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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

Proteasomal pathway participates in AA-induced degradation of ACSL4 protein. A: HepG2 cells were treated with 5 μg/ml CHX, 200 nM bortezomib (Bort), 50 nM bafilomycin A1 (Baf A1), or the combination of Bort and Baf A1 for 1 h prior to the addition of 150 μM AA. Cell lysates were isolated after 24 h of AA treatment. After Western blotting, for each sample, the signal of ACSL4 was normalized to signal of β-actin. The normalized ACSL4 signal without AA treatment was expressed as 100%. The data are representative of two separate experiments with similar results. The indicated value is for the blot shown. C, control. B, C: Plasmids encoding Flag-tagged ACSL4 and HA-Ubq were cotransfected into HEK293 cells. The empty vectors of pCMV-Entry and pCMV-HA were transfected as mock control. Two days post transfection, cells were treated with 20 μM MG132 to block proteasomal degradation for 6 h prior to cell lysis. Cell lysates were immunoprecipitated with anti-HA or anti-Flag antibodies, respectively. D, E: HEK293A cells were cotransfected with ACSL4-FLAG and HA-Ubq plasmids for 48 h. Then, cells were divided equally into two plates. After overnight culturing, cells were treated with 150 μM AA or vehicle for 8 h in the presence of MG132 before isolation of total cell lysates. Proteins (300 μg) from each lysate sample were subjected to IP with anti-FLAG. Total lysates were analyzed for ACSL4 and ubiquitinated proteins by immunoblotting using anti-ACSL4 and anti-Ubq antibody (D). IP complexes were analyzed for total ACSL4 with anti-ACSL4 antibody and ubiquitinated ACSL4 by anti-Ubq antibody (E). WB, Western blot.
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fig5: Proteasomal pathway participates in AA-induced degradation of ACSL4 protein. A: HepG2 cells were treated with 5 μg/ml CHX, 200 nM bortezomib (Bort), 50 nM bafilomycin A1 (Baf A1), or the combination of Bort and Baf A1 for 1 h prior to the addition of 150 μM AA. Cell lysates were isolated after 24 h of AA treatment. After Western blotting, for each sample, the signal of ACSL4 was normalized to signal of β-actin. The normalized ACSL4 signal without AA treatment was expressed as 100%. The data are representative of two separate experiments with similar results. The indicated value is for the blot shown. C, control. B, C: Plasmids encoding Flag-tagged ACSL4 and HA-Ubq were cotransfected into HEK293 cells. The empty vectors of pCMV-Entry and pCMV-HA were transfected as mock control. Two days post transfection, cells were treated with 20 μM MG132 to block proteasomal degradation for 6 h prior to cell lysis. Cell lysates were immunoprecipitated with anti-HA or anti-Flag antibodies, respectively. D, E: HEK293A cells were cotransfected with ACSL4-FLAG and HA-Ubq plasmids for 48 h. Then, cells were divided equally into two plates. After overnight culturing, cells were treated with 150 μM AA or vehicle for 8 h in the presence of MG132 before isolation of total cell lysates. Proteins (300 μg) from each lysate sample were subjected to IP with anti-FLAG. Total lysates were analyzed for ACSL4 and ubiquitinated proteins by immunoblotting using anti-ACSL4 and anti-Ubq antibody (D). IP complexes were analyzed for total ACSL4 with anti-ACSL4 antibody and ubiquitinated ACSL4 by anti-Ubq antibody (E). WB, Western blot.

Mentions: The marked reduction in ACSL4 protein half-life in AA-treated cells suggested that ACSL4 is subject to a rapid degradation process. The autophagy-lysosomal pathway and the Ubq-proteasome pathway are the two major cellular proteolytic systems that participate in intracellular protein degradation in eukaryotic cells. To determine which of these two pathways is primarily involved in AA-induced ACSL4 degradation, we pretreated HepG2 cells with bafilomycin A1 (a specific lysosomal inhibitor) and bortezomib (a proteasomal inhibitor), either separately or in combination and subsequently exposed to AA or vehicle (control). The protein synthesis inhibitor, CHX, was used as a negative control in these experiments. Figure 5A shows that bortezomib alone or combined with bafilomycin A1 largely blocked the AA-mediated degradation of ACSL4. On the contrary and as expected, treatment of cells with CHX was without any effect. These data suggested that AA mainly utilizes the Ubq-proteasome pathway to promote ACSL4 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)

Proteasomal pathway participates in AA-induced degradation of ACSL4 protein. A: HepG2 cells were treated with 5 μg/ml CHX, 200 nM bortezomib (Bort), 50 nM bafilomycin A1 (Baf A1), or the combination of Bort and Baf A1 for 1 h prior to the addition of 150 μM AA. Cell lysates were isolated after 24 h of AA treatment. After Western blotting, for each sample, the signal of ACSL4 was normalized to signal of β-actin. The normalized ACSL4 signal without AA treatment was expressed as 100%. The data are representative of two separate experiments with similar results. The indicated value is for the blot shown. C, control. B, C: Plasmids encoding Flag-tagged ACSL4 and HA-Ubq were cotransfected into HEK293 cells. The empty vectors of pCMV-Entry and pCMV-HA were transfected as mock control. Two days post transfection, cells were treated with 20 μM MG132 to block proteasomal degradation for 6 h prior to cell lysis. Cell lysates were immunoprecipitated with anti-HA or anti-Flag antibodies, respectively. D, E: HEK293A cells were cotransfected with ACSL4-FLAG and HA-Ubq plasmids for 48 h. Then, cells were divided equally into two plates. After overnight culturing, cells were treated with 150 μM AA or vehicle for 8 h in the presence of MG132 before isolation of total cell lysates. Proteins (300 μg) from each lysate sample were subjected to IP with anti-FLAG. Total lysates were analyzed for ACSL4 and ubiquitinated proteins by immunoblotting using anti-ACSL4 and anti-Ubq antibody (D). IP complexes were analyzed for total ACSL4 with anti-ACSL4 antibody and ubiquitinated ACSL4 by anti-Ubq antibody (E). WB, Western blot.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Proteasomal pathway participates in AA-induced degradation of ACSL4 protein. A: HepG2 cells were treated with 5 μg/ml CHX, 200 nM bortezomib (Bort), 50 nM bafilomycin A1 (Baf A1), or the combination of Bort and Baf A1 for 1 h prior to the addition of 150 μM AA. Cell lysates were isolated after 24 h of AA treatment. After Western blotting, for each sample, the signal of ACSL4 was normalized to signal of β-actin. The normalized ACSL4 signal without AA treatment was expressed as 100%. The data are representative of two separate experiments with similar results. The indicated value is for the blot shown. C, control. B, C: Plasmids encoding Flag-tagged ACSL4 and HA-Ubq were cotransfected into HEK293 cells. The empty vectors of pCMV-Entry and pCMV-HA were transfected as mock control. Two days post transfection, cells were treated with 20 μM MG132 to block proteasomal degradation for 6 h prior to cell lysis. Cell lysates were immunoprecipitated with anti-HA or anti-Flag antibodies, respectively. D, E: HEK293A cells were cotransfected with ACSL4-FLAG and HA-Ubq plasmids for 48 h. Then, cells were divided equally into two plates. After overnight culturing, cells were treated with 150 μM AA or vehicle for 8 h in the presence of MG132 before isolation of total cell lysates. Proteins (300 μg) from each lysate sample were subjected to IP with anti-FLAG. Total lysates were analyzed for ACSL4 and ubiquitinated proteins by immunoblotting using anti-ACSL4 and anti-Ubq antibody (D). IP complexes were analyzed for total ACSL4 with anti-ACSL4 antibody and ubiquitinated ACSL4 by anti-Ubq antibody (E). WB, Western blot.
Mentions: The marked reduction in ACSL4 protein half-life in AA-treated cells suggested that ACSL4 is subject to a rapid degradation process. The autophagy-lysosomal pathway and the Ubq-proteasome pathway are the two major cellular proteolytic systems that participate in intracellular protein degradation in eukaryotic cells. To determine which of these two pathways is primarily involved in AA-induced ACSL4 degradation, we pretreated HepG2 cells with bafilomycin A1 (a specific lysosomal inhibitor) and bortezomib (a proteasomal inhibitor), either separately or in combination and subsequently exposed to AA or vehicle (control). The protein synthesis inhibitor, CHX, was used as a negative control in these experiments. Figure 5A shows that bortezomib alone or combined with bafilomycin A1 largely blocked the AA-mediated degradation of ACSL4. On the contrary and as expected, treatment of cells with CHX was without any effect. These data suggested that AA mainly utilizes the Ubq-proteasome pathway to promote ACSL4 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