P G Mele

University of Buenos Aires, Buenos Aires, Buenos Aires F.D., Argentina

Are you P G Mele?

Claim your profile

Publications (17)44.85 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: It is well accepted that protein(s) with a short half-life are required in the pathway leading to steroid synthesis following stimulation by trophic hormones. A correlation between the disappearance of several proteins in different subcellular compartments and the inhibition of steroid synthesis produced by cycloheximide (CHx) has also been shown, In the present report we describe the effect of CHx in the stimulation of steroid synthesis using a cell-free assay. Mitochondrial progesterone (P4) production was studied by recombination of the different subcellular fractions of adrenal zona fasciculate and determined by radioimmunoassay. Soluble factors from ACTH-treated adrenals produced a four-fold stimulation of mitochondrial steroidogenesis (3.0 ± 0.6 vs. 13.3 ± 0.5 ng P4/tube for control and ACTH-treated adrenals respectively). Mitochondria obtained from CHx-ACTH-treated adrenals fail to respond to soluble ACTH-dependent factors. A permeable analogue of cholesterol (22(R)-OH cholesterol) could overcome the inhibition imposed by CHx, confinning the role of mitochondrial proteins in intramitochondiial cholesterol transport. The treatment of the adrenals with CHx 10 minutes before ACTH administration abolished also the stimulation induced by the cytosol on control mitochondria (2.6 ± 0.5 vs. 13.0 ± 1.0 ng P4/tube for CHx-ACTH-trcated cytosol vs. ACTH-treated cytosol). Arachidonic acid (AA) added to CHx-ACTH-treated cytosol subdued this inhibition (10.3 ± 1.2 ng P4/tube). CHx treatment had no effect on the stimulation by ACTH of the cAMP-dependent protein kinase. These results indicate the involvement of a cycloheximide-sensitive protein in the release of AA in adrenal steroidogenesis.
    Endocrine Research. 05/2012; 22(4).
  • [Show abstract] [Hide abstract]
    ABSTRACT: Although the role of arachidonic acid (AA) in angiotensin II (ANG II)- and potassium-stimulated steroid production in zona glomerulosa cells is well documented, the mechanism responsible for AA release is not fully described. In this study we evaluated the mechanism involved in the release of intramitochondrial AA and its role in the regulation of aldosterone synthesis by ANG II in glomerulosa cells. We show that ANG II and potassium induce the expression of acyl-coenzyme A (CoA) thioesterase 2 and acyl-CoA synthetase 4, two enzymes involved in intramitochondrial AA generation/export system well characterized in other steroidogenic systems. We demonstrate that mitochondrial ATP is required for AA generation/export system, steroid production, and steroidogenic acute regulatory protein induction. We also demonstrate the role of protein tyrosine phosphatases regulating acyl-CoA synthetase 4 and steroidogenic acute regulatory protein induction, and hence ANG II-stimulated aldosterone synthesis.
    Endocrinology 05/2012; 153(7):3284-94. · 4.72 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The phospho-dephosphorylation of intermediate proteins is a key event in the regulation of steroid biosynthesis. In this regard, it is well accepted that steroidogenic hormones act through the activation of serine/threonine (Ser/Thr) protein kinases. Although many cellular processes can be regulated by a crosstalk between different kinases and phosphatases, the relationship of Ser/Thr phosphorylation and tyrosine (Tyr)-dephosphorylation is a recently explored field in the regulation of steroid synthesis. Indeed in steroidogenic cells, one of the targets of hormone-induced Ser/Thr phosphorylation is a protein tyrosine phosphatase. Whereas protein tyrosine phosphatases were initially regarded as household enzymes with constitutive activity, dephosphorylating all the substrates they encountered, evidence is now accumulating that protein tyrosine phosphatases are tightly regulated by various mechanisms. Here, we will describe the role of protein tyrosine phosphatases in the regulation of steroid biosynthesis, relating them to steroidogenic acute regulatory protein, arachidonic acid metabolism and mitochondrial rearrangement.
    Molecular and Cellular Endocrinology 12/2010; 336(1-2):63-9. · 4.04 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Evidence has been introduced linking the lipoxygenase products and steroidogenesis in Leydig cells, thereby supporting that this pathway may be a common event in the hormonal control of steroid synthesis. On the other hand, it has also been reported that lipoxygenase products of arachidonic acid (AA) may not be involved in Leydig cells steroidogenesis. In this paper, we investigated the effects of PLA2 and lipoxygenase pathway inhibitors on steroidogenesis in rat testis Leydig cells. The effects of two structurally unrelated PLA2 inhibitors (4-bromophenacyl bromide (BPB) and quinacrine) were determined. BPB blocked the LH- and Bt2cAMP-stimulated testosterone production but had no effect on 22(4)-OH-cholesterol conversion to testosterone. Quinacrine caused a dose-dependent inhibition of LH- and Bt2cAMP-induced steroidogenesis. The effects of different lipoxygenase pathway inhibitors (nordihydroguaiaretic acid (NDGA), 5,8,11,14-eicosatetraynoic acid (ETYA), caffeic acid and esculetin) have also been determined. Both NDGA and ETYA inhibited LH- and Bt2cAMP-stimulated steroid synthesis in a dose-related manner. Furthermore caffeic acid and esculetin also blocked the LH-stimulated testosterone production. Moreover, exogenous AA induced a dose-dependent increase of testosterone secretion which was inhibited by NDGA. Our results strongly support the previous concept that the lipoxygenase pathway is involved in the mechanism of action of LH on testis Leydig cells.
    Endocrine Research 08/2009; 23(1-2):15-26. · 1.03 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Arachidonic acid (AA) and the lipooxygenase products have been shown to play an obligatory role in the mechanism of action of LH and ACTH, at a point after cAMP-dependent phosphorylation. We have demonstrated the presence of a phosphoprotein (p43) that responds to cAMP signals to induce steroid synthesis in adrenocortical tissue, an effect that is blocked by phospholipase A2 inhibitors. In this report we demonstrate that p43 exhibits autoproteolytic activity that is regulated by ACTH. Protein purified from ACTH-treated animals exhibited degradation in some of the isoforms resolved on two dimensional gel electrophoresis. Proteinase inhibitors (PMSF and 1,10 phenantroline) inhibited steroid synthesis induced by ACTH and 8-Br-cAMP in intact cells. Addition of exogenous AA reverted in part that inhibition. Here we present evidence for a hormone-regulated proteolytic activity of p43 and for the inhibition of steroidogenesis by proteinase inhibitors acting prior to the release of arachidonic acid.
    Endocrine Research 07/2009; 21(1-2):281-8. · 1.03 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Molecular and Cellular Endocrinology 03/2007; 265-266:113-20. · 4.04 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Although the role of arachidonic acid (AA) in the regulation of steroidogenesis is well documented, the mechanism for AA release is not clear. Therefore, the aim of this study was to characterize the role of an acyl-CoA thioesterase (ARTISt) and an acyl-CoA synthetase as members of an alternative pathway in the regulation of the intracellular levels of AA in steroidogenesis. Purified recombinant ARTISt releases AA from arachidonoyl-CoA (AA-CoA) with a Km of 2 micro m. Antibodies raised against recombinant acyl-CoA thioesterase recognize the endogenous protein in both adrenal tissue and Y1 adrenal tumor cells by immunohistochemistry and immunocytochemistry and Western blot. Stimulation of Y1 cells with ACTH significantly stimulated endogenous mitochondrial thioesterases activity (1.8-fold). Nordihydroguaiaretic acid (NDGA), an inhibitor of AA release known to affect steroidogenesis, affects the in vitro activity of recombinant ARTISt and also the endogenous mitochondrial acyl-CoA thioesterases. ACTH-stimulated steroid synthesis in Y1 cells was significantly inhibited by a synergistic effect of NDGA and triacsin C an inhibitor of the AA-CoA synthetase. The apparent IC50 for NDGA was reduced from 50 micro m to 25, 7.5 and 4.5 micro m in the presence of 0.1, 0.5 and 2 micro m triacsin C, respectively. Our results strongly support the existence of a new pathway of AA release that operates in the regulation of steroid synthesis in adrenal cells.
    European Journal of Biochemistry 12/2002; 269(22):5599-607. · 3.58 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Endocrine Research 12/2000; 26(4):653-62. · 1.03 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    European Journal of Biochemistry 09/1998; 256(1):60-6. · 3.58 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: It is well accepted that protein(s) with a short half-life are required in the pathway leading to steroid synthesis following stimulation by trophic hormones. A correlation between the disappearance of several proteins in different subcellular compartments and the inhibition of steroid synthesis produced by cycloheximide (CHx) has also been shown. In the present report we describe the effect of CHx in the stimulation of steroid synthesis using a cell-free assay. Mitochondrial progesterone (P4) production was studied by recombination of the different subcellular fractions of adrenal zona fasciculata and determined by radioimmunoassay. Soluble factors from ACTH-treated adrenals produced a four-fold stimulation of mitochondrial steroidogenesis (3.0 +/- 0.6 vs. 13.3 +/- 0.5 ng P4/tube for control and ACTH-treated adrenals respectively). Mitochondria obtained from CHx-ACTH-treated adrenals fail to respond to soluble ACTH-dependent factors. A permeable analogue of cholesterol (22(R)-OH cholesterol) could overcome the inhibition imposed by CHx, confirming the role of mitochondrial proteins in intramitochondrial cholesterol transport. The treatment of the adrenals with CHx 10 minutes before ACTH administration abolished also the stimulation induced by the cytosol on control mitochondria (2.6 +/- 0.5 vs. 13.0 +/- 1.0 ng P4/tube for CHx-ACTH-treated cytosol vs. ACTH-treated cytosol). Arachidonic acid (AA) added to CHx-ACTH-treated cytosol subdued this inhibition (10.3 +/- 1.2 ng P4/tube). CHx treatment had no effect on the stimulation by ACTH of the cAMP-dependent protein kinase. These results indicate the involvement of a cycloheximide-sensitive protein in the release of AA in adrenal steroidogenesis.
    Endocrine Research 12/1996; 22(4):533-9. · 1.03 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We have previously isolated and partially-sequenced a soluble phosphoprotein (p43) that acts as intermediary in the stimulation of steroid synthesis. In this report we have used synthetic peptides whose sequences match those obtained from p43 to generate antipeptide antibodies and show that these antibodies bind to purified p43 protein as determined by immunoblot analysis. The presence of p43 was detected by Western blot in both steroidogenic and non-steroidogenic tissues. One of the antibodies was also used to purify p43 on immunoaffinity chromatography columns. Proteins eluting from affinity columns produce a twelve-fold stimulation of progesterone synthesis. This effect was blocked by the use of an inhibitor of phospholipase A2. These results suggest the involvement of p43 in transducing the adrenocorticotropin signal to mitochondria in zona fasciculata cells. We also describe a partial cDNA clone with a predicted amino acid sequence that matches the sequences of the internal peptides of p43.
    Endocrine Research 12/1996; 22(4):521-32. · 1.03 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We have investigated the effect of the proteinase inhibitors 1,10-phenantroline (OP) and phenylmethylsulfonyl fluoride (PMSF) on steroidogenesis in rat adrenal cortex. Both PMSF and OP inhibited adrenocorticotropin (ACTH)- and 8-Br cAMP-induced stimulation of corticosterone synthesis. On the contrary, arachidonic acid-induced stimulation of corticosterone synthesis was only slightly inhibited by PMSF and unchanged by OP. Intra- and extracellular cAMP levels were determined by radioimmunoassay. While PMSF did not affect neither the intra- nor the extracellular cAMP levels, OP decreased the intra- and extracellular levels of unstimulated as well as ACTH-stimulated cells. The site of action of the proteinase inhibitors was also studied by recombination of mitochondria with the different subcellular fractions in vitro. Addition of PMSF abolished the stimulation achieved by in vitro activation of cytosol by cAMP and PKA. On the other hand, OP completely inhibited the activation of mitochondria. Our results provide evidence for the involvement of proteinases in ACTH-induced stimulation of steroidogenesis in adrenal cortex both prior to the release of arachidonic acid and at the level of cholesterol transport from the outer to the inner mitochondrial membrane.
    Biochimica et Biophysica Acta 03/1996; 1310(3):260-8. · 4.66 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: In previous reports we have demonstrated the presence of a soluble factor that responds to cAMP signals to induce steroid synthesis in adrenocortical tissue. Here, we describe the purification of this factor from adrenal zona fasciculata cells by using a five-step procedure that includes DEAE-cellulose, gel filtration, Mono Q HPLC and Superose HPLC, and elution of the protein from SDS/PAGE. This procedure results in the purification to homogeneity of a protein of 43-kDa that retains the capacity to stimulate steroid synthesis in an in vitro recombination assay. This activity is inhibited by the use of phospholipase A2 inhibitors. Antipeptide antibodies against the N-terminal region recognize p43 as a double band on SDS/PAGE that resolves in different spots on two-dimensional gel electrophoresis. Adrenocorticotropin treatment of adrenal glands results in the appearance of multiple spots that migrated towards a lower pH compared to controls, suggesting the presence of phosphorylated and dephosphorylated forms of p43. Sequencing of the N-terminal region and internal peptides reveals no significant similarities with other proteins, suggesting that p43 is a novel protein. We conclude from our data that the isolated protein (p43) is a novel, soluble protein that acts as intermediary in adrenocorticotropin-induced stimulation of arachidonic acid release and steroid synthesis.
    European Journal of Biochemistry 10/1994; 224(2):709-16. · 3.58 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In previous reports we have demonstrated the presence of a soluble factor that responds to cAMP signals to induce steroid synthesis in adrenocortical tissue. Here, we describe the purification of this factor from adrenal zona fasciculata cells by using a five-step procedure that includes DEAE-cellulose, gel filtration, Mono Q HPLC and Superose HPLC, and elution of the protein from SDS/PAGE. This procedure results in the purification to homogeneity of a protein of 43-kDa that retains the capacity to stimulate steroid synthesis in an in vitro recombination assay. This activity is inhibited by the use of phospholipase A2, inhibitors. Antipeptide antibodies against the N-terminal region recognize p43 as a double band on SDSPAGE that resolves in different spots on two-dimensional gel electrophoresis. Adrenocorticotropin treatment of adrenal glands results in the appearence of multiple spots that migrated towards a lower pH compared to controls, suggesting the presence of phosphorylated and dephosphorylated forms of p43. Sequencing of the N-terminal region and internal peptides reveals no significant similarities with other proteins, suggesting that p43 is a novel protein.We conclude from our data that the isolated protein (p43) is a novel, soluble protein that acts as intermediary in adrenocorticotropin-induced stimulation of arachidonic acid release and steroid synthesis.
    European Journal of Biochemistry. 08/1994; 224(2):709 - 716.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Vacuolar apical compartment (VAC) is a transient organelle originally observed in Madin-Darby canine kidney (MDCK) epithelial cells impaired from forming cell-cell contacts. VACs are large vacuoles which contain microvilli and apical plasma membrane markers (among others, a 184-kDa plasma membrane protein, AP2), but exclude basolateral membrane markers. Upon reestablishment of cell-cell contacts, VACs are rapidly (within 1 h) exocytosed toward intercellular spaces, after which the apical plasma membrane drifts toward its final destination (Vega-Salas, Salas, and Rodriguez-Boulan. 1988. J. Cell Biol. 107, 1717-1728). In this work, we studied the role of cAMP as a mediator for the exocytosis of VACs. We shifted confluent cells from low to normal calcium medium (thus reestablishing cell-cell contacts and causing VAC exocytosis), a shift which resulted in a significant rise of cellular levels of both total intracellular and protein-bound cAMP. The 8-Br analog of cAMP (8-Br-cAMP) (5-50 microM) caused externalization of the intracellular compartment of AP2 as measured by radioimmunoassay. A similar effect was observed with 3-isobutyl-1-methylxanthine. 8-Br-cAMP also caused the appearance of AP2-positive VAC images in nonpermeabilized cells, namely, VACs that become accessible to extracellular antibodies upon fusion with the plasma membrane. Lanthanum, which abolishes the peak of intracellular free calcium during a calcium switch, failed to block the exocytosis. On the other hand, 12-O-tetradecanoylphorbol-13-acetate induced only a modest exocytic response. Finally, 8-Br-cAMP induced VAC exocytosis in sparse MDCK cells grown in normal calcium medium. These data indicate that cAMP is a mediator between the extracellular signal provided by cell-cell contacts and VAC exocytosis.
    Experimental Cell Research 04/1993; 205(1):171-8. · 3.56 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The mitochondria, the microsomes and the cytosol have been described as possible sites of cAMP-dependent phosphorylation. However, there has been no direct demonstration of a cAMP-dependent kinase associated with the activation of the side-chain cleavage of cholesterol. We have investigated the site of action of the cAMP-dependent kinase using a sensitive cell-free assay. Cytosol derived from cells stimulated with ACTH or cAMP was capable of increasing progesterone synthesis in isolated mitochondria when combined with the microsomal fraction. Cytosol derived from cyclase or kinase of negative mutant cells did not. Cyclic AMP and cAMP-dependent protein kinase stimulated in vitro a cytosol derived from unstimulated adrenal cells. This cytosol was capable of stimulating progesterone synthesis in isolated mitochondria. Inhibitor of cAMP-dependent protein kinase abolished the effect of the cAMP. ACTH stimulation of cytosol factors is a rapid process observable with a half maximal stimulation at about 3 pM ACTH. The effect was also abolished by inhibitor of arachidonic acid release. The function of cytosolic phosphorylation is still unclear. The effect of inhibitors of arachidonic acid release, and the necessity for the microsomal compartment in order to stimulate mitochondrial steroidogenesis, suggest that the factor in the cytosol may play a role in arachidonic acid release.
    The Journal of Steroid Biochemistry and Molecular Biology 01/1992; 39(6):889-96. · 3.98 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Luteinizing hormone (LH) and human chorionic gonadotrophin (hCG) receptors are coupled to intracellular effector systems, most notably adenylate cyclase, through guanyl nucleotide-binding proteins or G-proteins. The molecular mechanism involved in the dynamic coupling of the LH/hCG receptor however, are not known. It has been postulated that receptor aggregation at the molecular level plays a critical role in this process. There have been attempts to understand the receptor association and dissociation phenomena at the molecular level. One of them involves the participation of the major histocompatibility complex (MHC) class I antigen in the mechanism of receptor activation and/or expression. One molecular basis for these mechanisms consists of a physical interaction between MHC proteins and receptors to form "compound receptors" able to transfer a hormonal signal to the cell. Using a photo-reactive probe we demonstrated that the LH/hCG receptors and the class I antigens are closely associated in the membrane. Thus, it is possible to form covalent complexes of hCG and class I antigens through the binding of the hormone to specific receptors. These findings imply that LH/hCG receptors and the MHC class I antigens may interact at the level of the plasma membrane in the mechanism of LH action. We also performed experiments using a single cell and limiting stimulation to a patch of membrane. The results stimulating the cell in a localized area suggested that even if all components are entirely free to float there is a constraint in the localization of the receptor, G-protein, and/or the effector, supporting the constraint dissociation model. Within a limited area subunits could dissociate, but they would not be free to diffuse throughout the membrane. Moreover the concept of compartmentalization that has been utilized to explain some inconsistencies in second-messenger action now can be proved by experimental design.
    The Journal of Steroid Biochemistry and Molecular Biology 02/1991; 40(1-3):441-51. · 3.98 Impact Factor