The endogenous subcellular localisations of the long chain fatty acid-activating enzymes ACSL3 and ACSL4 in sarcoma and breast cancer cells

Radif, Yassmeen; Ndiaye, Haarith; Kalantzi, Vasiliki; Jacobs, Ruth; Hall, Andrew; Minogue, Shane; Waugh, Mark G.

doi:10.1007/s11010-018-3332-x

Cited by 42 publications

(39 citation statements)

References 44 publications

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“…It is, of course, possible that low levels of sigma-1 receptors are expressed in these MCF7 cells obtained from ATCC. Indeed, Western blotting and immunocytochemical analysis of MCF7 cells obtained from ECACC (Radif et al, 2018) demonstrated the presence of sigma-1 receptors but to our knowledge, no direct comparison of MCF7 cells from these different sources has been made. The inclusion of (+) pentazocine in binding assays using MCF7 cells caused a rightward shift in the K d of [³H] DTG binding, but had no effect on B max calculations.…”

Section: Discussionmentioning

confidence: 94%

“…We would suggest that ideally, determination of binding parameters for the sigma-2 receptor would be best performed using [³H] DTG and cell preparations devoid of sigma-1 receptors, thereby avoiding the need for a masking agent. This could include, for example, MCF7 cell membranes, although caution should be applied as we have found some of these to contain sigma-1 receptors (Spruce et al, 2004;Radif et al, 2018).…”

Section: Discussionmentioning

confidence: 99%

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Hazards of Using Masking Protocols When Performing Ligand Binding Assays: Lessons From the Sigma-1 and Sigma-2 Receptors

et al. 2020

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Sigma-1 and sigma-2 receptors are emerging therapeutic targets. Although the molecular identity of the sigma-2 receptor has recently been determined, receptor quantitation has used, and continues to use, the sigma-1 selective agents (+) pentazocine or dextrallorphan to mask the sigma-1 receptor in radioligand binding assays. Here, we have assessed the suitability of currently established saturation and competition binding isotherm assays that are used to quantify parameters of the sigma-2 receptor. We show that whilst the sigma-1 receptor mask (+) pentazocine has low affinity for the sigma-2 receptor (K i 406 nM), it can effectively compete at this site with [³H] di-O-tolyl guanidine (DTG) at the concentrations frequently used to mask the sigma-1 receptor (100 nM and 1 μM). This competition influences the apparent affinity of DTG and other ligands tested in this system. A more troublesome issue is that DTG can displace (+) pentazocine from the sigma-1 receptor, rendering it partly unmasked. Indeed, commonly used concentrations of (+) pentazocine, 100 nM and 1 μM, allowed 37 and 11% respectively of sigma-1 receptors to be bound by DTG (300 nM), which could result in an overestimation of sigma-2 receptor numbers in assays where sigma-1 receptors are also present. Similarly, modelled data for 1 μM dextrallorphan show that only 71-86% of sigma-1 receptors would be masked in the presence of 300 nM DTG. Therefore, the use of dextrallorphan as a masking agent would also lead to the overestimation of sigma-2 receptors in systems where sigma-1 receptors are present. These data highlight the dangers of using masking agents in radioligand binding studies and we strongly recommend that currently used masking protocols are not used in the study of sigma-2 receptors. In order to overcome these problems, we recommend the use of a cell line apparently devoid of sigma-1 receptors [e.g., MCF7 (ATCC HTB-22)] in the absence of any masking agent when determining the affinity of agents for the sigma-2 receptor. In addition, assessing the relative levels of sigma-1 and sigma-2 receptors can be achieved using [³H] DTG saturation binding followed by two-site analysis of (+) pentazocine competition binding with [³H] DTG.

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Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

Hazards of Using Masking Protocols When Performing Ligand Binding Assays: Lessons From the Sigma-1 and Sigma-2 Receptors

et al. 2020

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“…Among the five isoforms of ACSL, ACSL4 located in peroxisomes has substrate specificity for polyunsaturated fatty acids C20:4 and C20:5, whereas ACSL1 and ACSL5 have a preference for long chain saturated and unsaturated fatty acids of 16 to 20 carbons. ACLS4 is known to be involved in steroidogenesis by regulating the arachidonate level and is closely linked to inflammation, eicosanoid synthesis, and tumourigenesis . Because ACSL4 plays a major role in maintaining lipid homeostasis, dysfunction or dysregulation of ACLS4 could result in severe metabolic disorders.…”

Section: Discussionmentioning

confidence: 99%

“…ACLS4 is known to be involved in steroidogenesis by regulating the arachidonate level 32 and is closely linked to inflammation, 33 eicosanoid synthesis, 34 and tumourigenesis. 35…”

Section: Discussionmentioning

confidence: 99%

Suppression of ABCD2 dysregulates lipid metabolism via dysregulation of miR‐141:ACSL4 in human osteoarthritis

Park

Kim

et al. 2018

Cell Biochemistry & Function

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Even though increasing evidence indicates the importance of peroxisomal lipid metabolism in regulating biological and pathological events, its involvement in cartilage development has not been well studied. Here, we identified the importance of peroxisomal function, particularly the functional integrity of ABCD2, in the pathogenesis of osteoarthritis (OA). Knockdown of ABCD2 in OA chondrocytes induced the accumulation of very long chain fatty acids (VLCFAs) and apoptotic cell death. Moreover, knockdown of ABCD2 altered profiles of miRNAs that affect the expression level of ACSL4, a known direct regulator of lipid metabolism. Suppression of ACSL4 in human chondrocytes‐induced VLCFA accumulation, MMP‐13 expression, and apoptotic cell death. In vivo morph‐down of the ACSL4 homologue in zebrafish resulted in significant defects in cartilage development and in vivo knockdown of ACSL4 in cartilage tissue of an OA model mice promoted severe cartilage degradation. In summary, to the best of our knowledge, this is the first report suggesting that the regulatory network among peroxisomal ABCD2:ACSL4:VLCFA serves as a novel regulator of cartilage homeostasis, and these data may provide novel insights into the role of peroxisomal fatty acid metabolism in pathogenesis of human OA. Significance of the study Our study indicates that peroxisomal dysfunction is closely related to OA pathogenesis. Particularly, the functional integrity of ABCD2 may play an important role in OA pathogenesis via the accumulation of VLCFAs and stimulation of apoptotic death through altering profiles of miRNAs that target ACSL4. Our findings suggest that targeting the regulatory network among the peroxisomal ABCD2:ACSL4:VLCFA axis may provide a new potential therapeutic strategy for OA pathogenesis.

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“…Fatty acids from different sources (diet, endogenous de novo synthesis and adipose tissue hydrolysis) can cross the plasma membrane and enter the muscle cell by fatty acid transport proteins (FATPs), fatty acid translocase (FAT/CD36), caveolins and plasma membrane fatty acid-binding proteins (FABPpm) [30]. Once in the cell, long-chain fatty acids are activated to fatty acyl-CoAs by a family of acyl-CoA synthetases (ACS) identified at the plasma membrane, mitochondria, and lipid droplets [31]. FATPs have also ACS activity [32].…”

Section: Role Of Carnitine In Mitochondrial Fatty Acid Transport and mentioning

confidence: 99%

Carnitine in Human Muscle Bioenergetics: Can Carnitine Supplementation Improve Physical Exercise?

et al. 2020

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l-Carnitine is an amino acid derivative widely known for its involvement in the transport of long-chain fatty acids into the mitochondrial matrix, where fatty acid oxidation occurs. Moreover, l-Carnitine protects the cell from acyl-CoA accretion through the generation of acylcarnitines. Circulating carnitine is mainly supplied by animal-based food products and to a lesser extent by endogenous biosynthesis in the liver and kidney. Human muscle contains high amounts of carnitine but it depends on the uptake of this compound from the bloodstream, due to muscle inability to synthesize carnitine. Mitochondrial fatty acid oxidation represents an important energy source for muscle metabolism particularly during physical exercise. However, especially during high-intensity exercise, this process seems to be limited by the mitochondrial availability of free l-carnitine. Hence, fatty acid oxidation rapidly declines, increasing exercise intensity from moderate to high. Considering the important role of fatty acids in muscle bioenergetics, and the limiting effect of free carnitine in fatty acid oxidation during endurance exercise, l-carnitine supplementation has been hypothesized to improve exercise performance. So far, the question of the role of l-carnitine supplementation on muscle performance has not definitively been clarified. Differences in exercise intensity, training or conditioning of the subjects, amount of l-carnitine administered, route and timing of administration relative to the exercise led to different experimental results. In this review, we will describe the role of l-carnitine in muscle energetics and the main causes that led to conflicting data on the use of l-carnitine as a supplement.

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The endogenous subcellular localisations of the long chain fatty acid-activating enzymes ACSL3 and ACSL4 in sarcoma and breast cancer cells

Cited by 42 publications

References 44 publications

Hazards of Using Masking Protocols When Performing Ligand Binding Assays: Lessons From the Sigma-1 and Sigma-2 Receptors

Hazards of Using Masking Protocols When Performing Ligand Binding Assays: Lessons From the Sigma-1 and Sigma-2 Receptors

Suppression of ABCD2 dysregulates lipid metabolism via dysregulation of miR‐141:ACSL4 in human osteoarthritis

Carnitine in Human Muscle Bioenergetics: Can Carnitine Supplementation Improve Physical Exercise?

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