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

Chin Fung Kelvin Kan,Amar Bahadur Singh,Diana M Stafforini,Salman Azhar,Jingwen Liu

doi:10.1194/jlr.m045971

Chin Fung Kelvin Kan, Amar Bahadur Singh + Show 3 more

Open Access

https://doi.org/10.1194/jlr.m045971

Copy DOI

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.

Highlights

ACSL4 is a member of the long-chain acyl-CoA synthetase (ACSL) family with a marked preference for arachidonic acid (AA) as its substrate
Utilizing a highly specific anti-hamster ACSL3 antibody [17], we showed that in contrast to ACSL4, ACSL3 protein levels remained unchanged in response to high-fat diet (HFD) feeding. quantitative RT-PCR (qRT-PCR) analyses of four hepatic ACSL isoforms showed that ACSL4 mRNA levels were 40% lower in the HFD group as compared with the normal chow diet (NCD) group
The mRNA levels of ACSL1 and ACSL5 were unchanged, while ACSL3 mRNA levels were reduced by 50% upon HFD feeding (Fig. 1B) despite the unchanged ACSL3 protein levels

Summary

Introduction

ACSL4 is a member of the long-chain acyl-CoA synthetase (ACSL) family with a marked preference for arachidonic acid (AA) as its substrate. We provide evidence that AA selectively downregulates ACSL4 protein expression in hepatic cells. Our studies have identified a novel substrate-induced posttranslational regulatory mechanism by which AA downregulates ACSL4 protein expression in hepatic cells.—Kan, C. Liu. Arachidonic acid downregulates acyl-CoA synthetase 4 expression by promoting its ubiquitination and proteasomal degradation. Five isoforms of ACSLs (ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6) have been identified and characterized in human, mouse, and rat tissues [5] These isoforms catalyze similar enzymatic reactions, they exhibit variable cellular functions and generate distinct metabolic outcomes in an isoform-specific and tissue/cell type-specific manner. The current hypothesis is that substrate specificity, subcellular localization, tissue-specific expression, and upstream signaling regulatory pathways all contribute to the unique functions of the individual ACSLs. Arachidonic acid (AA) (20:4, n-6) is an essential PUFA.

Methods

Results

Conclusion