The Human Variome Project country node of Argentina in the first two years of activity: past, present and future / El nodo Argentino del Proyecto Varioma Humano en los primeros dos años de actividad: pasado, presente y futuro

The Human Variome Project (HVP) is an international effort aiming systematically to collect and share information on all human genetic variants. It has been working for years in collaboration with local scientific societies by establishing systems to collect every genetic variant reported in a country and to store these variants within a database repository: LOVD (Argentinian chapter: ar.lovd.org). Formally established in 2017 in the Argentinian Node, up to June 2019 we collected more than 25,000 genetic variants deposited by 17 different laboratories. Nowadays the HVP country nodes represent more than 30 countries. In Latin America there are four country nodes: Argentina, Brazil, Mexico and Venezuela; the first two interacted recently launching the LatinGen database. In the present work we want to share our experience in applying the HVP project focusing on its organization, rules and nomenclature to reach the goal of sharing genetic variants and depositing them in the Leiden Open Variation Database. Contributing laboratories are seeking to share variant data to gain access all over the country. It is one of our goals to stimulate the highest quality by organizing courses, applying current nomenclature rules, sponsoring lectures in national congresses, distributing newsletter to serve the Argentinian genomics community and to stimulate the interaction among Latin America countries.

El Proyecto Varioma Humano (HVP) es un esfuerzo internacional que tiene como objetivo recopilar y compartir sistemáticamente información sobre todas las variantes genéticas humanas. Hemos estado trabajando durante tres años en colaboración con sociedades científicas locales, mediante el establecimiento de sistemas para recolectar todas las variantes genéticas reportadas en el país y almacenarlas dentro de la base de datos LOVD (capítulo argentino: ar.lovd.org). En el año 2017 fue establecido formalmente el Nodo Argentino del HVP, habiéndose recolectado más de 25.000 variantes genéticas depositadas por 17 laboratorios diferentes hasta junio de 2019. Hoy en día existen al menos 30 nodos del HVP, correspondientes a diferentes países. En América Latina hay cuatro nodos: Argentina, Brasil, México y Venezuela; Los dos primeros interactuaron recientemente lanzando la base de datos LatinGen. En el presente trabajo queremos compartir nuestra experiencia en la aplicación del proyecto HVP centrándonos en su organización, reglas y nomenclatura para alcanzar el objetivo de compartir variantes genéticas y depositarlas en la base de datos de variaciones abiertas de Leiden (LOVD). Es uno de nuestros objetivos estimular la más alta calidad mediante la organización de cursos, aplicación de las reglas de nomenclatura actuales, patrocinio de conferencias en congresos nacionales, distribución de boletines informativos para la comunidad de genómica argentina, y estimulación de la interacción entre los países de América Latina.

Valproic acid alters mitochondrial cholesterol transport in Y1 adrenocortical cells.

Several reports suggest putative interactions between valproic acid (VPA) treatment and the hypothalamus-pituitary-adrenal axis. Given that VPA alters mitochondrial functions, an action of this drug on a mitochondrial process such as steroid synthesis in adrenal cells should be expected. In order to disclose a putative action of VPA on the adrenocortical cell itself we evaluated VPA effects on regulatory steps of the acute stimulation of steroidogenesis in Y1 adrenocortical cells. This study demonstrates that VPA increases progesterone production in non-stimulated cells without inducing the levels of Steroidogenic Acute Regulatory (StAR) protein, which facilitates cholesterol transport. This result suggests that VPA increases mitochondrial cholesterol transport through a StAR-independent mechanism and is further supported by the fact that in isolated mitochondria VPA stimulates exogenous cholesterol metabolization to progesterone. VPA also reduces the cAMP-mediated increase of the StAR protein, mRNA levels, promoter activity and progesterone production. In summary, the present data show that VPA can alter steroid production in adrenal cells by a complex mechanism that mainly involves an action on cholesterol access to the inner mitochondrial membrane. The VPA-mediated increase of basal steroidogenesis could be linked to the increase of basal cortisolemia described in patients under VPA treatment.

Enzymes involved in arachidonic acid release in adrenal and Leydig cells.

Stimulation of receptors and subsequent signal transduction results in the activation of arachidonic acid (AA) release. Once AA is released from phospholipids or others esters, it may be metabolized via the cycloxygenase or the lipoxygenase pathways. How the cells drive AA to these pathways is not elucidated yet. It is reasonable to speculate that each pathway will have different sources of free AA triggered by different signal transduction pathways. Several reports have shown that AA and its lipoxygenase-catalyzed metabolites play essential roles in the regulation of steroidogenesis by influencing cholesterol transport from the outer to the inner mitochondrial membrane, the rate-limiting step in steroid hormone biosynthesis. Signals that stimulate steroidogenesis also cause the release of AA from phospholipids or other esters by mechanisms that are not fully understood. This review focuses on the enzymes of AA release that impact on steroidogenesis.

Penetrance and clinical manifestations of non-hotspot germline RET mutation, C630R, in a family with medullary thyroid carcinoma.

Germline mutations in specific hot spot-codons of the RET proto-oncogene are associated with multiple endocrine neoplasia type 2 (MEN 2). Clinical RET gene testing has been routine for the last 10 years in some countries. In Argentina, RET testing excluding MEN 2B was always reported with a mutation at codon 634, with one exception: we described a novel mutation T > C transition at codon 630 (C630R), the family to which we extend the study in the present report. This family comprised 29 members in four generations including 6 individuals affected with medullary thyroid cancer (MTC), positive for the C630R mutation and normal adrenaline/ noradrenaline and ionized calcium/parathyroid hormone levels. Two asymptomatic mutation carriers aged 5 and 11 years underwent total thyroidectomy. The histopathologic examination showed C-cell hyperplasia and microcarcinoma foci, while preoperative basal calcitonins were normal for both. Our report emphasizes the importance of testing for non-hot spot RET mutations in apparently mutation negative MEN 2 families. Furthermore, it would appear that C630R mirrors C634R in penetrance (100% in this family) and in early age of onset of MTC, although paradoxically, no pheochromocytomas and hyperparathyroidism have developed. In addition to recommending RET testing before 5 years of age; we also can postulate that codon 630 may be the key point along the extracellular domain, important in the tissue-specific penetrance.

Adrenocorticotropin induces heme oxygenase-1 expression in adrenal cells.

Heme oxygenase (HO) catalyzes the first and rate-controlling step of heme catabolism into biliverdin, iron and carbon monoxide. Three isoforms of HO have been identified so far: the inducible HO-1 and the constitutive HO-2 and HO-3. Both HO-1 and HO-2 were expressed in zona fasciculata (ZF) adrenal cells and in a mouse adrenocortical cell line (Y1). HO-1 but not HO-2 expression was upregulated by adrenocorticotropic hormone (ACTH) and accumulation of HO-1 protein correlated with an increase in HO activity in Y1 cells. ACTH induced HO-1 expression in a time- and dose-dependent manner with a maximum after 5 h of treatment and a threshold concentration of 0.1 mIU/ml. Actinomycin D and cycloheximide completely blocked the effect of ACTH on HO-1 mRNA expression whereas mRNA stability was not affected by ACTH. Permeable analogs of cAMP mimicked the effect of ACTH on HO-1 expression and ACTH induction was prevented by the protein kinase A (PKA) inhibitor H89. Steroid production was significantly increased when both HO-1 and HO-2 activities were inhibited by Sn-protoporphyrin IX (SnPPIX). The lipid peroxidation and increase in carbonyl content triggered by hydrogen peroxide was prevented by treatment of Y1 cells with bilirubin and ACTH.

Arachidonic acid regulation of steroid synthesis: new partners in the signaling pathway of steroidogenic hormones.

Although the role of arachidonic acid (AA) in trophic hormone-stimulated steroid production in various steroidogenic cells is well documented, the mechanism responsible for AA release remains unknown. We have previously shown evidence of an alternative pathway of AA generation in steroidogenic tissues. Our results are consistent with the hypothesis that, in steroidogenic cells, AA is released by the action of a mitochondrial acyl-CoA thioesterase (MTE-I). We have shown that recombinant MTE-I hydrolyses arachidonoyl-CoA to release free AA. An acyl-CoA synthetase specific for AA, acyl-CoA synthetase 4, has also been described in steroidogenic tissues. In the present study we investigate the new concept in the regulation of intracellular levels of AA, in which trophic hormones can release AA by mechanisms different from the classical PLA2-mediated pathway. Inhibition of ACS4 and MTE-I activity by triacsin C and NDGA, respectively results in a reduction of StAR mRNA and protein abundance. When both inhibitors are added together there is a synergistic effect in the inhibition of StAR mRNA, StAR protein levels and ACTH-stimulated steroid synthesis. The inhibition of steroidogenesis produced by the NDGA and triacsin C can be overcome by the addition of exogenous AA. In summary, results shown here demonstrate a critical role of the acyl-CoA synthetase and the acyl-CoA thioesterase in the regulation of AA release, StAR induction, and steroidogenesis. This further suggests a new concept in the regulation of intracellular distribution of AA through a mechanism different from the classical PLA2-mediated pathway that involves a hormone-induced acyl-CoA synthetase and a hormone-regulated acyl-CoA thioesterase.

Molecular events triggered by heat shock in Y1 adrenocortical cells.

Several stimuli, including stress conditions, promote the activation of MAP kinases family members (ERK1/2, JNK, p38). In turn, these enzymes regulate several cellular functions. Given that MAPK activation requires the phosphorylation of these proteins, their inactivation depends on the activity of specific phosphatases. MAPK phosphatase-1 (MKP-1), a phosphatase specifically involved in the inactivation of MAPK family members, is induced by mitogenic stimuli and stress conditions. Here we describe the effect of heat shock (HS), 10 min, 45 degrees C, on MAPKs activities and MKP-1 mRNA and protein levels in Y1 adrenocortical cells. Western blot analysis performed with antibodies against the phosphorylated forms of ERK1/2 and JNK revealed that HS produced the rapid activation of these kinases. Their inactivation was also a rapid event and occurred together with the increase of MKP-1 protein levels detected by Western blot analysis. In addition, the effect of HS on MKP-1 protein levels seems to be exerted at the transcriptional level, since the amount of its mRNA in heat shocked cells was higher than in nonheated cells. Comparison of the temporal profiles of MKP-1 protein induction and MAPKs phospho-dephosphorylation suggests that MKP-1 induction could contribute to ERK1/2 and JNK inactivation after HS.

Protein tyrosine phosphatases are involved in LH/chorionic gonadotropin and 8Br-cAMP regulation of steroidogenesis and StAR protein levels in MA-10 Leydig cells.

The LH signal transduction pathway features the activation of protein tyrosine phosphatases (PTPs) as one of the components of a cascade that includes other well characterized events such as cAMP-dependent protein kinase A (PKA) activation. Moreover, the action of PTPs is required to increase the rate-limiting step in steroid biosynthesis, namely the cAMP-regulated transfer of cholesterol to the inner mitochondrial membrane. Since both PKA activity and steroidogenic acute regulatory (StAR) protein induction are obligatory steps in this transfer of cholesterol, the present study was performed to investigate the role of PTPs in the regulation of PKA activity and StAR expression in response to LH/chorionic gonadotropin (CG) and 8Br-cAMP in MA-10 cells. While the exposure of MA-10 cells to the PTP inhibitor, phenylarsine oxide (PAO), did not modify PKA activity, it partially inhibited the effect of human CG and cAMP analog on StAR protein levels. Time-course studies demonstrated that PAO inhibited cAMP induction of StAR protein and mRNA. At 30 min, the effect on cAMP-stimulated StAR protein levels was a 35% inhibition, progressing to up to 90% inhibition at 120 min of stimulation. The maximal inhibitory effect on cAMP-induced StAR mRNA level was obtained at 60 min (85%). In summary, these results demonstrate that inhibition of PTP activity affected both StAR protein and mRNA synthesis and suggest that the activity of hormone-regulated PTPs is a requirement in the LH signaling cascade that results in the up-regulation of StAR protein and, subsequently, increased steroid synthesis.

The obligatory action of protein tyrosine phosphatases in ACTH-stimulated steroidogenesis is exerted at the level of StAR protein.

A key regulatory step in the steroidogenic hormones signaling pathway is the synthesis of steroidogenic acute regulatory protein (StAR). This protein facilitates the delivery of cholesterol to the inner mitochondrial membrane, the rate-limiting step in steroidogenesis. ACTH and LH pathway also includes tyrosine dephosphorylation processes. Indeed, our previous studies have demonstrated that both hormones increase protein tyrosine phosphatase (PTP) activity by a PKA-dependent mechanism and that the action of PTPs is required for the stimulation of steroid biosynthesis in adrenal and Leydig cells. In order to test the putative relationship between PTP activity and StAR protein induction in adrenocortical cells, in the present study we evaluated steroid production and StAR protein level in Y1 adrenocortical cells under PTP inhibition. Phenylarsine oxide (PAO), a powerful cell permeable PTP inhibitor, reduced ACTH-stimulated steroidogenesis in a concentration-dependent fashion. A concentration of 2.5 microM of this compound inhibited steroid synthesis in a 56% (ACTH = 318 +/- 30, ACTH + PAO = 145 +/- 18 ng progesterone/mL, P < 0.001) and also abrogated StAR protein induction. Phenylarsine oxide reduced the protein level after 60 min and this effect still remained at 120 min. A second PTP inhibitor, benzyl phosphonic acid, acting by a different mechanism, reproduced PAO effects on both steroidogenesis and StAR protein. Taken together, these results indicate that PTP activity participates in StAR protein induction and led us to attribute to the PKA-mediated PTP activation in steroidogenic systems a functional role, as mediator of StAR protein induction.

Human umbilical vein: involvement of cyclooxygenase-2 pathway in bradykinin B1 receptor-sensitized responses.

In isolated human umbilical vein (HUV), the contractile response to des-Arg9-bradykinin (des-Arg9-BK), selective BK B1 receptor agonist, increases as a function of the incubation time. Here, we evaluated whether cyclooxygenase (COX) pathway is involved in BK B1-sensitized response obtained in 5-h incubated HUV rings. The effect of different concentrations of indomethacin, sodium salicylate, ibuprofen, meloxicam, lysine clonixinate or NS-398 administrated 30 min before concentration-response curves (CRC) was studied. All treatments produced a significant rightward shift of the CRC to des-Arg9-BK in a concentration-dependent manner, which provides pharmacological evidence that COX pathway is involved in the BK B1 responses. Moreover, in this tissue, the NS-398 pKb (5.2) observed suggests that COX-2 pathway is the most relevant. The strong correlation between published pIC50 for COX-2 and the NSAIDs' pKbs estimated further supports the hypothesis that COX-2 metabolites are involved in BK B1 receptor-mediated responses. In other rings, indomethacin (30, 100 micromol/l) or NS-398 (10, 30 micromol/l) produced a significant rightward shift of the CRC to BK, selective BK B2 agonist, and its pKbs were similar to the values to inhibit BK B1 receptor responses, suggesting that COX-2 pathway also is involved in BK B2 receptor responses. Western blot analysis shows that COX-1 and COX-2 isoenzymes are present before and after 5-h in vitro incubation and apparently COX-2 does not suffer additional induction.

LH/chorionic gonadotropin signaling pathway involves protein tyrosine phosphatase activity downstream of protein kinase A activation: evidence of an obligatory step in steroid production by Leydig cells.

Our recent reports indicate that protein tyrosine phosphorylation is an obligatory component of the mechanism of action of ACTH in its stimulatory action of corticosteroid production in adrenal zona fasciculata (ZF). The role of protein tyrosine phosphatase (PTP) activity in the regulation of steroidogenesis by LH/chorionic gonadotropin (CG) was tested using cell-permeable PTP inhibitors. Thus, PTP inhibition blocks LH- and 8-bromo-cAMP-stimulated testosterone production by Leydig cells without affecting 22(R)OH-cholesterol-supported steroidogenesis, similar results to those obtained in the adrenal ZF/ACTH system, leading us to propose that PTP action is an obligatory and common step in the cascade triggered by both hormones. Then, we continued the study testing whether LH modulates PTP activity in MA-10 cells, a Leydig cell line. In this regard, we observed by an in-gel PTP assay two PTPs of 110 and 50 kDa that are activated by hormone and 8-bromo-cAMP activation of the cells. Moreover, there is a transient increase by the second messenger in total PTP activity that correlates with the higher activity displayed by the 110 and 50 kDa proteins in the in-gel assay. In accordance with these results, analysis of tyrosine phosphorylated proteins showed the LH-induced dephosphorylation of proteins of 120, 68 and 50 kDa. The results of this study indicate that PTPs play an important role in the regulation of Leydig cell functions and that there exists a cross talk between serine/threonine phosphorylation and tyrosine dephosphorylation mediated by hormone-activated cAMP-dependent protein kinase and PTPs. These results are the first evidence of PTP having a role in LH/CG-stimulated steroidogenesis.

Induction of nitric oxide synthase activity in adrenal cells.

The induction of nitric oxide synthase (NOS) II by bacterial lypopolysaccharide (LPS) was studied in a steroidogenic mouse tumor adrenal cell line (Y1). Conditioned media from LPS-stimulated peritoneal macrophages induced an increase in NOS II expression as shown by western and northern blot analysis. Accordingly, in the presence of conditioned media an increase in nitrite levels was observed. In addition, steroid production was significantly decreased. In conclusion, NOS II expression could be induced in steroidogenic cells with a concomitant inhibition of steroid production.

An ACTH-activated protein tyrosine phosphatase (PTP) is modulated by PKA-mediated phosphorylation.

In adrenal cortex, ACTH regulation of steroidogenesis depends on PKA-dependent serine/threonine phosphorylation and also on the activity of protein tyrosine phosphatases (PTPs). In addition, ACTH increases total PTPs involving at least three soluble PTPs (50, 82 and 115 kDa). Serine/threonine phosphorylation of these enzymes themselves could be a regulatory mechanism of their activity since the increase of total PTP activity is dependent on PKA-activation. In this report we analyzed the effect of in vitro phospho-dephosphorylation processes on the activity displayed by the ACTH-activated PTP of 115 kDa. Using an in-gel PTP assay we demonstrate that dephosphorylation catalyzed by potato acid phosphatase (PAP) reduces the activity of the 115 kDa PTP present in ZF from ACTH-treated animals and PKA-mediated phosphorylation reverses this effect.

Regulation of arachidonic acid release in steroidogenesis: role of a new acyl-CoA thioestrase (ARTISt).

It has been well established that arachidonic acid (AA) and its metabolism to leukotrienes plays an obligatory role in steroid production. The release of AA is regulated by hormone stimulation and protein phosphorylation. We have cloned a cDNA of a phosphoprotein with a molecular mass of 43 kDa (p43), purified from the cytosol of stimulated adrenal glands. This protein acts as intermediary in the stimulation of steroid synthesis through AA release, and has been found to be a member of a recently described acyl-CoA thioesterase family. In view of the mandatory role of this protein in the activation of AA-mediated steroidogenesis, the term Arachidonic acid-Related Thioesterase Involved in Steroidogenesis (ARTISt), is proposed for p43. The present study describes the production of the recombinant protein by cDNA expression in Escherichia coli and its functional characterization. Recombinant acyl-CoA thioesterase was capable to release AA from the respective acyl-CoA, and this activity was affected by well-recognized inhibitors of AA release and metabolism: 4-bromophenacyl bromide (BPB) and nordihydroguariaretic acid (NDGA). In addition, the inhibition of acyl-CoA thioesterase activity by NDGA correlates with the inhibition of steroid synthesis produced by this compound in adrenal cortex cells. Moreover, the recombinant protein was phosphorylated in vitro by PKA. These results provide the first evidence linking acyl-CoA thioesterases with the regulation of steroidogenesis, and support a regulatory role for acyl-CoA thioesterases in steroidogenic tissues, suggesting an alternative pathway for AA release in signal transduction.

Corticotropin increases protein tyrosine phosphatase activity by a cAMP-dependent mechanism in rat adrenal gland.

Corticotropin signal transduction pathway involves serine/threonine protein phosphorylation. Recent reports suggest that protein tyrosine dephosphorylation may also be an integral component of that pathway. The present study was performed to investigate the role played by protein tyrosine phosphatases (PTPs) on acute response to corticotropin and the hypothetical regulation of PTPs by this hormone. We have used two powerful cell permeant PTP inhibitors, phenylarsine oxide (PAO) and pervanadate (PV), in order to examine the relevance of PTP activity on hormone-stimulated and 8-bromo-adenosine 3',5'-phosphate (8Br-cAMP is a permeant analogue of adenosine 3',5'-phosphate)-stimulated steroidogenesis in adrenal zona fasciculata (ZF) cells. In both cases, PAO and PV inhibited the steroid production in a dose-dependent fashion, and had no effect on steroidogenesis supported by a permeant analogue of cholesterol. The effect of hormonal stimulation on PTP activity was analyzed in rat adrenal ZF. In vivo corticotropin treatment reduced phosphotyrosine content in endogenous proteins and produced a transient increase of PTP activity in the cytosolic fraction, reaching a maximum (twofold) after 15 min. Incubation of adrenal ZF with 8Br-cAMP also produced PTP activation, suggesting that it can be mediated by cAMP-dependent protein kinase (PKA)-dependent phosphorylation. Detection of PTP activity in an in-gel assay showed three corticotropin-stimulated soluble PTPs with molecular masses of 115, 80 and 50 kDa. In summary, we report for the first time a hormone-dependent PTP activation in a steroidogenic tissue and provide evidence that PTP activity plays an important role in corticotropin signal pathway, acting downstream of PKA activation and upstream of cholesterol transport across the mitochondrial membrane.

Activation of a thioesterase specific for very-long-chain fatty acids by adrenergic agonists in perfused hearts.

We have recently described an acyl-CoA thioesterase specific for very-long-chain fatty acids, named ARTISt, that regulates steroidogenesis through the release of arachidonic acid in adrenal zona fasciculata cells. In this paper we demonstrate the presence of the protein as a 43 kDa band and its mRNA in cardiac tissue. The activity of the protein was measured using an heterologous cell-free assay in which it is recombined with adrenal microsomes and mitochondria to activate mitochondrial steroidogenesis. Isoproterenol and phenylephrine activate the enzyme in a dose-dependent manner (10(-10)-10(-6) M). Both propranolol (10(-5) M) and prazosin (10(-5) M) block the action of isoproterenol and phenylephrine respectively. Antipeptide antibodies against the serine lipase motif of the protein and the Cys residue present in the catalytic domain also block the activity of the protein. Taken together, our results confirm the presence of ARTISt in heart and provide evidence for a catecholamine-activated regulatory pathway of the enzyme in that tissue.

Effects of L-arginine in rat adrenal cells: involvement of nitric oxide synthase.

The effects of L-arginine on corticosterone production, cGMP, and nitrite levels were examined in zona fasciculata adrenal cells. L-Arginine significantly decreased both basal and ACTH-stimulated corticosterone production. This effect was still evident when steroidogenesis was induced by 8-bromo-cAMP and 22(R)-hydroxycholesterol, but not in the presence of exogenously added pregnenolone. L-Arginine increased cGMP and nitrite levels,; these effects were blocked by the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl-ester. Transport of L-[3H]arginine was rapid, saturable, and monophasic, with an apparent Km of 163+/-14 microM and a maximum velocity of 53+/-6 pmol/min x 10(5) cells. The basic amino acids L-lysine and L-ornithine, but not D-arginine or the nitric oxide synthase inhibitors N(G)-nitro-L-arginine methyl-ester and N(G)-nitro-L-arginine, impaired L-arginine uptake. Taken together, these results suggest that steroidogenesis in zona fasciculata adrenal cells may be negatively modulated by L-arginine-derived nitric oxide.

Effect of nitric oxide on rat adrenal zona fasciculata steroidogenesis.

The present study was designed to investigate the role of nitric oxide (NO) in the regulation of adrenocortical function. Different NO donors, such as sodium nitroprusside (SNP), S-nitroso-L-acetyl penicillamine, diethylamine/NO complex sodium salt and diethylenetriamine NO adduct, significantly decreased corticosterone production both in unstimulated and in corticotropin-stimulated zone fasciculata adrenal cells, in a dose-dependent manner. The effect of SNP was reversed by ferrous hemoglobin. A selective inhibitor of NO synthase, L-NG-nitro-arginine significantly increased corticosterone secretion. The effect of SNP was not mediated by cGMP as permeable cGMP analogs did not reproduce its inhibitory effect. SNP significantly inhibited the steroidogenesis stimulated by 8Br-cAMP and 22(R)-OH-cholesterol, but was ineffective when corticosterone was produced in the presence of exogenously added pregnenolone. Moreover, the conversion of [3H]cholesterol to [3H]pregnenolone and the production of pregnenolone or progesterone (assessed by RIA) were significantly decreased by SNP. Taken together, these results suggest that NO may be a negative modulator of adrenal zona fasciculata steroidogenesis.

An adrenocorticotropin-regulated phosphoprotein intermediary in steroid synthesis is similar to an acyl-CoA thioesterase enzyme.

We have previously reported the purification of a phosphoprotein (p43) intermediary in steroid synthesis from adrenal zona fasciculata [Paz C., Dada, L. A., Cornejo Maciel, M. F., Mele, P. G., Cymeryng, C. B., Neuman, I., Mendez, C. F., Finkielstein, C. V., Solano, A. R., Park, M., Fischer, W. H., Towbin, H., Scartazzini, R. & Podestá, E. J. (1994) Eur J. Biochem. 224, 709-716]. Here, we describe the cloning and sequencing of a cDNA encoding p43 as well as the hormonal regulation of the p43 transcript. The protein resulted homologous to a very recently described mitochondrial peroxisome-proliferator-induced very-long-chain acyl-CoA thioesterase (MTE-I). The deduced amino acid sequence of the protein shows consensus sites for phosphorylation by different protein kinases, and a lipase serine motif. Antibodies raised against a synthetic peptide that includes the lipase serine motif and against the N-terminal region of p43 block the action of the protein. The transcript of p43 was detected in ovary of pseudopregnant rats, rat adrenal zona fasciculata and glomerulosa, mouse Leydig tumor cell line (MA-10), rat brain and human placenta. Inhibition of adrenocorticotropin hormone (ACTH) release and steroid synthesis by dexamethasone produced a dose-dependent decrease in the abundance of the adrenal transcript. The transcript was induced by in vivo stimulation of the adrenals with ACTH. The effect had a rapid onset (5 min), reached maximal stimulation (62%) at 15 min, and returned to basal levels at 30 min. The effect of ACTH on the p43 transcript was inhibited by actinomycin D and enhanced by cycloheximide. Our results provide the first evidence linking acyl-CoA thioesterases with very-long-chain specificities, and a protein intermediary in steroid synthesis, thereby supporting a regulatory role for acyl-CoA thioesterases in steroidogenic tissues.

A novel arachidonic acid-related thioesterase involved in acute steroidogenesis.

We have reported the purification of a phosphoprotein (p43) intermediary in arachidonic acid release and steroid synthesis. Here we describe the cloning and sequencing of a cDNA encoding p43 as well as the hormonal regulation of the p43 transcript. The protein is homologous to a family of novel acyl-CoA thioesterases and identical to a peroxisome proliferator-inducible mitochondrial acyl-CoA thioesterase that shows highest substrate specificity for very-long-chain fatty acids such as arachidoyl- and palmitoyl-CoA. The deduced amino acid sequence of the protein has consensus sites for phosphorylation by different protein kinases, and a putative lipase serine motif. This motif is conserved in several species such as mouse, rat and human. Antibodies raised against a synthetic peptide that includes the lipase serine motif block the action of the protein. The transcript was induced by in vivo stimulation of the adrenals with ACTH. The effect of ACTH was rapid (5 min), reached a maximum (62%) at 15 min and returned to basal levels at 30 min. The effect was inhibited by actinomycin D and enhanced by cycloheximide. Our results provide the first evidence linking acyl-CoA thioesterases, with specificity for very-long-chain acids, and a protein intermediary in steroid synthesis, thereby supporting a regulatory role for acyl-CoA thioesterases in steroidogenic tissues. Given the obligatory role of the protein in the activation of steroidogenesis through arachidonic acid release, we propose the name Arachidonic acid- Related Thioesterase Involved in Steroidogenesis (ARTISt) for p43.