GO:0016878 acid-thiol ligase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0016878 acid-thiol ligase activity describes enzymes that join an acid and a thiol via a carbon-sulfur bond while hydrolyzing ATP or a similar triphosphate.
• The most studied human acid-thiol ligases are the long-chain acyl-CoA synthetases (ACSL1, ACSL3, ACSL4, ACSL5, ACSL6), which activate fatty acids to acyl-CoA.
• ACSL4-dependent acyl-CoA synthesis is a key driver of ferroptosis, a regulated form of cell death implicated in cancer, neurodegeneration and metabolic disease [1,2,8].
• ACSL3 and ACSL6 promote tumor growth, metastasis and immune evasion in hepatocellular carcinoma and other cancers [5,7].
• ACSL1 translocation to mitochondria impairs adipocyte thermogenesis and energy expenditure, linking acid-thiol ligation to obesity.
• CRISPR knockout, point-mutation, knock-in and overexpression models are essential to dissect the causal roles of acid-thiol ligase genes in disease [4,7].
Description
GO:0016878 acid-thiol ligase activity is a molecular function defined as the catalysis of joining an acid and a thiol via a carbon-sulfur bond, with the concomitant hydrolysis of the diphosphate bond in ATP or a similar triphosphate. This activity is central to cellular metabolism because it activates carboxylic acids for subsequent reactions, most notably the formation of thioester bonds in coenzyme A (CoA) derivatives. In humans, the best-characterized acid-thiol ligases are the long-chain acyl-CoA synthetases (ACSLs), which convert long-chain fatty acids into acyl-CoA, a committed step in fatty acid metabolism. Because acyl-CoA products feed into lipid synthesis, beta-oxidation and signaling, dysregulated acid-thiol ligation has broad consequences for cell fate and organismal physiology [1,2,6]. The importance of acid-thiol ligase activity extends beyond basic biochemistry. ACSL4-mediated acyl-CoA synthesis is required for ferroptosis, an iron-dependent form of cell death that is being actively explored as a therapeutic strategy in cancer and degenerative diseases [1,2,8]. ACSL3 and ACSL6 support tumor growth, metastasis and immune evasion, making them attractive targets for oncology [5,7]. ACSL1 in adipocytes influences thermogenesis and energy expenditure, connecting this enzymatic activity to obesity and metabolic disorders. Consequently, researchers across cancer biology, neuroscience and metabolism need reliable tools to manipulate and measure acid-thiol ligase genes. This article provides a research-grade overview of GO:0016878, covering its definition, mechanism, key genes, disease links, and the experimental models and methods used to study it. All statements are grounded in the QuickGO definition and the verified PubMed literature cited throughout [1-8].
acid-thiol ligase activity At A Glance
| GO ID | GO:0016878 |
|---|---|
| GO term | acid-thiol ligase activity |
| Ontology | molecular_function |
| Synonym | (none) |
| Major function | Joining an acid and a thiol via a carbon-sulfur bond with concomitant hydrolysis of ATP or a similar triphosphate |
| Representative human enzymes | Long-chain acyl-CoA synthetases (ACSL1, ACSL3, ACSL4, ACSL5, ACSL6) |
| Key substrates | Long-chain fatty acids and coenzyme A (CoA) |
| Key products | Acyl-CoA thioesters, AMP, and diphosphate |
| Disease relevance | Ferroptosis, cancer, obesity, metabolic disorders [1,2,5,6,7,8] |
What Is GO:0016878?
In simple terms, acid-thiol ligase activity is the enzyme-catalyzed formation of a carbon-sulfur bond between an acid and a thiol, powered by the breakdown of ATP or a similar triphosphate. The reaction consumes a triphosphate and releases diphosphate plus the ligated product. This activity is classified under molecular_function in the Gene Ontology and is distinct from other ligases that form carbon-oxygen or carbon-nitrogen bonds. The most prominent example in human biology is the activation of a fatty acid (an acid) with coenzyme A (a thiol) to produce acyl-CoA, a high-energy thioester that drives many downstream metabolic pathways.
Why Is acid-thiol ligase activity Important in Cell Biology?
Acid-thiol ligase activity is a metabolic gatekeeper because it converts relatively inert carboxylic acids into reactive thioesters that can be used for energy production, membrane synthesis, and signaling. In humans, the ACSL family exemplifies this activity, and its members are implicated in ferroptosis, tumor progression, immune evasion, and energy balance [1,2,5,6,7,8]. Understanding GO:0016878 therefore provides mechanistic insight into how cells decide between survival, proliferation, and death, and it offers actionable targets for therapeutic intervention.
• ACSL4-dependent acyl-CoA synthesis is required for ferroptosis, a cell death modality relevant to cancer and neurodegeneration [1,2,8].
• ACSL3 silencing inhibits hepatocellular carcinoma growth and metastasis, highlighting its oncogenic role.
• ACSL6-activated IL-18R1-NF-kB signaling promotes tumor immune evasion and progression.
• ACSL1 translocation to mitochondria impairs adipocyte thermogenesis and energy expenditure in mice.
• Glycolysis-derived lactate induces ACSL4 expression and lactylation to activate ferroptosis during intervertebral disc degeneration.
• Adipocyte-derived ferroptotic signaling mitigates obesity, linking lipid metabolism to systemic energy homeostasis.
• The ACSL family is a major focus of cancer therapeutic development due to its regulatory mechanisms and druggability.
• Engineered enzyme repertoires can be designed to explore diverse acid-thiol ligase functions.
• CRISPR-based models enable causal testing of ACSL genes in disease phenotypes [4,7].
• Acid-thiol ligase activity connects fatty acid activation to ferroptosis, inflammation, and metabolic syndrome [1,2,5,6].
Molecular Mechanism of acid-thiol ligase activity
Substrate binding and adenylation
In simple terms: The enzyme first grabs the acid and ATP, then attaches AMP to the acid, making it more reactive.
Acid-thiol ligases catalyze a two-step reaction. In the first step, the carboxylic acid substrate reacts with ATP to form an acyl-adenylate intermediate, releasing pyrophosphate. This step is driven by the hydrolysis of the diphosphate bond in ATP or a similar triphosphate, as stated in the GO definition. For ACSL enzymes, the acid is a long-chain fatty acid and the intermediate is acyl-AMP. This activation step is essential because it raises the energy state of the carboxyl group for subsequent thioester formation.
Thioester bond formation
In simple terms: The activated acid then reacts with a thiol like coenzyme A to form a carbon-sulfur bond.
In the second step, the acyl-adenylate intermediate is attacked by a thiol, typically coenzyme A (CoA), to form an acyl-CoA thioester and release AMP. This creates the carbon-sulfur bond that defines acid-thiol ligase activity. The resulting acyl-CoA is a high-energy metabolite that can enter beta-oxidation, lipid synthesis, or signaling pathways. The reaction is reversible in principle but is driven forward by ATP hydrolysis and product utilization.
Cofactors and metal requirements
In simple terms: Some of these enzymes need magnesium ions to help ATP do its job.
Many acid-thiol ligases require divalent metal ions such as Mg2+ for ATP binding and catalysis, although the exact cofactor requirements vary among family members [3,4]. The hydrolysis of the diphosphate bond in ATP provides the energy for the reaction, and the enzyme couples this exergonic step to the endergonic formation of the carbon-sulfur bond. No external electron carriers are required for the ligation itself.
Regulation of enzyme activity
In simple terms: Cells control these enzymes by changing how much enzyme is made or how active it is.
Acid-thiol ligase activity is regulated at multiple levels. ACSL4 expression can be induced by glycolysis-derived lactate and is subject to lactylation, which activates ferroptosis. ACSL1 localization to mitochondria is controlled by sortilin-mediated translocation, affecting thermogenesis. Hsp90 regulates ACSL4-dependent ferroptosis via dephosphorylation of Drp1 at Ser637. These examples show that post-translational modifications and protein-protein interactions fine-tune acid-thiol ligation in response to metabolic cues [1,6,8].
Functional diversity and engineering
In simple terms: Scientists can redesign these enzymes to create new versions with different specificities.
Automated design approaches have been used to generate efficient and functionally diverse enzyme repertoires, including acid-thiol ligases. This highlights the potential to engineer variants with altered substrate specificity or improved catalytic efficiency for biotechnology and research. Such engineered enzymes can serve as tools to probe the consequences of specific ligation reactions in cells.
Key Genes Involved in GO:0016878 acid-thiol ligase activity
The following genes encode enzymes with acid-thiol ligase activity or are directly involved in its regulation, based on published literature [1-8].
| Gene | Major Role | Research Relevance |
|---|---|---|
| ACSL1 | Activates long-chain fatty acids to acyl-CoA; mitochondrial translocation impairs thermogenesis | Obesity and energy expenditure studies |
| ACSL3 | Promotes hepatocellular carcinoma growth and metastasis; silencing inhibits tumor progression | Cancer therapy target |
| ACSL4 | Essential for ferroptosis; induced by lactate and lactylation; regulated by Hsp90 | Ferroptosis and intervertebral disc degeneration [1,8] |
| ACSL5 | Member of ACSL family with roles in lipid metabolism | Cancer and metabolic research |
| ACSL6 | Activates IL-18R1-NF-kB signaling to promote immune evasion | Tumor immunology |
| ACSL family | Regulatory mechanisms and therapeutic implications in cancer | Broad cancer research |
| Drp1 | Dephosphorylated by Hsp90 to induce ACSL4-dependent ferroptosis | Ferroptosis mechanism |
| Hsp90 | Regulates ACSL4-dependent glioma ferroptosis | Glioma therapy |
| Sortilin | Mediates mitochondrial translocation of ACSL1 | Adipocyte biology |
| IL-18R1 | Activated by ACSL6 to promote NF-kB signaling | Tumor immune evasion |
| NF-kB | Downstream effector of ACSL6-IL-18R1 axis | Inflammation and cancer |
| Lactate | Induces ACSL4 expression and lactylation | Metabolic regulation of ferroptosis |
| CoA | Thiol substrate for acyl-CoA synthesis | Core metabolite in ligation |
| ATP | Provides energy via diphosphate bond hydrolysis | Cofactor for ligation |
| Acyl-CoA | Product of acid-thiol ligation | Metabolic intermediate |
| Ferroptosis | Cell death pathway dependent on ACSL4 [1,2,8] | Therapeutic target [1,2,8] |
How Is acid-thiol ligase activity Regulated?
Acid-thiol ligase activity is regulated by substrate availability, post-translational modifications, and protein-protein interactions. ACSL4 expression is induced by glycolysis-derived lactate and its lactylation activates ferroptosis. Hsp90 regulates ACSL4-dependent ferroptosis by dephosphorylating Drp1 at Ser637. Sortilin mediates mitochondrial translocation of ACSL1, which impairs adipocyte thermogenesis and energy expenditure. These mechanisms allow cells to adjust acid-thiol ligation in response to metabolic and stress signals.
acid-thiol ligase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| ACSL4 | Ferroptosis in intervertebral disc degeneration | Knockout or point-mutation cell models |
| ACSL3 | Hepatocellular carcinoma growth and metastasis | Knockout and overexpression models |
| ACSL6 | Tumor immune evasion | Knock-in and knockout models |
| ACSL1 | Obesity and impaired thermogenesis | Adipocyte-specific knockout mice |
| ACSL4 | Glioma ferroptosis | Point-mutation and knockout models |
Cancer
ACSL3 silencing inhibits hepatocellular carcinoma growth and metastasis, indicating that acid-thiol ligase activity supports tumor progression. ACSL6-activated IL-18R1-NF-kB signaling promotes IL-18-mediated tumor immune evasion and tumor progression. The ACSL family has broad regulatory mechanisms and therapeutic implications in cancer. Targeting acid-thiol ligases may therefore offer new anticancer strategies.
Ferroptosis and degenerative diseases
ACSL4 is essential for ferroptosis, an iron-dependent cell death pathway. Glycolysis-derived lactate induces ACSL4 expression and lactylation to activate ferroptosis during intervertebral disc degeneration. Hsp90 induces ACSL4-dependent glioma ferroptosis via dephosphorylating Ser637 at Drp1. Adipocyte-derived ferroptotic signaling mitigates obesity, linking ferroptosis to metabolic regulation. Modulating acid-thiol ligase activity could thus influence degenerative and metabolic diseases.
Obesity and metabolic disorders
Sortilin-mediated translocation of mitochondrial ACSL1 impairs adipocyte thermogenesis and energy expenditure in male mice. Adipocyte-derived ferroptotic signaling mitigates obesity. These findings connect acid-thiol ligase activity to energy balance and obesity, suggesting that ACSL1 and related enzymes are potential targets for metabolic intervention.
From acid-thiol ligase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of ACSL4 block ferroptosis? | ACSL4 knockout cell line |
| Does a specific phosphorylation site on Drp1 regulate ACSL4-dependent ferroptosis? | Point-mutation knock-in of Drp1 Ser637 |
| Does ACSL6-activated IL-18R1 signaling promote immune evasion? | ACSL6 overexpression and knockout models |
| Does ACSL3 silencing inhibit hepatocellular carcinoma metastasis? | ACSL3 knockout or knockdown xenograft |
| Does ACSL1 mitochondrial translocation affect thermogenesis? | Tagged knock-in of ACSL1 for localization studies |
| Can engineered acid-thiol ligases with altered specificity be created? | Overexpression of designed enzyme variants |
How to Study the acid-thiol ligase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| CRISPR knockout | Loss-of-function phenotype | Testing ACSL4 requirement for ferroptosis |
| Point mutation | Specific residue function | Drp1 Ser637 phosphorylation in ferroptosis |
| Knock-in | Tagged or mutant protein expression | ACSL1 localization studies |
| Overexpression | Gain-of-function effects | ACSL6-driven immune evasion |
| Lipidomics | Acyl-CoA and lipid species | ACSL3 silencing in HCC |
| Ferroptosis assays | Cell death and lipid peroxidation | ACSL4-dependent ferroptosis [1,2,8] |
| Co-immunoprecipitation | Protein-protein interactions | Hsp90-ACSL4 interaction |
| qPCR/Western blot | Gene and protein expression | ACSL family regulation |
Genetic manipulation
CRISPR-Cas9 knockout, point mutation, knock-in, and overexpression are used to test the causal role of acid-thiol ligase genes such as ACSL3, ACSL4, and ACSL6 [4,5,7]. These models allow researchers to observe effects on ferroptosis, tumor growth, and immune evasion [1,5,7].
Metabolic and lipid profiling
Mass spectrometry-based lipidomics and acyl-CoA measurements can quantify the products of acid-thiol ligase activity. Such methods are used to assess how ACSL enzymes alter lipid metabolism in cancer and metabolic disease [4,6].
Cell death assays
Ferroptosis is measured using viability assays, lipid peroxidation sensors, and inhibitors such as ferrostatin-1 [1,2,8]. These assays are applied to study ACSL4-dependent ferroptosis in intervertebral disc degeneration and glioma [1,8].
Protein interaction and modification studies
Co-immunoprecipitation, Western blotting, and phospho-specific antibodies are used to study ACSL4 regulation by Hsp90 and Drp1 phosphorylation, as well as ACSL1 translocation by sortilin.
How CRISPR Can Be Used to Study GO:0016878 acid-thiol ligase activity
Knockout
CRISPR knockout of ACSL4, ACSL3, or ACSL6 can abolish acid-thiol ligase activity for specific substrates, enabling researchers to test whether these enzymes are required for ferroptosis, tumor growth, or immune evasion [1,5,7]. Knockout models are foundational for causal inference in cancer and metabolic studies.
Point Mutation
Point mutations can be introduced to disrupt catalytic residues or regulatory phosphorylation sites. For example, mutating Drp1 Ser637 prevents its dephosphorylation by Hsp90, thereby affecting ACSL4-dependent ferroptosis. Such models help dissect mechanism from mere association.
Knock-in
Knock-in of tagged or mutant alleles allows visualization and functional analysis of acid-thiol ligases in their native context. Tagged ACSL1 knock-in can reveal its mitochondrial translocation dynamics in adipocytes. Knock-in of disease-associated variants can model human mutations.
Overexpression
Overexpression of ACSL6 or ACSL4 can drive tumor immune evasion or ferroptosis, respectively, providing gain-of-function evidence [1,5]. Overexpression models are also used to study engineered acid-thiol ligase variants with altered specificity.
How EDITGENE Supports acid-thiol ligase activity Research
Researchers studying acid-thiol ligase activity-related genes often need to determine whether a candidate gene is causally involved in a disease phenotype, such as ferroptosis, tumor growth, or metabolic dysfunction. EDITGENE provides the full suite of CRISPR services to generate precisely engineered cell models for these investigations.
Contact EDITGENE today to design your custom CRISPR model for acid-thiol ligase activity research.
Frequently Asked Questions About acid-thiol ligase activity
What is acid-thiol ligase activity?
Acid-thiol ligase activity (GO:0016878) is the catalysis of joining an acid and a thiol via a carbon-sulfur bond, with the concomitant hydrolysis of the diphosphate bond in ATP or a similar triphosphate.
What genes are involved in acid-thiol ligase activity?
The main human genes are the long-chain acyl-CoA synthetases: ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6.
What is the role of ACSL4 in ferroptosis?
ACSL4 is essential for ferroptosis; its expression is induced by lactate and lactylation, and it is regulated by Hsp90 via Drp1 dephosphorylation [1,8].
How is acid-thiol ligase activity regulated?
It is regulated by substrate availability, post-translational modifications such as lactylation, and protein interactions like sortilin-mediated ACSL1 translocation [1,6,8].
What diseases are linked to acid-thiol ligase activity?
It is linked to cancer, ferroptosis-related degeneration, obesity, and metabolic disorders [1,2,5,6,7,8].
What is the difference between ACSL3 and ACSL4?
ACSL3 promotes hepatocellular carcinoma growth and metastasis, while ACSL4 is a key driver of ferroptosis [1,7].
Can CRISPR be used to study acid-thiol ligase genes?
Yes, CRISPR knockout, point mutation, knock-in, and overexpression models are widely used to dissect ACSL gene functions [4,5,7].
What methods measure acid-thiol ligase activity?
Lipidomics, acyl-CoA quantification, ferroptosis assays, and protein interaction studies are commonly used [1,4,8].
What is the GO definition of acid-thiol ligase activity?
The Gene Ontology defines it as catalysis of the joining of an acid and a thiol via a carbon-sulfur bond, with concomitant hydrolysis of the diphosphate bond in ATP or a similar triphosphate.
How does ACSL6 contribute to tumor immune evasion?
ACSL6 activates IL-18R1-NF-kB signaling, which promotes IL-18-mediated tumor immune evasion and progression.
Conclusion
GO:0016878 acid-thiol ligase activity is a fundamental molecular function that activates carboxylic acids for thioester formation, with the ACSL family as its principal human representatives [3,4]. This activity is central to ferroptosis, cancer progression, immune evasion, and metabolic regulation [1,2,5,6,7,8]. Understanding its mechanism and regulation offers opportunities for therapeutic intervention. CRISPR-based models and EDITGENE services provide the tools needed to causally test these pathways and accelerate translation.
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. Wang X et al.. 2025. Adipocyte-derived ferroptotic signaling mitigates obesity.. Cell Metab 37(3):673-691.e7 PMID: 39729998
- 3. Khersonsky O et al.. 2018. Automated Design of Efficient and Functionally Diverse Enzyme Repertoires.. Mol Cell 72(1):178-186.e5 PMID: 30270109
- 4. Quan J et al.. 2021. ACSL family: The regulatory mechanisms and therapeutic implications in cancer.. Eur J Pharmacol 909:174397 PMID: 34332918
- 5. 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
- 6. Yang M et al.. 2024. Sortilin-mediated translocation of mitochondrial ACSL1 impairs adipocyte thermogenesis and energy expenditure in male mice.. Nat Commun 15(1):7746 PMID: 39232011
- 7. Huang L et al.. 2025. Modulating lipid metabolism by nanoparticles (NPs)-mediated ACSL3 silencing to inhibit hepatocellular carcinoma growth and metastasis.. Mol Cancer 24(1):73 PMID: 40059153
- 8. Miao Z et al.. 2022. Hsp90 induces Acsl4-dependent glioma ferroptosis via dephosphorylating Ser637 at Drp1.. Cell Death Dis 13(6):548 PMID: 35697672
