Regulation of arachidonic acid release in steroidogenesis: role of a new acyl-CoA thioesterase ( Artist)

MALOBERTI PAULA; MELE PABLO; CORNEJO MACIEL F; CANO FLORENCIA; BEY PAULA; CRISTINA PAZ; PODESTA ERNESTO JORGE

Congreso; IXth Adrenal Cortex Conference; 2000

It is well established that protein phosphorylation plays on obligatory role in the mechanism of action of both adrenocorticotropin and luteinizing hormone in adrenal and Leydig cells, respectively. Searching for the protein involved in steroid synthesis through the release of arachidonic acid (AA), we identified a phosphopeotein (p43). Recent cloning of a eDNA encoding pa revealed that the protein is homologous to a novel acyl-CoA thioesterase. In view of the obligatory role of this protein in the activation of steroidogenesis through the release of AA. we proposed the name Arachidonie acid-Related Thioesterase Involved in Steroidogenesis (ARTISt) for p43. Expression of ARTISt cDNA in Escherichla coli using the expression vector pGEX results in the production of an immunoreactive protein. Consistent with the finding of consensus sites for pbosphorylation by cAMP?depend protein kinase (PKA) in its sequence, recombinant ARTISt was phosphorylated in vitro by PKA. The acyl-CoA thioesterase activity of the recombinant protein was also assayed using 1-(14C) arachidonoyl-CoA as substrate. The enzyme catalyzed the release of AA with an apparent Km of 2.1 uM. Phospholipase A2 inhibitors such as 4- bromophcnacyl bromide (BPB) and nordihydroguayeretic acid (NDGA) inhibit enzyme activity in a dose-dependent fashion. Moreover, the effect of NDGA on ARTISt activity correlates with the inhibition of steroid synthesis produced in adrenal cortex cells. The recent finding of a novel arachidonate-preferring aryl-CoA synthetase in steroidogenic cells of rat adrenal, ovary and testis, provides further evidence for the presence of a new regulatory pathway that controls the release of AA in steroidogenic tissues. This late finding is consistent with the, now established, acyl-CoA transport role of the diazepam binding inhibitor (DBI) and the acylCoA thioesterase activity of ARTISt. AA will, in its turn, regulate the transcription of the steroidogenic acute regulatory protein (StAR) for full steroidogenic activity.