ACSF2 Human

What is the biochemical function of ACSF2 and how has it been characterized?

ACSF2 functions as a medium-chain acyl-CoA synthetase that preferentially activates saturated fatty acids containing 6 to 10 carbons. This has been established through overexpression studies in COS-1 cells, where the enzyme demonstrated an apparent Km of 24.4 μM for C10:0 fatty acid and Vmax of 385 nmol/20min/mg protein . To validate this function, researchers have employed RNA interference techniques showing that ACSF2 is responsible for most medium-chain ACS activity in Neuro2a cells .

Methodologically, ACSF2 activity can be assessed through:

• Radioisotope-based assays using 14C-labeled fatty acids

• HPLC detection of CoA derivatives

• Coupled enzymatic assays measuring AMP or PPi production

• Comparative analysis with known ACS family members

What are the structural characteristics of ACSF2 protein?

ACSF2 contains a conserved AMP-binding domain essential for its catalytic activity. A critical lysine residue (K599 in mouse ACSF2) has been identified as crucial for enzyme function; mutation of this residue to alanine abolishes activity completely .

Multiple splice variants have been identified across species. In geese, four alternative splice variants (ACSF2-1, ACSF2-2, ACSF2-3, and ACSF2-4) have been characterized with coding regions of 1770, 1692, 1599, and 1917 bp, encoding proteins of 589, 563, 532, and 638 amino acids respectively, all retaining the conserved AMP-binding sites .

For structural studies, researchers commonly employ:

• Site-directed mutagenesis using PCR-based overlap extension

• Sequence alignment across species

• Domain prediction software

• Homology modeling based on related ACS enzymes

How is ACSF2 expressed across different tissues and cell types?

ACSF2 shows broad tissue distribution with variable expression levels. mRNA expression has been detected in most tissues, although immunohistochemical analysis reveals differences in protein expression between various cell types within each tissue .

In heart tissue, ACSF2 is highly expressed along with other ACS genes including ACSL1, ACSL4, ACSS1, and ACSS2 . The expression pattern of ACSF2 variants can be tissue-specific; for example, in geese, the ACSF2-2 transcript was not detected in hypothalamus, pituitary gland, and granulosa cells, while other variants showed broader expression .

Methodological approaches for studying tissue distribution include:

• RT-PCR or RNA-seq for mRNA expression analysis

• Western blotting for protein quantification

• Immunohistochemistry for cellular and subcellular localization

• Comparative expression analysis across developmental stages

Where is ACSF2 localized within cells?

ACSF2 exhibits variable subcellular localization depending on the cell type. In Neuro2a and P19 cells, endogenous ACSF2 localizes to the Golgi region, and this localization is disrupted by nocodazole treatment, confirming Golgi association . In contrast, MA-10, HepG2, and skin fibroblasts show mitochondrial localization of ACSF2 . In goose cells, subcellular localization studies have identified ACSF2 as a mitochondrial matrix protein .

Computational prediction tools such as WoLF PSORT have also classified ACSF2 as mitochondrial in various species .

Methods for determining subcellular localization include:

• Immunofluorescence with organelle-specific markers

• Subcellular fractionation followed by Western blotting

• Expression of tagged fusion proteins

• Organelle disruption studies (e.g., nocodazole treatment)

What experimental approaches are optimal for modulating ACSF2 expression and activity?

Several approaches have been validated for effectively modulating ACSF2 function in experimental models:

Genetic modulation:

• RNA interference: siRNA targeting bp 8-27 of mouse ACSF2 coding sequence has proven effective for transient knockdown

• Stable knockdown: shRNA expression vectors with antibiotic selection (e.g., Hygromycin at 300 mg/ml) for generating stable cell lines

• Overexpression: Full-length cDNA clones in appropriate mammalian expression vectors

• Site-directed mutagenesis: Particularly targeting the critical lysine residue (K599) for structure-function studies

Functional assessment:

• Enzyme activity assays comparing wild-type and modified ACSF2

• Metabolic profiling using lipidomics or metabolomics

• Phenotypic analysis in relevant cell models (e.g., neurite outgrowth in Neuro2a cells)

• Isotope labeling to track metabolic flux changes

What is ACSF2's role in neuronal development and function?

ACSF2 plays a significant role in neuronal development, particularly in neurite outgrowth and potentially synapse formation. When Neuro2a cells are induced to differentiate with retinoic acid, ACSF2 migrates to nodes and points of neurite-neurite contact, co-localizing with the presynaptic marker synaptophysin .

ACSF2-deficient Neuro2a cells exhibit significantly blunted neurite outgrowth in response to retinoic acid treatment, with quantitative assessment showing reduced neurite extension . This suggests ACSF2 may be essential for proper neuronal development, possibly by providing activated fatty acids for membrane synthesis or protein modification in growing neurites.

Methodological approaches for studying ACSF2 in neuronal contexts include:

• Retinoic acid-induced differentiation of neuroblastoma cell lines

• Quantitative neurite outgrowth assessment using concentric circle analysis

• Co-localization studies with neuronal markers

• Live-cell imaging of ACSF2 trafficking during differentiation

How does ACSF2 contribute to immune function and cancer biology?

Recent research has revealed potential roles for ACSF2 in immune regulation and cancer biology. In hepatocellular carcinoma, ACSF2 expression correlates significantly with immune cell infiltration, including B cells, CD8+ T cells, and CD4+ T cells . This correlation suggests ACSF2 may influence the tumor microenvironment through metabolic pathways affecting immune cell function.

The mechanisms potentially linking ACSF2 to immune function include:

• Provision of activated fatty acids for energy metabolism in immune cells

• Influence on membrane lipid composition affecting receptor signaling

• Modulation of lipid mediator production involved in inflammation

Research approaches to explore these connections include:

• Expression correlation analysis in cancer databases

• Immune cell infiltration assessment in tissues with varying ACSF2 expression

• Co-culture systems with immune and cancer cells

• Metabolic profiling of immune cells after ACSF2 modulation

What is known about ACSF2 in reproductive biology?

ACSF2 has demonstrated interesting associations with reproductive function in animal models. In geese, a single nucleotide polymorphism (SNP) in the ACSF2 intron was identified as linked to egg-laying performance . Comparative analysis revealed that the high egg production (HEP) group showed significantly lower ACSF2 mRNA levels compared to the low egg production (LEP) group .

Further experimentation demonstrated that:

• Overexpression of ACSF2 increased caspase-3 mRNA levels in granulosa cells

• ACSF2 knockdown decreased caspase-3 mRNA levels

• ACSF2 mRNA levels positively correlated with caspase-3 mRNA levels in vivo (R = 0.86, P < 0.01)

How does ACSF2 relate to other members of the acyl-CoA synthetase family?

ACSF2 belongs to the acyl-CoA synthetase superfamily but is phylogenetically distinct from the well-characterized subfamilies. Comparative genomic analysis has classified ACSF2 as an independent member of the ACS superfamily, separate from the common ACSS, ACSM, ACSL, ACSVL, and ACSBG subfamilies .

Methodological approaches for comparative analysis include:

• Phylogenetic analysis using multiple sequence alignment

• Substrate specificity assays across ACS family members

• Domain structure and conserved motif comparisons

• Expression pattern analysis across tissues

What are the critical knowledge gaps in ACSF2 research?

Despite progress in understanding ACSF2, several significant knowledge gaps remain:

• Physiological substrates: While in vitro studies show preference for medium-chain fatty acids, the physiologically relevant substrates in different tissues remain poorly defined.

• Regulatory mechanisms: How ACSF2 expression and activity are regulated under different physiological and pathological conditions.

• Human disease associations: Limited data exists on ACSF2 mutations or expression changes in human diseases.

• Cell-type specific functions: The reason for variable subcellular localization across cell types and its functional implications.

• Interaction partners: Comprehensive characterization of proteins interacting with ACSF2 in different cellular contexts.

What emerging technologies could advance ACSF2 research?

Several emerging technologies offer promising approaches for addressing knowledge gaps:

• CRISPR-Cas9 gene editing: For generating conditional knockout models and introducing specific mutations

• Single-cell transcriptomics and proteomics: To understand cell-type specific expression patterns

• Advanced metabolomics and lipidomics: For comprehensive profiling of ACSF2-dependent metabolic changes

• Proximity labeling techniques (BioID, APEX): To identify context-specific protein interaction partners

• Cryo-EM structural studies: To determine ACSF2 protein structure at atomic resolution

• Patient-derived organoids: To study ACSF2 function in physiologically relevant human tissue models