GO:0004467 long-chain fatty acid-CoA ligase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0004467 describes the enzymatic activity that attaches coenzyme A to long-chain fatty acids (13-22 carbons) in an ATP-dependent two-step reaction, producing a long-chain fatty acyl-CoA.
• The reaction is catalyzed by long-chain acyl-CoA synthetases (ACSLs), a family of five enzymes (ACSL1, ACSL3, ACSL4, ACSL5, ACSL6) that channel fatty acids into beta-oxidation, lipid synthesis, and membrane remodeling.
• ACSL4 is the most studied member in disease; its activity promotes ferroptosis by incorporating polyunsaturated fatty acids into phospholipids, and it is a key driver of cancer metastasis and inflammation.
• Dysregulated long-chain fatty acid-CoA ligase activity is implicated in triple-negative breast cancer, pulmonary hypertension, intervertebral disc degeneration, and metabolic disorders.
• Research tools include CRISPR knockout, point-mutation knock-in, overexpression models, and lipidomics to dissect ACSL isoform-specific functions.
• Targeting ACSL enzymes is a promising therapeutic strategy for ferroptosis-related diseases and cancers.
Description
Long-chain fatty acid-CoA ligase activity (GO:0004467) is a fundamental enzymatic function that activates fatty acids for diverse metabolic fates. This activity, catalyzed by the acyl-CoA synthetase long-chain (ACSL) family, converts long-chain fatty acids (13-22 carbons) into their corresponding acyl-CoA thioesters, a prerequisite for both energy production and lipid biosynthesis. The reaction consumes ATP and CoA, yielding AMP and diphosphate, and is essential for maintaining cellular lipid homeostasis. Researchers study this activity because it sits at the crossroads of fatty acid oxidation, membrane phospholipid remodeling, and signaling, with profound implications for cancer, ferroptosis, and metabolic diseases. Understanding the molecular details of GO:0004467 provides a foundation for developing targeted therapies and for interpreting lipidomic and metabolic data.
long-chain fatty acid-CoA ligase activity At A Glance
| GO ID | GO:0004467 |
|---|---|
| GO term | long-chain fatty acid-CoA ligase activity |
| Ontology | molecular_function |
| Synonym | acyl-CoA synthetase activity; long-chain acyl-CoA synthetase activity; LCFA synthetase activity; fatty acid thiokinase (long-chain) activity; lignoceroyl-CoA synthase activity; stearoyl-CoA synthetase; pristanoyl-CoA synthetase |
| Major function | ATP-dependent activation of long-chain fatty acids (13-22 carbons) to fatty acyl-CoA thioesters |
| Reaction | a long-chain fatty acid + ATP + CoA = a long-chain fatty acyl-CoA + AMP + diphosphate |
| Enzyme family | ACSL family (ACSL1, ACSL3, ACSL4, ACSL5, ACSL6) |
| Cellular roles | Fatty acid beta-oxidation, phospholipid remodeling, lipid signaling, ferroptosis regulation |
| Disease relevance | Cancer, ferroptosis, pulmonary hypertension, intervertebral disc degeneration, metabolic disorders |
What Is GO:0004467?
GO:0004467, long-chain fatty acid-CoA ligase activity, is defined as the catalysis of the reaction: a long-chain fatty acid + ATP + CoA = a long-chain fatty acyl-CoA + AMP + diphosphate. A long-chain fatty acid has an aliphatic tail containing 13 to 22 carbons. This activity is also known as acyl-CoA synthetase, acyl-activating enzyme, or LCFA synthetase, and it performs the first step in fatty acid utilization by forming a thioester bond between the fatty acid carboxyl group and coenzyme A.
Why Is long-chain fatty acid-CoA ligase activity Important in Cell Biology?
Long-chain fatty acid-CoA ligase activity is a metabolic gatekeeper that determines the fate of fatty acids, directing them toward oxidation for energy or incorporation into complex lipids. Its dysregulation is increasingly linked to major human diseases, including cancer, where ACSL4 promotes metastasis and ferroptosis sensitivity, and pulmonary hypertension, where ferroptosis-mediated inflammation drives pathology. Because this activity controls lipid peroxidation and membrane dynamics, it is a prime target for therapeutic intervention in oncology and inflammatory diseases.
• Essential for fatty acid beta-oxidation and energy production.
• Required for phospholipid remodeling and membrane composition.
• Regulates ferroptosis sensitivity by controlling PUFA incorporation into membranes.
• Drives cancer metastasis in triple-negative breast cancer via integrin β1 activation.
• Promotes tumor progression through epigenetic regulation of SNAIL.
• Implicated in pulmonary hypertension through ferroptosis-mediated inflammation.
• Linked to intervertebral disc degeneration via lactate-induced ACSL4 expression.
• Provides a target for therapeutic modulation in metabolic and neoplastic diseases.
• Serves as a biomarker for lipid metabolism reprogramming in cancer.
• Enables CRISPR-based functional genomics to dissect isoform-specific roles.
What Happens During long-chain fatty acid-CoA ligase activity?
Substrate binding and activation
In simple terms: The enzyme grabs a long fatty acid and uses ATP to prime it for reaction.
The reaction begins with the binding of a long-chain fatty acid (13-22 carbons) and ATP to the enzyme's active site. The enzyme catalyzes the formation of an acyl-AMP intermediate, releasing pyrophosphate. This step activates the fatty acid's carboxyl group for nucleophilic attack by coenzyme A.
Acyl-CoA thioester formation
In simple terms: Coenzyme A attaches to the activated fatty acid, forming a fatty acyl-CoA.
In the second step, coenzyme A attacks the acyl-AMP intermediate, displacing AMP and forming a thioester bond between the fatty acid and CoA. The products are long-chain fatty acyl-CoA, AMP, and diphosphate. This thioester is a high-energy compound that can be used in various metabolic pathways.
Channeling into metabolic pathways
In simple terms: The fatty acyl-CoA is sent to different cellular processes, like burning fat or building membranes.
Once formed, long-chain fatty acyl-CoAs are directed to beta-oxidation in mitochondria or peroxisomes for energy production, or to glycerolipid and phospholipid synthesis in the endoplasmic reticulum. ACSL isoforms exhibit distinct subcellular localizations that influence these fates.
Role in ferroptosis and lipid peroxidation
In simple terms: Certain ACSL enzymes, especially ACSL4, make membranes more susceptible to oxidative damage, leading to a form of cell death called ferroptosis.
ACSL4 preferentially activates polyunsaturated fatty acids (PUFAs), which are then incorporated into phospholipids. These PUFA-containing phospholipids are prone to peroxidation, and when peroxidation exceeds cellular antioxidant capacity, ferroptosis is triggered. This process is regulated by phosphorylation of ACSL4 by PKCβII, which amplifies lipid peroxidation. ACSL4-mediated ferroptosis is also modulated by intercellular interactions via NF2-YAP signaling.
Key Genes Involved in GO:0004467 long-chain fatty acid-CoA ligase activity
The following genes encode enzymes with long-chain fatty acid-CoA ligase activity or directly regulate this activity.
| Gene | Major Role | Research Relevance |
|---|---|---|
| ACSL1 | Activates long-chain fatty acids for beta-oxidation and lipid synthesis | Metabolic disorders, fatty liver disease, ferroptosis |
| ACSL3 | Activates fatty acids for phospholipid synthesis | Cancer, lipid droplet formation, ferroptosis |
| ACSL4 | Activates PUFAs, promotes ferroptosis and metastasis | Triple-negative breast cancer, ferroptosis, pulmonary hypertension |
| ACSL5 | Activates fatty acids in intestine and liver | Metabolic syndrome, cancer |
| ACSL6 | Activates fatty acids in brain and muscle | Neurological disorders, lipid metabolism |
| PKCβII | Phosphorylates ACSL4 to enhance lipid peroxidation | Ferroptosis regulation |
| NF2 | Regulates ferroptosis via YAP signaling | Cancer, ferroptosis |
| YAP | Transcription factor downstream of NF2 | Cancer, ferroptosis |
| SNAIL | Upregulated by ACSL4-mediated histone acetylation | TNBC metastasis |
| Integrin β1 | Activated by ACSL4-mediated membrane remodeling | TNBC metastasis |
| Lactate | Induces ACSL4 expression and lactylation | Intervertebral disc degeneration |
| ACSL4 (lactylation) | Post-translational modification enhancing ACSL4 activity | Ferroptosis in disc degeneration |
How Is long-chain fatty acid-CoA ligase activity Regulated?
Long-chain fatty acid-CoA ligase activity is regulated at multiple levels. ACSL4 is phosphorylated by PKCβII at Thr679, which enhances its enzymatic activity and promotes lipid peroxidation. In intervertebral disc degeneration, glycolysis-derived lactate induces ACSL4 expression and lactylation, activating ferroptosis. Additionally, intercellular interactions via NF2-YAP signaling modulate ferroptosis sensitivity by affecting ACSL4 function. These regulatory mechanisms highlight the integration of metabolic and signaling cues in controlling this activity.
long-chain fatty acid-CoA ligase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| ACSL4 | Triple-negative breast cancer metastasis | CRISPR knockout 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 |
| ACSL4 | Ferroptosis | Point mutation of phosphorylation site (T679A) |
| ACSL3 | Cancer lipid metabolism | Overexpression in cancer cell lines |
Cancer and metastasis
ACSL4-mediated membrane phospholipid remodeling activates integrin β1, facilitating triple-negative breast cancer metastasis. ACSL4 also promotes H3K9 and H3K27 hyperacetylation, upregulating SNAIL to drive metastasis. The ACSL family is broadly implicated in cancer progression, making it a therapeutic target.
Ferroptosis-related diseases
ACSL4 is a key driver of ferroptosis by incorporating PUFAs into phospholipids. PKCβII phosphorylation of ACSL4 amplifies lipid peroxidation, inducing ferroptosis. Ferroptosis-mediated inflammation promotes pulmonary hypertension, where ACSL4 activity contributes to disease pathology. In intervertebral disc degeneration, lactate-induced ACSL4 expression triggers ferroptosis.
Metabolic disorders
Dysregulation of long-chain fatty acid-CoA ligase activity is linked to metabolic syndrome, fatty liver disease, and obesity. ACSL isoforms control fatty acid partitioning between oxidation and storage, influencing insulin sensitivity and lipid profiles.
From long-chain fatty acid-CoA ligase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does ACSL4 loss reduce ferroptosis? | ACSL4 knockout cell lines (e.g., HT-1080) |
| Does ACSL4 phosphorylation enhance lipid peroxidation? | ACSL4 T679A point-mutation knock-in |
| Does ACSL4 overexpression promote metastasis? | ACSL4 overexpression in TNBC cells |
| Does ACSL4 lactylation affect its activity? | Knock-in of lactylation-deficient mutant |
| Does ACSL4 interact with integrin β1? | Tagged knock-in (e.g., HA-ACSL4) for co-IP |
| Does ACSL4 regulate SNAIL via histone acetylation? | ACSL4 knockout with H3K9ac/H3K27ac ChIP |
How to Study the long-chain fatty acid-CoA ligase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Lipidomics | Acyl-CoA and phospholipid species | Assessing ACSL4-mediated PUFA incorporation |
| CRISPR knockout screening | Gene essentiality and ferroptosis regulators | Identifying ACSL4 as a ferroptosis driver |
| Phosphoproteomics | Phosphorylation sites on ACSL enzymes | Mapping PKCβII-ACSL4 signaling |
| Co-immunoprecipitation | Protein-protein interactions | Detecting ACSL4-integrin β1 interaction |
| ChIP-seq | Histone modifications and transcription factor binding | Linking ACSL4 to SNAIL upregulation |
| RNA-seq | Transcriptional changes upon ACSL modulation | Identifying downstream pathways |
| Metabolic flux analysis | Fatty acid oxidation and synthesis rates | Measuring ACSL1-dependent beta-oxidation |
Lipidomics and metabolomics
Mass spectrometry-based lipidomics quantifies acyl-CoA species and phospholipid composition, revealing the impact of ACSL activity on cellular lipidomes.
CRISPR screening
Genome-wide CRISPR knockout screens identify genes that modulate ferroptosis sensitivity, including ACSL4 and related metabolic enzymes.
Phosphoproteomics
Phosphoproteomics can identify phosphorylation sites on ACSL enzymes, such as PKCβII-mediated ACSL4 phosphorylation, linking signaling to activity.
Imaging and subcellular localization
Fluorescence microscopy of tagged ACSL isoforms reveals their distinct subcellular localizations, which dictate fatty acid channeling.
How CRISPR Can Be Used to Study GO:0004467 long-chain fatty acid-CoA ligase activity
Knockout
CRISPR knockout of ACSL4 in cancer cell lines abolishes ferroptosis sensitivity and reduces metastasis, validating its role in these processes. Knockout models are essential for distinguishing isoform-specific functions.
Point Mutation
Point mutations, such as ACSL4 T679A, prevent phosphorylation and impair lipid peroxidation, allowing dissection of post-translational regulation. Lactylation site mutations can similarly test the role of metabolic modifications.
Knock-in
Knock-in of tagged ACSL4 (e.g., HA or GFP) enables localization and interaction studies. Knock-in of disease-associated variants can model human mutations affecting enzyme activity.
Overexpression
Overexpression of ACSL4 in TNBC cells promotes metastasis and integrin β1 activation, providing gain-of-function evidence. Overexpression models are useful for testing therapeutic inhibitors.
How EDITGENE Supports long-chain fatty acid-CoA ligase activity Research
Researchers studying long-chain fatty acid-CoA ligase activity-related genes often need to determine whether a candidate gene is causally involved in ferroptosis, cancer metastasis, or metabolic reprogramming. EDITGENE provides comprehensive CRISPR-based services to generate precisely engineered cell models, enabling functional validation and mechanistic studies.
Contact EDITGENE today to design your custom CRISPR model for long-chain fatty acid-CoA ligase activity research.
Frequently Asked Questions About long-chain fatty acid-CoA ligase activity
What is long-chain fatty acid-CoA ligase activity?
It is the enzymatic activity (GO:0004467) that activates long-chain fatty acids (13-22 carbons) by attaching coenzyme A, forming fatty acyl-CoA, in an ATP-dependent reaction.
What genes are involved in long-chain fatty acid-CoA ligase activity?
The ACSL family genes: ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6, each with distinct substrate preferences and tissue distributions.
How is long-chain fatty acid-CoA ligase activity regulated?
It is regulated by phosphorylation (e.g., PKCβII phosphorylates ACSL4), lactylation, and signaling pathways such as NF2-YAP.
What diseases are associated with ACSL4?
ACSL4 is linked to triple-negative breast cancer metastasis, ferroptosis-related diseases like pulmonary hypertension, and intervertebral disc degeneration.
What is the role of ACSL4 in ferroptosis?
ACSL4 incorporates polyunsaturated fatty acids into phospholipids, making membranes susceptible to lipid peroxidation and ferroptosis.
How can I study long-chain fatty acid-CoA ligase activity in the lab?
Use CRISPR knockout, point mutation, overexpression models, lipidomics, and CRISPR screens to dissect its functions.
What are the substrates of long-chain fatty acid-CoA ligase?
Long-chain fatty acids with 13-22 carbons, ATP, and coenzyme A.
What is the reaction catalyzed by GO:0004467?
A long-chain fatty acid + ATP + CoA = a long-chain fatty acyl-CoA + AMP + diphosphate.
Which ACSL isoform is most studied in cancer?
ACSL4 is heavily studied in cancer, particularly in triple-negative breast cancer metastasis and ferroptosis.
Can CRISPR be used to target ACSL genes?
Yes, CRISPR knockout, knock-in, and point mutation models are widely used to study ACSL gene functions.
Conclusion
Long-chain fatty acid-CoA ligase activity (GO:0004467) is a central metabolic function that governs fatty acid fate and cellular lipid homeostasis. Its dysregulation contributes to cancer, ferroptosis, and inflammatory diseases, making it a compelling therapeutic target. Continued research using advanced CRISPR models and multi-omics approaches will further elucidate its mechanistic roles and translational potential.
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. Sinha A et al.. 2024. ACSL4-mediated H3K9 and H3K27 hyperacetylation upregulates SNAIL to drive TNBC metastasis.. Proc Natl Acad Sci U S A 121(52):e2408049121 PMID: 39700137
- 8. Wu J et al.. 2019. Intercellular interaction dictates cancer cell ferroptosis via NF2-YAP signalling.. Nature 572(7769):402-406 PMID: 31341276
