Redox signaling in ischemic postconditioning protection involves PKCε and Erk1/2 pathways and converges indirectly in Nrf2 activation.

Ischemic-postconditioning (iPostC) exerts cardioprotection by preserving redox homeostasis in the reperfused heart. This protective effect has been associated with the activation of endogenous antioxidant response driven by transcription factor Nrf2 and with the activation of 'reperfusion injury salvage kinases' (RISK) as PI3K, PKC and Erk1/2. Redox homeostasis is essential for normal cell physiology since reactive oxygen species (ROS) are crucial for processes that involve protein signaling. Thus, it has become clear that not only the perturbation of redox balance to oxidative state is deleterious but also towards a reductive state contributing to pathogenesis of diseases. However, there is still a scarce knowledge about the role of ROS in the cardioprotective signals mediated by RISK in postconditioned hearts. Therefore, we studied the role of ROS as initiator of RISK signaling molecules in iPostC-conferred cardioprotection. With the aim to study the relationship between redox-dependent RISK activation and the downstream activation of the transcription factor Nrf2, we evaluated the effect of redox signaling disruption by the effect of ascorbic acid in iPostC hearts. Our results showed that PKCε and Erk1/2 activation is redox-dependent and that concurs downstream with Nrf2 deficient activation. Besides, using inhibitors we found that neither PI3K nor Erk1/2 are directly related with Nrf2 activation, indicating that these kinases have other targets. We conclude that redox signaling participates in cardioprotection triggered by iPostC through the action of kinase-dependent and -independent mechanisms and concurred with the downstream regulation of Nrf2-mediated antioxidant response to prolonged redox balance during long reperfusion.

Functional and structural roles of conserved cysteine residues in the carboxyl-terminal domain of the follicle-stimulating hormone receptor in human embryonic kidney 293 cells.

The carboxyl-terminal segment of G protein-coupled receptors has one or more conserved cysteine residues that are potential sites for palmitoylation. This posttranslational modification contributes to membrane association, internalization, and membrane targeting of proteins. In contrast to other members of the glycoprotein hormone receptor family (the LH and thyroid-stimulating hormone receptors), it is not known whether the follicle-stimulating hormone receptor (FSHR) is palmitoylated and what are the effects of abolishing its potential palmitoylation sites. In the present study, a functional analysis of the FSHR carboxyl-terminal segment cysteine residues was carried out. We constructed a series of mutant FSHRs by substituting cysteine residues with alanine, serine, or threonine individually and together at positions 629 and 655 (conserved cysteines) and 627 (nonconserved). The results showed that all three cysteine residues are palmitoylated but that only modification at Cys629 is functionally relevant. The lack of palmitoylation does not appear to greatly impair coupling to G(s) but, when absent at position 629, does significantly impair cell surface membrane expression of the partially palmitoylated receptor. All FSHR Cys mutants were capable of binding agonist with the same affinity as the wild-type receptor and internalizing on agonist stimulation. Molecular dynamics simulations at a time scale of approximately 100 nsec revealed that replacement of Cys629 resulted in structures that differed significantly from that of the wild-type receptor. Thus, deviations from wild-type conformation may potentially contribute to the severe impairment in plasma membrane expression and the modest effects on signaling exhibited by the receptors modified in this particular position.

Role of the intracellular domains of the human FSH receptor in G(alphaS) protein coupling and receptor expression.

The human (h) follicle-stimulating hormone receptor (FSHR) belongs to the superfamily of G protein-coupled receptors (GPCRs). This receptor consists of 695 amino acid residues and is preferentially coupled to the G(s) protein. This receptor is highly conserved among species (overall homology, 85%), with a 25-69% homology drop when compared to the human LH and TSH receptors. Although studies in prototypical rhodopsin/beta-adrenergic receptors suggest that multiple domains in the intracellular loops (iL) and the carboxyl-terminus (Ctail) of these receptors contribute to G protein coupling and receptor expression, there is a paucity of structure/function data on the role of these domains in FSHR function. Employing point mutations we have found that several residues present in the iL2 of the hFSHR are important for both coupling the receptor to the G(s) protein and maintaining the receptor molecule in an inactive conformation. In fact, HEK-293 cells expressing several hFSHR mutants with substitutions at R(450) (central to the highly conserved ERW triplet motif) and T(453) (a potential target for phosphorylation) failed to mediate ligand-provoked G(s) protein activation but not agonist binding, whereas substitutions at the hydrophobic L(460) (a conserved residue present in all glycoprotein hormone receptors) conferred elevated basal cAMP to the transfected cells. Thus, this particular loop apparently acts as a conformational switch for allowing the receptor to adopt an active conformation upon agonist stimulation. Residues in both ends of the iL3 are important for signal transduction in a number of GPCRs, including the FSHR. We have recently explored the importance of the reversed BBXXB motif (BXXBB; where B represents a basic residue and X a non-basic residue) present in the juxtamembrane region of the hFSHR iL3. A hFSHR mutant with all basic amino acids present in the iL3 BXXBB motif replaced by alanine failed to bind agonist and activate effector, and was expressed as an immature < or =62kDa form of the receptor. Individual substitutions of basic residues resulted in mutants that bound agonist normally but failed to activate effector when replaced at R(552) or R(556). Triple mutations in the same motif located in the NH(2)-end of the Ctail resulted in a complete inability of the receptor to bind agonist and activate effector, whereas individual substitutions resulted in decreased or virtually abolished agonist binding and cAMP accumulation, with both functions correlating with the detected levels of mature (80kDa) forms of the receptor. Thus, the BXXBB motif at the iL3 of the FSHR is essential for coupling the activated receptor to the G(s) protein, whereas the same motif in the Ctail is apparently more important for membrane expression. The role of cysteine residues present in the Ctail of the FSHR is an enigma since there are no conserved cysteines amongst LHR, FSHR and TSHR. C(629) and C(655) are conserved in the gonadotropin receptors but not in the TSHR. Alanine replacement of C(627) had no effect on hFSHR expression and function, whereas the same mutation at C(629) altered membrane expression and signal transduction. Serine or threonine substitutions of C(655) did not modify any of the parameters analyzed. In the hFSHR, C(629) may be a target for palmitoylation, and apparently it is the only cysteine residue in the Ctail domain that might play an important role in receptor function.

Effects of FSH and 17beta-estradiol on the transactivation of estrogen-regulated promoters and cell proliferation in L cells.

In the present study, we analyzed human follicle-stimulating hormone (FSH)-induced cell proliferation and transactivation of estrogen-sensitive reporter genes-in L cells stably expressing the human FSH receptor [L-(hFSHR(+)) cells]. In order to dissect the signaling pathways involved in this process, L-(hFSHR(+)) cells were transiently transfected with either the 3X-ERE-TAT-Luc or the ERE-VitA2-TK-CAT reporter genes and treated with FSH or PKA activators (cholera toxin, forskolin and 8-Br-cAMP) in the presence or absence of various kinase inhibitors. We found that FSH and all PKA activators, specifically induced transactivation of both reporter genes. Transactivation of estrogen-sensitive genes by FSH or PKA activators were blocked (approximately 90%) by H89 (PKA inhibitor) and LY294002 but not by Wortmannin (PI3-K inhibitors), 4-OH-tamoxifen, ICI182,780 or SB203580 (p38 MAPK inhibitor); PD98059 (ERK1/2 inhibitor) partially (approximately 30%) blocked the FSH-mediated effect. The combination of FSH and estradiol resulted in a synergistic effect on transactivation as well as on cell proliferation, and this enhancement was attenuated by antiestrogens. We additionally analyzed the participation of the coactivators SRC-1 and cAMP response element binding protein (CREB)-binding protein (CBP) in FSH-evoked estrogen receptor (ER)-dependent transactivation; we found that CBP but not SRC-1 potentiated FSH-induced transcriptional activation of both ER-sensitive reporters, being this effect stronger on the ERE-VitA2-TK-CAT than on the 3X-ERE-TAT-Luc reporter. Thus, in L-(hFSHR(+)) cells FSH induces transcriptional activation of estrogen-sensitive genes through an A-kinase-triggered signaling pathway, using also to a lesser extent the ERK1/2 and p38 pathways. PI3-K is not apparently involved in this FSH-mediated process since LY294002, but not Wortmannin, specifically binds ERs and completely blocks estrogen action. Presumably, CBP cooperates with the ER on genes that contain estrogen responsive elements through mechanisms involving the participation of other proteins and/or basal transcription factors (e.g. CREB), which in turn mediate the transcriptional response of estrogen-sensitive reporter genes to FSH stimulation.

Purification, characterization and kinetic properties of the multifunctional thioredoxin-glutathione reductase from Taenia crassiceps metacestode (cysticerci).

The multifunctional enzyme thioredoxin-glutathione reductase (TGR) was purified to homogeneity from the soluble fraction of Taenia crassiceps metacestode (cysticerci). Specific activities of 17.5 and 4.7 U mg(-1) were obtained with Plasmodium falciparum thioredoxin and GSSG, respectively, at pH 7.75. Under the same conditions, Km values of 17, 15, and 3 microM were respectively calculated for thioredoxin, GSSG and NADPH. The kcat/Km ratio of T. crassiceps TGR for both thioredoxin and GSSG falls in the range observed for typical thioredoxin reductases and glutathione reductases. Purified enzyme also showed glutaredoxin activity, with a specific activity of 19.2 U mg(-1) with hydroxyethyl disulfide as substrate. Both thioredoxin and GSSG disulfide reductase activities were fully inhibited by nanomolar concentrations of the gold compound auranofin, supporting the existence of an essential selenocysteine residue. Relative molecular mass of native enzyme was 136,000 +/- 3000, while the corresponding value per subunit, obtained under denaturing conditions, was 66,000 +/- 1000. These results suggest TGR exists as a dimeric protein. Isoelectric point of the enzyme was at pH 5.2. Moderate or high concentrations of GSSG, but neither thioredoxin nor NADPH, resulted in a markedly hysteretic kinetic, characterized by a lag time before the steady state velocity was reached. The magnitude of the lag time was dependent on GSSG and enzyme concentration. Preincubation of the enzyme with micromolar concentrations of GSH or DTT abolished the hysteresis, suggesting that a thiol-disulfide exchange mechanism is involved.

Contact sites from human placental mitochondria: characterization and role in progesterone synthesis.

To understand the functional compartmentalization of human placental mitochondria, we analyzed the composition and steroidogenic activity of contact sites. Several fractions containing contact sites were isolated using osmotic shock treatment and sucrose gradient centrifugation. These fractions contained various proteins and marker enzymes associated with mitochondrial membranes. The fractions containing the cytochrome P450 side chain cleavage system, cholesterol, nicotinamide adenine dinucleotide phosphate-isocitrate dehydrogenase, porin, and adenosine 5(')-triphosphate-diphosphohydrolase activity showed the capacity to synthesize progesterone. Our observations indicate that all necessary elements and enzymes for steroidogenesis are present and functional in placental mitochondrial contact sites. This organization may facilitate the metabolism of cholesterol delivered to the outer mitochondrial membrane into steroid hormones by the inner mitochondrial membrane cholesterol side chain cleavage system.

Calcium modulates the ATP and ADP hydrolysis in human placental mitochondria.

This study evaluated the effect of Ca2+ on the extramitochondrial hydrolysis of ATP and ADP by the extramitochondrial ATPase in isolated mitochondria and submitochondrial particles (SMPs) from human term placenta. The effect of different oxidizable substrates on the hydrolysis of ATP and ADP in the presence of sucrose or K+ was evaluated. Ca2+ increased phosphate release from ATP and ADP, but this stimulation showed different behavior depending on the oxidizable substrate present in the incubation media. Ca2+ stimulated the hydrolysis of ATP and ADP in the presence of sucrose. However, Ca2+ did not stimulate the hydrolysis of ADP in the medium containing K+. Ca2+ showed inhibition depending on the respiratory substrate. This study suggests that the energetic state of mitochondria controls the extramitochondrial ATPase activity, which is modulated by Ca2+ and respiratory substrates.

Differential effects of magnesium on the hydrolysis of ADP and ATP in human term placenta. Effect of substrates and potassium.

This study evaluates the effect of Mg2+ on the extramitochondrial hydrolysis of ATP and ADP by human term placental mitochondria (HPM) and submitochondrial particle (SMP). Extramitochondrial ATPase and ADPase activities were evaluated in the presence or absence of K+, and different oxidizable substrates. Mg2+ increased both ATP and ADP hydrolysis according to the experimental conditions, and this stimulation was related to the mitochondrial intactness. The ADPase activity in intact mitochondria is 100-fold higher in presence of K+, succinate and 1mM Mg2+ while this activity is only increased by two-fold on the SMP when compared to the sample without Mg2+. It is clearly demonstrated that up-regulation of these enzyme activities occur in intact mitochondria and not on the enzyme itself. The results suggest that the regulation of ATP and ADP hydrolysis is complex, and Mg2+ plays an important role in the modulation of the extramitochondrial ATPase and ADPase activities in HPM

Metabolismo energético y esteroidogénico de la plancenta humana / Energetic and steroidogenic metabolism at the human placenta

Las células del sinciciotrofoblasto obtienen el ATP a través de la glucólisis anaerobia. Sin embargo, aunque las mitocondrias de la placenta sintetizan ATP, éste no participa en los procesos citoplasmáticos. Nuestros datos muestran la presencia de una ATP-difosfohidrolsa (apirasa) asociada a las mitocondrias de la placenta, que se inhibe por vanadato y FSBA. En este trabajo proponemos la hipótesis de que la apirasa y el ATP que sintetizan las mitocondrias de las células del sinciciotrofoblasto están asociados al transporte de colesterol necesario para la síntesis de progesterona, y que el uso del ATP y la actividad de la apirasa están asociados a los puntos de unión mitocondriales.

El colesterol es esencial en el desarrollo embrionario y en el crecimiento celular / Cholesterol as an essential factor for embryonic and cellular growth

El colesterol es indispensable para el desarrollo y crecimiento celular. En varios organismos, como en los insectos, el colesterol se considera una vitamina, ya que debe ser ingerido en los alimentos. En Drosophila, el colesterol es esencial para la embriogénesis y en su ausencia estas moscas no desarrollan las alas. Esto se debe a la interacción entre el colesterol y las proteínas hedgehog. También es necesaria la presencia de colesterol en la regulación de varias enzimas, sobre todo en las que están en relación con su síntesis. Finalmente, se ha observado que algunos intermediarios de la síntesis del colesterol modulan la velocidad del ciclo celular al interactuar con el DNA nuclear y, al parecer, esta asociación también está relacionada con los procesos cancerígenos.