The acyl-CoA synthetases (ACS) are enzymes that catalyze the production of acyl-CoA from fatty acids. The length of the carbon chain of the fatty acid species defines the substrate specificity for the different ACS.
The presence of an ACS specific for arachidonate had been shown by enzymatic characterizations in 1985 (Laposata et al. 1985), and later the primary structure, enzymatic properties, and tissue expression of this newly identified ACS enzyme, designated ACS4 (later named ACSL4), were described by Kang et al. (1997). It belongs to the large family of mammalian long-chain acyl-CoA synthetases (ACSL), which activate fatty acids with chain lengths of 12–20 carbon atoms. The human and mouse genes for the ACSLs are termed ACSL1,3-6 and Acsl1,3-6, respectively. Each isoform has a substrate preference, subcellular localization, and tissue distribution and has been suggested to participate in the modulation of various pathophysiological events. Among the proteins in the family, ACSL4 has a strong preference for polyunsaturated fatty acids, in particular arachidonic acid (AA) (Soupene and Kuypers 2008).
Structure and Activity
ACSL4 catalyzes the formation of fatty acyl-CoA in a two-step reaction: the formation of a fatty acyl-AMP molecule as an intermediate and the formation of a fatty acyl-CoA.
In the first step, an acyl-AMP intermediate is formed from ATP. AMP is then exchanged with CoA to produce the activated acyl-CoA. The release of AMP in this reaction defines the superfamily of AMP-forming enzymes. This is a required step before free fatty acids can participate in most catabolic and anabolic reactions (Soupene and Kuypers 2008).
In humans, ACSL4 gene is localized in chromosome Xq22.3-q23, spans approximately 90 kb, and consists of 16 exons. One, two, and three ACSL4 spliced isoforms have been identified in rats, humans, and mice, respectively. Mouse variant 1 corresponds to human variant 2 and mouse variant 3 corresponds to human variant 1. Mouse variant 2 corresponds to the single variant identified in rat. Mouse variants 2 and 3 encode the same short isoform with only a difference of three bases at a splice site in the first encoding exon. This exon contains two in-frame AUGs. AUG1 is the initiator codon for a long isoform 1 and can be removed by using an alternatively spliced acceptor site present in this exon between the two AUGs. The downstream in-frame AUG2 is used for a shorter isoform 2. The three nucleotides, AAG, missing in variant 3, indicate the presence of a second alternatively spliced acceptor site, AAGAAG/AAA, which is located three nucleotides downstream of the site used in variant 2: AAG/AAGAAA. The alternative splicing event of mouse variant 3 is identical to the one occurring for human variant 1, whereas the splicing site of mouse variant 2 is identical to the one producing the rat variant. This suggests that the equivalent of the three mouse variants might also exist in human and rat and have yet to be identified (Soupene and Kuypers 2008).
The long isoform of ACSL4 is predicted to carry a leader peptide. The long isoform of ACSL4 has been proposed to be a brain-specific form, and the leader sequence has been speculated to target this form specifically to this tissue. However, there are inconsistencies in the identification of the different spliced isoforms and their cellular localization.
The mammalian structure of this protein has not been solved but homology to a bacterial form, whose structure has been determined, points at specific structural features that are important for these enzymes across species. The bacterial form acts as a dimer and has a conserved short motif, called the fatty acid gate domain, that seems to determine substrate specificity (Soupene and Kuypers 2008).
Cell Localization and Tissue Distribution
ACSL4 is a peripheral membrane protein, located mainly on the mitochondrial-associated membrane fraction (MAM), on peroxisomal membrane, and on microsomes (Lewin et al. 2002). In relation to the tissue distribution of ACSL4, its mRNA is present in several tissues, such as the adrenal gland, epididymis, brain, lung, ovary, placenta, liver, and testis. It is notable for its abundance in steroidogenic cells, especially in cells of fasciculata and reticularis zones of the adrenal gland, Leydig cells of testis, and luteal cells of the ovary (Kang et al. 1997). ACSL4 is expressed in the brain in the early stages of development with a significant amount of mRNA detected in embryos at stage E7 (Cao et al. 2000). In addition, ACSL4 is expressed in adult brain and newborn mouse brain especially in dentular gyrus granule cells, pyramidal neurons of the hippocampal layer CA1, and the granular cell layer and Purkinje cells of the cerebellum (Cao et al. 2000). While there is relatively little or no expression of ACSL4 in other adult tissues, on the contrary it is overexpressed in tumor tissue and is not expressed in adjacent normal tissue in samples of breast, prostate, colon, and liver tumors (Cao et al. 2001; Sung et al. 2003; Monaco et al. 2010; Orlando et al. 2015; Wu et al. 2015).
ACSL4 Relevance in Physiological Processes
This mechanism could then regulate biological processes that occur in cells, the transport of lipids across membranes, and the addressing of the AA to a specific compartment of the cell. Lipoxygenase pathway is located in the membranes of the endoplasmic reticulum and nucleus and is also associated with mitochondria. Contacts between mitochondria and the endoplasmic reticulum play an important role in cell metabolism; for example, they ensure a direct transmission of calcium from the endoplasmic reticulum mitochondria. This could also be the case to ensure a direct transmission of the AA to the site of action of lipoxygenase and may be an explanation of how cell can direct the AA via the cyclooxygenase or lipoxygenase pathway.
Regarding the function of ACSL4 regulating AA metabolism in other cell types, sustained downregulation of ACSL4 results in markedly reduced PGE2 release in human arterial smooth muscular cells, indicating that ACSL4 plays an important role as a regulator of eicosanoid synthesis and secretion, which might regulate smooth muscle cell proliferation, release of inflammatory mediators, or other processes in the vascular wall (Golej et al. 2011).
Efficient membrane fusion has been successfully mimicked in vitro using artificial membranes and a number of cellular proteins that are currently known to participate in membrane fusion. However, these proteins are not sufficient to promote efficient fusion between biological membranes, indicating that critical fusogenic factors remain unidentified. It was identified a TIP30 protein complex containing TIP30, ACSL4, and endophilin B1 (Endo B1) that promotes the fusion of endocytic vesicles with Rab5a vesicles, which transport endosomal acidification enzymes vacuolar (H+)-ATPases (V-ATPases) to the early endosomes in vivo. TIP30 protein complex facilitates the fusion of endocytic vesicles with Rab5a vesicles in vitro. Fusion of the two vesicles also depends on AA, coenzyme A, and the synthesis of arachidonyl-CoA by ACSL4. Moreover, the TIP30 complex is able to transfer arachidonyl groups onto phosphatidic acid (PA), producing a new lipid species that is capable of inducing close contact between membranes. Together, it was suggested that the TIP30 complex facilitates biological membrane fusion through modification of PA on membrane (Zhang et al. 2011).
Long-chain polyunsaturated fatty acids and their metabolites play an important role in embryonic development and cell biology. It showed that Acsl4a, the homologous enzyme to ACSL4 in zebra fish, is essential for the appropriate development in dorsoventral pattern. Acsl4a loss affects the BMP (bone morphogenetic protein) signaling pathway (Miyares et al. 2013). Acsl4a modulates the activity of Smad transcription factors, mediators downstream of BMP. Acsl4a promotes the inhibition of p38 (mitogen-activated protein kinase) and the inhibition mediated by Akt of glycogen synthase kinase 3 inhibitors critics of Smad activity. These results reveal a critical role for Acsl4a in the modulation of BMP-Smad activity and provide a pathway for potential of unsaturated long-chain fatty acids to influence a variety of processes of development (Miyares et al. 2013).
Studies using Drosophila melanogaster as model were performed in order to study ACSL4 functions on development and on nervous system. dAcsl enzyme is highly homologous to human ACSL3 and ACSL4. It was shown that dAcsl and ACSL4 are highly conserved in function since ACSL4 can replace the functions of dAcsl in terms of viability in the lethal mutant, lipid storage, and synaptic connections of the visual center. In development, Dpp (decapentaplegic, a protein similar to BMP) production decreases specifically in the brain of the mutant dAcsl larvae. This results in a decrease of glial cells and neurons and axonal alterations in the visual cortex. All of these effects are reversed by ACSL4 enzyme (Zhang et al. 2009; Liu et al. 2011). ACSL4 participates in BMP signal in the development of synapses and regulates axonal transport of synaptic vesicles that are necessary for the synaptic development and transmission (Zhang et al. 2009). Moreover, the knockdown of ACSL4 in neurites reduces dendritic spine formation, suggesting a role for ACSL4 in the maturation and remodeling of neurons and providing a link to the X-linked mental retardation associated with human mutations in ACSL4 gene (Meloni et al. 2009).
Recently it was described that ACSL4 is involved in a new recognized form of regulated cell death. Ferroptosis is a form of regulated necrotic cell death controlled by glutathione peroxidase 4 (GPX4). It is characterized morphologically by the presence of smaller than normal mitochondria with condensed mitochondrial membrane densities, reduction or vanishing of mitochondria cristae, and outer mitochondrial membrane rupture. It can be induced by experimental compounds (e.g., erastin, Ras-selective lethal small molecule 3, and buthionine sulfoximine) or clinical drugs (e.g., sulfasalazine, sorafenib, and artesunate) in cancer cells and certain normal cells (e.g., kidney tubule cells, neurons, fibroblasts, and T cells). Misregulated ferroptosis has been implicated in multiple physiological and pathological processes, including cancer cell death, neurotoxicity, neurodegenerative diseases, acute renal failure, drug-induced hepatotoxicity, hepatic and heart ischemia/reperfusion injury, and T cell (Yang and Stockwell 2016). Two independent approaches, a genome-wide CRISPR-based genetic screen and microarray analysis of ferroptosis-resistant cell lines, uncover ACSL4 as an essential component for ferroptosis execution. Mechanistically, ACSL4 enriched cellular membranes with long polyunsaturated ω6 fatty acids. Pharmacological targeting of ACSL4 ameliorated tissue demise in a mouse model of ferroptosis, suggesting that ACSL4 inhibition is a viable therapeutic approach to preventing ferroptosis-related diseases (Doll et al. 2016).
Regulation of ACSL4 Expression
ACSL4 expression levels are regulated by hormone action in steroidogenic cells. It has been demonstrated that the hormones ACTH, LH, angiotensin, and epidermal growth factor (EGF) induce the expression of the mRNA as ACSL4 protein. The role of tyrosine phosphatases in the mechanism of hormonal regulation of the synthesis of steroid hormones has been demonstrated (Castillo et al. 2008). Inhibitors of tyrosine phosphatases inhibit the StAR protein induction and the synthesis of hormones steroids (Paz et al. 2016). One of the tyrosine phosphatases involved is the enzyme SHP2 whose activation regulates the induction of ACSL4 (Cooke et al. 2010).
At transcriptional level, murine ACSL4 promoter is regulated by LH and cAMP action and through transcription factors SP1 and CREB (Orlando et al. 2013). It was reported as the regulation of ACSL4 by epigenetic mechanism. Different micro-RNAs have recently been demonstrated to regulate the expression of ACSL4. For example, mir34a has been shown to regulate, among other genes, the expression of ACSL4 in colon cancer cells. Loss of p53 function produces inhibition of the expression of mir34a which leads to genes regulated by this miRNA increasing their expression levels (Kaller et al. 2011). Studying the mechanism of hepatitis B virus in liver cells demonstrated the regulation and role of ACSL4 in this mechanism through the regulation of the enzyme by the mir-205 (Cui et al. 2014).
Also, posttranslation modification such as ubiquitination and phosphorylation could be implied in the expression regulation of ACSL4. For example, recent work demonstrated in liver cells that AA regulates the stability of ACSL4 protein by an ubiquitination mechanism (Kan et al. 2014).
ACSL4 Involvement in Pathological Processes
ACSL4 was found to associate with different pathologies. Both point mutations and deletions have been reported in the gene. Related to ACSL4 role in the nervous system, mutations have been associated with severe non-syndromic X-linked intellectual disability (Meloni et al. 2002). Affected males show nonspecific, nonprogressive intellectual disability, ranging from severe to moderate without seizures, whereas carrier females showed highly variable cognitive capacities, ranging from moderate intellectual disability to normal intelligence. ACSL4 deletions have also been associated in patients with Alport syndrome, elliptocytosis, and intellectual disability. Reduction of ACSL4 activity may lead to deranged fatty acid metabolism in neurons, causing defects of neuron outgrowth, synaptogenesis, and other developmental functions important for normal brain development.
The expression of ACSL4 has been shown to be associated with aggressiveness of several types of cancer such as colon, prostate, breast, and hepatocellular carcinoma (Cao et al. 2001; Sung et al. 2003; Monaco et al. 2010; Orlando et al. 2015; Wu et al. 2015). Particularly, it was reported that this enzyme is overexpressed in estrogen receptor (ER)-negative, androgen receptor (AR)-negative breast tumors and cell lines, and in AR-negative prostate tumors and cell lines (Maloberti et al. 2010; Monaco et al. 2010).
Functionally, ACSL4 is part of the mechanism responsible for increased breast cancer cell proliferation, invasion, and migration, demonstrated both in vitro and in vivo (Maloberti et al. 2010; Orlando et al. 2015). The sole transfection of MCF-7 cells, a model of nonaggressive ER-positive breast cancer cells, with ACSL4 cDNA, transforms them into a highly aggressive phenotype, and their injection into nude mice has resulted in the development of growing tumors with marked nuclear polymorphism, a high mitotic index, and low expression of ER (Orlando et al. 2012). In addition, targeting ACSL4 in cells and in tumors has indeed proven to reverse the loss of ER expression (Orlando et al. 2012, 2015). These results are in agreement with the negative correlation between ACSL4 and ER expression in human breast tumor samples (Monaco et al. 2010).
In addition, a synergistic effect in the inhibition of cell growth was shown by a combination of ACSL4 and ER inhibitors. The combination was effective in inhibiting cell proliferation and tumor growth in a very aggressive triple negative breast cancer cell line, MDA-MB-231, which does not express ER and overexpresses ACSL4. These results suggest that ACSL4, in combination with ER inhibitors, could be an interesting target to be used in combination with other inhibitors and which might prevent the side effects of supramaximal doses and generate more positive effects than single-drug therapy.
Recently, it also demonstrated a functional role of ACSL4 in prostate cancer. Ectopic expression of ACSL4 in prostate cancer cells with negative expression of this gene has been shown to increase cell proliferation, migration, and invasion. Moreover, ablation of ACSL4 expression in metastatic prostate cancer cells endogenously expressing ACSL4 reduces cell proliferation, migration, and invasion (Wu et al. 2015). In addition, expression of ACSL4 produces resistance to chemotherapeutic treatment with Casodex (Wu et al. 2015).
Summary: Future and Perspective
The role of ACSL4 in the metabolism of AA reveals its importance in numerous physiological and pathological processes. Given its role in cancer, ACSL4 may constitute a new therapeutic target and biomarked for different types of tumors. Breast, colon, and prostate cancers and hepatocellular carcinoma have in common the peculiarity of having high aggressiveness and high capacity to generate metastases. In these four metastatic cancer types, due to their resistance to chemotherapeutic and/or hormonal treatment as the case may be, there has been a growing need to find therapeutic targets that serve as adjuvant therapies. A role of ACSL4 and mTOR was described for these types of tumors. As ACSL4 is a novel regulator of mTOR pathway, the combined inhibition of an upstream mechanism such as ACSL4 activity and mTOR seems to be a potential target to be used in order to avoid compensatory feedback. Therefore, as ACSL4 regulates ERα expression and the mTOR pathway, this enzyme could be also an interesting target, in combination with 4-OHTAM, to restore tumor hormone dependence in tumors with poor prognosis and low survival rates. Moreover, as the expression of ACSL4 is negatively regulated by estrogen and in turn regulates the levels of ERα, the presence of ACSL4 could be a prognostic factor for hormone resistance in ERα-positive breast cancer tissues that begin to express it. A combined therapy of ACSL4 and ERα inhibitors could thus be very useful in actually preventing the appearance of hormone resistance. New roles for ACSL4 have recently been described, such as in ferroptosis. More studies are needed to evaluate its implication in the development and the regulated cellular death, as well as in pathological processes whose expression is dysregulated.
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