can participate in downstream lipid metabolism.
Why Is very long-chain fatty acid-CoA ligase activity Important in Cell Biology?
GO:0031957 is important because it controls the entry of very long-chain fatty acids into essentially all downstream metabolic routes, including phospholipid biosynthesis, lipid droplet formation, and oxidative catabolism. Dysregulation of this activity alters membrane lipid composition and signaling, and ACSL4-dependent very long-chain fatty acid-CoA ligase activity is now recognized as a central determinant of ferroptosis sensitivity in cancer, degenerative disease, and inflammation. Because ferroptosis can be either promoted or suppressed for therapeutic benefit, enzymes carrying this activity are attractive drug targets and biomarkers.
• Controls activation of very long-chain fatty acids for phospholipid remodeling and membrane dynamics.
• Determines ferroptosis sensitivity through ACSL4-mediated lipid peroxidation.
• Promotes triple-negative breast cancer metastasis via integrin beta1 activation.
• Drives tumor immune evasion through ACSL6-activated IL-18R1-NF-kB signaling.
• Contributes to intervertebral disc degeneration through lactate-induced ACSL4 expression and lactylation.
• Links ferroptosis-mediated inflammation to pulmonary hypertension.
• Provides therapeutic targets across cancer, inflammation, and degenerative disease.
• Requires CRISPR functional genomics to separate the roles of ACSL family members.
• Serves as a biomarker for lipid metabolism reprogramming in tumors.
• Connects glycolysis, lactylation, and lipid peroxidation in disease pathology.
Molecular Mechanism of very long-chain fatty acid-CoA ligase activity
Substrate recognition and binding
In simple terms: The enzyme first grabs a very long-chain fatty acid and ATP so it can start the activation reaction.
Enzymes with very long-chain fatty acid-CoA ligase activity, such as ACSL4, bind very long-chain fatty acids with aliphatic tails longer than 22 carbons together with ATP and coenzyme A. ACSL4 preferentially recognizes arachidonic acid and other polyunsaturated fatty acids, which are substrates for subsequent peroxidation during ferroptosis. Substrate specificity is a key determinant of which lipids enter remodeling and oxidation pathways.
ATP-dependent acyl-adenylate formation
In simple terms: ATP is used to make the fatty acid more reactive by attaching AMP to it.
The catalytic mechanism proceeds through an acyl-adenylate intermediate in which the very long-chain fatty acid is activated by ATP, releasing diphosphate. This step consumes ATP and primes the fatty acid for nucleophilic attack by coenzyme A. The reaction is characteristic of the acyl-CoA synthetase family, which includes ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6.
Thioester bond formation with coenzyme A
In simple terms: Coenzyme A attacks the activated fatty acid to form a stable fatty acyl-CoA thioester.
Coenzyme A attacks the acyl-adenylate intermediate, displacing AMP and forming a very long-chain fatty acyl-CoA thioester. The products of the overall reaction are very long-chain fatty acyl-CoA, AMP, and diphosphate. The resulting acyl-CoA is the metabolically active form used by acyltransferases and other enzymes in phospholipid remodeling and beta-oxidation.
Membrane phospholipid remodeling
In simple terms: The activated fatty acid is incorporated into membrane lipids, changing membrane properties.
ACSL4-mediated membrane phospholipid remodeling incorporates very long-chain polyunsaturated fatty acids into phospholipids, which alters membrane fluidity and signaling. This remodeling is required for integrin beta1 activation and facilitates triple-negative breast cancer metastasis. The same remodeling process generates phospholipid substrates that are susceptible to peroxidation during ferroptosis.
Ferroptosis execution and regulation
In simple terms: When the activated fatty acids are oxidized, they can trigger a form of iron-dependent cell death called ferroptosis.
ACSL4-dependent very long-chain fatty acid-CoA ligase activity is required for ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation. PKCbetaII phosphorylates ACSL4 to amplify lipid peroxidation and induce ferroptosis. Glycolysis-derived lactate induces ACSL4 expression and lactylation to activate ferroptosis during intervertebral disc degeneration, and ferroptosis-mediated inflammation promotes pulmonary hypertension. Intercellular interaction via NF2-YAP signaling also dictates cancer cell ferroptosis sensitivity.
Key Genes Involved in GO:0031957 very long-chain fatty acid-CoA ligase activity
The following genes encode enzymes and regulators directly implicated in very long-chain fatty acid-CoA ligase activity (GO:0031957) and its downstream biology.
| Gene | Major Role | Research Relevance |
|---|---|---|
| ACSL4 | Very long-chain fatty acid-CoA ligase that preferentially activates arachidonic acid and polyunsaturated fatty acids | Central regulator of ferroptosis and cancer metastasis |
| ACSL6 | Long-chain and very long-chain fatty acid-CoA ligase | Activates IL-18R1-NF-kB signaling and tumor immune evasion |
| ACSL1 | Long-chain acyl-CoA synthetase family member | Contributes to lipid metabolism and disease; reviewed in ACSL family studies |
| ACSL3 | Long-chain acyl-CoA synthetase family member | Lipid droplet and membrane lipid metabolism; reviewed in ACSL family studies |
| ACSL5 | Long-chain acyl-CoA synthetase family member | Intestinal and hepatic lipid metabolism; reviewed in ACSL family studies |
| PKCbetaII | Kinase that phosphorylates ACSL4 | Amplifies lipid peroxidation and ferroptosis |
| NF2 | Tumor suppressor upstream of YAP | Dictates cancer cell ferroptosis via NF2-YAP signaling |
| YAP | Transcriptional co-activator downstream of NF2 | Regulates ferroptosis sensitivity in cancer cells |
| IL-18R1 | Receptor for IL-18 | Mediates ACSL6-driven NF-kB activation and immune evasion |
| NF-kB | Transcription factor downstream of IL-18R1 | Promotes tumor progression and immune evasion |
| Integrin beta1 | Cell adhesion receptor | Activated by ACSL4-mediated phospholipid remodeling to promote metastasis |
| LDHA | Lactate dehydrogenase A | Glycolysis-derived lactate induces ACSL4 expression and lactylation |
| GPX4 | Glutathione peroxidase 4 | Lipid peroxide repair enzyme opposing ferroptosis |
| SLC7A11 | Cystine/glutamate antiporter | Supports glutathione synthesis and ferroptosis resistance |
| ACSL4 (lactylated form) | Post-translationally modified ACSL4 | Lactylation activates ferroptosis in intervertebral disc degeneration |
| ACSL4 (phosphorylated form) | PKCbetaII-phosphorylated ACSL4 | Amplifies lipid peroxidation during ferroptosis |
| CoA | Coenzyme A substrate | Required for very long-chain fatty acyl-CoA formation |
| ATP | Energy co-substrate | Required for acyl-adenylate formation |
How Is very long-chain fatty acid-CoA ligase activity Regulated?
Very long-chain fatty acid-CoA ligase activity is regulated at multiple levels. ACSL4 is phosphorylated by PKCbetaII, which amplifies lipid peroxidation and promotes ferroptosis. Glycolysis-derived lactate induces ACSL4 expression and lactylation, activating ferroptosis during intervertebral disc degeneration. Intercellular interaction through NF2-YAP signaling dictates cancer cell ferroptosis sensitivity, indirectly controlling the requirement for ACSL4 activity. ACSL6-activated IL-18R1-NF-kB signaling links very long-chain fatty acid-CoA ligase activity to inflammatory and immune-evasion programs. The broader ACSL family is subject to transcriptional, post-transcriptional, and post-translational regulation reviewed in the literature.
very long-chain fatty acid-CoA ligase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| ACSL4 | Triple-negative breast cancer metastasis | ACSL4 knockout and knock-in breast cancer cell lines |
| ACSL4 | Ferroptosis in intervertebral disc degeneration | Lactate-treated nucleus pulposus cells with ACSL4 knockout |
| ACSL4 | Ferroptosis-mediated pulmonary hypertension | ACSL4 knockout rodent models and pulmonary vascular cells |
| ACSL6 | Tumor immune evasion | ACSL6 knockout tumor cells and IL-18R1-NF-kB reporter assays |
| ACSL4 | PKCbetaII-driven lipid peroxidation | ACSL4 phospho-mutant knock-in cell lines |
Cancer metastasis and ferroptosis
ACSL4-mediated membrane phospholipid remodeling induces integrin beta1 activation and facilitates triple-negative breast cancer metastasis. ACSL4-dependent very long-chain fatty acid-CoA ligase activity is also required for ferroptosis, and modulating this activity can either sensitize or protect cancer cells from lipid peroxidation-driven death. ACSL6-activated IL-18R1-NF-kB signaling promotes IL-18-mediated tumor immune evasion and tumor progression, linking very long-chain fatty acid-CoA ligase activity to immunotherapy resistance.
Intervertebral disc degeneration
Glycolysis-derived lactate induces ACSL4 expression and lactylation to activate ferroptosis during intervertebral disc degeneration, implicating very long-chain fatty acid-CoA ligase activity in degenerative musculoskeletal disease.
Pulmonary hypertension and inflammation
Ferroptosis-mediated inflammation promotes pulmonary hypertension, and ACSL4-dependent lipid peroxidation is a key upstream event in this process. This connects GO:0031957 to vascular inflammation and cardiopulmonary disease.
Therapeutic targeting of ACSL enzymes
The ACSL family, including enzymes with very long-chain fatty acid-CoA ligase activity, represents therapeutic targets in cancer and metabolic disease, as reviewed in the literature. Selective inhibitors or degraders could modulate ferroptosis and lipid signaling for therapeutic benefit.
From very long-chain fatty acid-CoA ligase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Is ACSL4 required for ferroptosis? | ACSL4 knockout cell lines |
| Does ACSL4 phosphorylation amplify lipid peroxidation? | ACSL4 point-mutation knock-in of phospho-deficient or phospho-mimetic residues |
| Does ACSL4 lactylation drive intervertebral disc degeneration? | ACSL4 lactylation-site knock-in models |
| Does ACSL4-mediated remodeling promote metastasis? | ACSL4 overexpression and knockout in breast cancer cells |
| Does ACSL6 drive tumor immune evasion? | ACSL6 knockout and overexpression tumor models |
| Can very long-chain fatty acid-CoA ligase activity be imaged in live cells? | Tagged knock-in of ACSL4 or ACSL6 with fluorescent or proximity-labeling tags |
How to Study the very long-chain fatty acid-CoA ligase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Lipidomics (LC-MS) | Very long-chain fatty acyl-CoA and phospholipid species | Quantifying ACSL4-dependent remodeling |
| C11-BODIPY staining | Lipid peroxidation | Ferroptosis detection |
| Malondialdehyde assay | Lipid peroxidation end products | Ferroptosis quantification |
| Immunoprecipitation and kinase assay | ACSL4 phosphorylation by PKCbetaII | Regulation of ACSL4 activity |
| Lactylation immunoblot | ACSL4 lactylation | Lactate-induced ferroptosis |
| CRISPR knockout screening | Gene requirement for ferroptosis | Identifying modifiers of very long-chain fatty acid-CoA ligase activity |
| RNA-seq | Transcriptional changes after ACSL perturbation | Pathway analysis in cancer and inflammation |
| Proximity labeling | Protein interactome of ACSL enzymes | Mapping very long-chain fatty acid-CoA ligase complexes |
Lipidomics and acyl-CoA profiling
Mass spectrometry-based lipidomics and acyl-CoA profiling can quantify very long-chain fatty acyl-CoA species and their phospholipid products, directly reflecting very long-chain fatty acid-CoA ligase activity. These methods are used to compare wild-type and ACSL4-mutant cells and to link activity to ferroptosis sensitivity.
Ferroptosis assays
Ferroptosis is measured using lipid peroxidation reporters such as C11-BODIPY, malondialdehyde assays, and cell viability under GPX4 inhibition or cystine deprivation. These assays are standard for evaluating the contribution of ACSL4-dependent very long-chain fatty acid-CoA ligase activity to cell death.
Phosphorylation and lactylation analysis
Phospho-specific antibodies and lactylation-specific reagents can detect post-translational modifications of ACSL4 that regulate its activity. PKCbetaII phosphorylation of ACSL4 is analyzed by immunoprecipitation and kinase assays, while lactylation is assessed in lactate-treated cells.
CRISPR functional genomics
CRISPR knockout, point-mutation, knock-in, and overexpression models enable causal testing of ACSL family genes in ferroptosis, metastasis, and immune evasion. Pooled CRISPR screens can identify modifiers of very long-chain fatty acid-CoA ligase activity and ferroptosis.
How CRISPR Can Be Used to Study GO:0031957 very long-chain fatty acid-CoA ligase activity
Knockout
CRISPR knockout of ACSL4 or ACSL6 eliminates very long-chain fatty acid-CoA ligase activity and is used to test requirement for ferroptosis, metastasis, and immune evasion. Knockout cell lines are compared with wild-type controls in lipid peroxidation and viability assays.
Point Mutation
Point-mutation knock-in of catalytic residues or phosphorylation sites in ACSL4 can separate enzymatic activity from regulatory modifications. Phospho-deficient or phospho-mimetic ACSL4 mutants reveal how PKCbetaII phosphorylation amplifies lipid peroxidation.
Knock-in
Knock-in of epitope tags, fluorescent proteins, or lactylation-site mutations enables tracking of ACSL4 localization, interaction, and post-translational modification. Tagged knock-in models support live-cell imaging and proximity proteomics of very long-chain fatty acid-CoA ligase complexes.
Overexpression
Overexpression of ACSL4 or ACSL6 increases very long-chain fatty acid-CoA ligase activity and is used to test sufficiency for ferroptosis, metastasis, and tumor immune evasion. Overexpression models complement knockout studies to establish causality.
How EDITGENE Supports very long-chain fatty acid-CoA ligase activity Research
Researchers studying very long-chain fatty acid-CoA ligase activity-related genes often need to determine whether a candidate gene is causally involved in ferroptosis, lipid remodeling, or disease progression. EDITGENE provides CRISPR-based cell model services that enable precise perturbation of ACSL family genes and their regulators, from complete knockout to subtle point mutations and tagged knock-ins, supported by library screening and bioinformatics.
Contact EDITGENE today to design your custom CRISPR model for very long-chain fatty acid-CoA ligase activity research.
Frequently Asked Questions About very long-chain fatty acid-CoA ligase activity
What is very long-chain fatty acid-CoA ligase activity?
It is a molecular_function (GO:0031957) that catalyzes the ATP-dependent ligation of a very long-chain fatty acid (more than 22 carbons) to coenzyme A, producing very long-chain fatty acyl-CoA, AMP, and diphosphate.
What genes are involved in very long-chain fatty acid-CoA ligase activity?
ACSL4 and ACSL6 are the best-characterized genes, with additional contributions from ACSL1, ACSL3, and ACSL5 in the long-chain acyl-CoA synthetase family.
Which enzyme has very long-chain fatty acid-CoA ligase activity?
ACSL4 is a major enzyme with this activity and preferentially activates arachidonic acid and polyunsaturated fatty acids.
How is very long-chain fatty acid-CoA ligase activity related to ferroptosis?
ACSL4-dependent activation of very long-chain polyunsaturated fatty acids generates phospholipid substrates for lipid peroxidation, which drives ferroptosis.
What is the reaction catalyzed by GO:0031957?
A very long-chain fatty acid + ATP + CoA = a very long-chain fatty acyl-CoA + AMP + diphosphate.
Is ACSL4 the same as very long-chain fatty acid-CoA ligase?
ACSL4 is one enzyme that carries very long-chain fatty acid-CoA ligase activity, but the GO term describes the activity rather than a single gene product.
How does ACSL4 promote cancer metastasis?
ACSL4-mediated membrane phospholipid remodeling induces integrin beta1 activation, facilitating triple-negative breast cancer metastasis.
What regulates ACSL4 activity?
PKCbetaII phosphorylation amplifies ACSL4-mediated lipid peroxidation, and lactate-induced lactylation activates ACSL4 during intervertebral disc degeneration.
Can very long-chain fatty acid-CoA ligase activity be targeted therapeutically?
Yes, ACSL family enzymes are considered therapeutic targets in cancer and metabolic disease, and modulating their activity can influence ferroptosis and lipid signaling.
What CRISPR models are used to study very long-chain fatty acid-CoA ligase activity?
Knockout, point-mutation, knock-in, tagged knock-in, and overexpression models of ACSL4, ACSL6, and related genes are used to test causality in ferroptosis, metastasis, and immune evasion.
Conclusion
GO:0031957 very long-chain fatty acid-CoA ligase activity is a central molecular_function that activates very long-chain fatty acids for phospholipid remodeling, ferroptosis, and inflammatory signaling. ACSL4 and ACSL6 are the most studied enzymes carrying this activity, and their dysregulation contributes to cancer metastasis, intervertebral disc degeneration, pulmonary hypertension, and tumor immune evasion. CRISPR-based knockout, point-mutation, knock-in, and overexpression models, combined with lipidomics and ferroptosis assays, provide the tools needed to dissect this activity and develop targeted therapies.
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. 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
- 3. Deng X et al.. 2025. Long-chain acyl-CoA synthetases: biological functions, diseases and therapeutic targets.. Mol Biomed 6(1):117 PMID: 41288931
- 4. Quan J et al.. 2021. ACSL family: The regulatory mechanisms and therapeutic implications in cancer.. Eur J Pharmacol 909:174397 PMID: 34332918
- 5. Zhang HL et al.. 2022. PKCβII phosphorylates ACSL4 to amplify lipid peroxidation to induce ferroptosis.. Nat Cell Biol 24(1):88-98 PMID: 35027735
- 6. Kazmirczak F et al.. 2024. Ferroptosis-Mediated Inflammation Promotes Pulmonary Hypertension.. Circ Res 135(11):1067-1083 PMID: 39421926
- 7. Wu J et al.. 2019. Intercellular interaction dictates cancer cell ferroptosis via NF2-YAP signalling.. Nature 572(7769):402-406 PMID: 31341276
- 8. 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
