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Arachidonic acid downregulates acyl-CoA synthetase 4 expression by promoting its ubiquitination and proteasomal degradation.

Kan CF, Singh AB, Stafforini DM, Azhar S, Liu J - J. Lipid Res. (2014)

Bottom Line: AA treatment decreased the half-life of ACSL4 protein in HepG2 cells by approximately 4-fold (from 17.3 ± 1.8 h to 4.2 ± 0.4 h) without causing apoptosis.We further show that ACSL4 is intrinsically ubiquitinated and that AA treatment can enhance its ubiquitination.Collectively, our studies have identified a novel substrate-induced posttranslational regulatory mechanism by which AA downregulates ACSL4 protein expression in hepatic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304.

ABSTRACT
ACSL4 is a member of the long-chain acyl-CoA synthetase (ACSL) family with a marked preference for arachidonic acid (AA) as its substrate. Although an association between elevated levels of ACSL4 and hepatosteatosis has been reported, the function of ACSL4 in hepatic FA metabolism and the regulation of its functional expression in the liver remain poorly defined. Here we provide evidence that AA selectively downregulates ACSL4 protein expression in hepatic cells. AA treatment decreased the half-life of ACSL4 protein in HepG2 cells by approximately 4-fold (from 17.3 ± 1.8 h to 4.2 ± 0.4 h) without causing apoptosis. The inhibitory action of AA on ACSL4 protein stability could not be prevented by rosiglitazone or inhibitors that interfere with the cellular pathways involved in AA metabolism to biologically active compounds. In contrast, treatment of cells with inhibitors specific for the proteasomal degradation pathway largely prevented the AA-induced ACSL4 degradation. We further show that ACSL4 is intrinsically ubiquitinated and that AA treatment can enhance its ubiquitination. Collectively, our studies have identified a novel substrate-induced posttranslational regulatory mechanism by which AA downregulates ACSL4 protein expression in hepatic cells.

No MeSH data available.


Related in: MedlinePlus

Detection of ubiquitination of endogenous ACSL4 in HepG2 cells without and with AA treatment. HepG2 cells were treated for 8 h with 50 μM AA or control in the presence or absence of proteasome inhibitor MG132 (20 μM). Proteins (600 μg) from each lysate sample were subjected to IP with anti-ACSL4 antibody or control antibody, rabbit IgG. A: Total lysates were analyzed for ACSL4 and ubiquitinated proteins by immunoblotting using anti-ACSL4 and anti-Ubq antibody. B: IP complexes were analyzed for ubiquitinated ACSL4 with anti-ACSL4 antibody and anti-Ubq antibody. WB, Western blot.
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fig6: Detection of ubiquitination of endogenous ACSL4 in HepG2 cells without and with AA treatment. HepG2 cells were treated for 8 h with 50 μM AA or control in the presence or absence of proteasome inhibitor MG132 (20 μM). Proteins (600 μg) from each lysate sample were subjected to IP with anti-ACSL4 antibody or control antibody, rabbit IgG. A: Total lysates were analyzed for ACSL4 and ubiquitinated proteins by immunoblotting using anti-ACSL4 and anti-Ubq antibody. B: IP complexes were analyzed for ubiquitinated ACSL4 with anti-ACSL4 antibody and anti-Ubq antibody. WB, Western blot.

Mentions: Finally, we examined the ubiquitination status of endogenous ACSL4 in HepG2 cells. HepG2 cells were treated for 8 h with 50 μM AA or control in the presence or absence of proteasome inhibitor MG132 (20 μM). Equal amounts of whole cell lysates were subjected to IP with anti-ACSL4 antibody or a control antibody (rabbit IgG), followed by Western blotting using anti-ACSL4 or anti-Ubq antibody. Detection of ACSL4 and Ubq in total cell lysates showed that the AA treatment reduced ACSL4 protein amount compared with the control, and this reduction was abolished by MG132 (Fig. 6A, compare lane 2 to lane 4). Cellular levels of ubiquitinated proteins were barely seen in the absence of MG132, but were readily detectable in the MG132-treated sample, and the signal intensity was slightly higher by cotreatment with AA (Fig. 6A, lane 3 vs. lane 4). Importantly, after ACSL4 IP, the amount of pulled down unubiquitinated ACSL4, as shown by anti-ACSL4 Western blot, was lower in the precipitates of AA-treated sample than that of the control sample (Fig. 6B, compare lane 3 with lane 2); however, the AA-treated sample clearly had a higher amount of polyubiquitinated ACSL4 than the untreated control. In contrast with anti-ACSL4 IP, Western blotting with anti-ACSL4 or anti-Ubq antibodies did not detect specific bands in control IgG immunoprecipitates (Fig. 6B, lanes 4 to 6). These data are highly consistent with the results obtained in ACSL4-overexpressing cells (Fig. 5). Altogether, these results provide direct evidence that AA exposure leads to enhanced ACSL4 ubiquitination and possibly channeling of ACSL4 toward its proteasomal degradation.


Arachidonic acid downregulates acyl-CoA synthetase 4 expression by promoting its ubiquitination and proteasomal degradation.

Kan CF, Singh AB, Stafforini DM, Azhar S, Liu J - J. Lipid Res. (2014)

Detection of ubiquitination of endogenous ACSL4 in HepG2 cells without and with AA treatment. HepG2 cells were treated for 8 h with 50 μM AA or control in the presence or absence of proteasome inhibitor MG132 (20 μM). Proteins (600 μg) from each lysate sample were subjected to IP with anti-ACSL4 antibody or control antibody, rabbit IgG. A: Total lysates were analyzed for ACSL4 and ubiquitinated proteins by immunoblotting using anti-ACSL4 and anti-Ubq antibody. B: IP complexes were analyzed for ubiquitinated ACSL4 with anti-ACSL4 antibody and anti-Ubq antibody. WB, Western blot.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109760&req=5

fig6: Detection of ubiquitination of endogenous ACSL4 in HepG2 cells without and with AA treatment. HepG2 cells were treated for 8 h with 50 μM AA or control in the presence or absence of proteasome inhibitor MG132 (20 μM). Proteins (600 μg) from each lysate sample were subjected to IP with anti-ACSL4 antibody or control antibody, rabbit IgG. A: Total lysates were analyzed for ACSL4 and ubiquitinated proteins by immunoblotting using anti-ACSL4 and anti-Ubq antibody. B: IP complexes were analyzed for ubiquitinated ACSL4 with anti-ACSL4 antibody and anti-Ubq antibody. WB, Western blot.
Mentions: Finally, we examined the ubiquitination status of endogenous ACSL4 in HepG2 cells. HepG2 cells were treated for 8 h with 50 μM AA or control in the presence or absence of proteasome inhibitor MG132 (20 μM). Equal amounts of whole cell lysates were subjected to IP with anti-ACSL4 antibody or a control antibody (rabbit IgG), followed by Western blotting using anti-ACSL4 or anti-Ubq antibody. Detection of ACSL4 and Ubq in total cell lysates showed that the AA treatment reduced ACSL4 protein amount compared with the control, and this reduction was abolished by MG132 (Fig. 6A, compare lane 2 to lane 4). Cellular levels of ubiquitinated proteins were barely seen in the absence of MG132, but were readily detectable in the MG132-treated sample, and the signal intensity was slightly higher by cotreatment with AA (Fig. 6A, lane 3 vs. lane 4). Importantly, after ACSL4 IP, the amount of pulled down unubiquitinated ACSL4, as shown by anti-ACSL4 Western blot, was lower in the precipitates of AA-treated sample than that of the control sample (Fig. 6B, compare lane 3 with lane 2); however, the AA-treated sample clearly had a higher amount of polyubiquitinated ACSL4 than the untreated control. In contrast with anti-ACSL4 IP, Western blotting with anti-ACSL4 or anti-Ubq antibodies did not detect specific bands in control IgG immunoprecipitates (Fig. 6B, lanes 4 to 6). These data are highly consistent with the results obtained in ACSL4-overexpressing cells (Fig. 5). Altogether, these results provide direct evidence that AA exposure leads to enhanced ACSL4 ubiquitination and possibly channeling of ACSL4 toward its proteasomal degradation.

Bottom Line: AA treatment decreased the half-life of ACSL4 protein in HepG2 cells by approximately 4-fold (from 17.3 ± 1.8 h to 4.2 ± 0.4 h) without causing apoptosis.We further show that ACSL4 is intrinsically ubiquitinated and that AA treatment can enhance its ubiquitination.Collectively, our studies have identified a novel substrate-induced posttranslational regulatory mechanism by which AA downregulates ACSL4 protein expression in hepatic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304.

ABSTRACT
ACSL4 is a member of the long-chain acyl-CoA synthetase (ACSL) family with a marked preference for arachidonic acid (AA) as its substrate. Although an association between elevated levels of ACSL4 and hepatosteatosis has been reported, the function of ACSL4 in hepatic FA metabolism and the regulation of its functional expression in the liver remain poorly defined. Here we provide evidence that AA selectively downregulates ACSL4 protein expression in hepatic cells. AA treatment decreased the half-life of ACSL4 protein in HepG2 cells by approximately 4-fold (from 17.3 ± 1.8 h to 4.2 ± 0.4 h) without causing apoptosis. The inhibitory action of AA on ACSL4 protein stability could not be prevented by rosiglitazone or inhibitors that interfere with the cellular pathways involved in AA metabolism to biologically active compounds. In contrast, treatment of cells with inhibitors specific for the proteasomal degradation pathway largely prevented the AA-induced ACSL4 degradation. We further show that ACSL4 is intrinsically ubiquitinated and that AA treatment can enhance its ubiquitination. Collectively, our studies have identified a novel substrate-induced posttranslational regulatory mechanism by which AA downregulates ACSL4 protein expression in hepatic cells.

No MeSH data available.


Related in: MedlinePlus