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A proposed treatment for malaria is a combination of fosmidomycin and clindamycin. Both compounds inhibit the methylerythritol 4-phosphate (MEP) pathway, the parasitic source of farnesyl and geranylgeranyl pyrophosphate (FPP and GGPP, respectively). Both FPP and GGPP are crucial for the biosynthesis of several essential metabolites such as ubiquinone and dolichol, as well as for protein prenylation. Dietary prenols, such as farnesol (FOH) and geranylgeraniol (GGOH), can rescue parasites from MEP inhibitors, suggesting the existence of a missing pathway for prenol salvage via phosphorylation. In this study, we identified a gene in the genome of P. falciparum, encoding a transmembrane prenol kinase (PolK) involved in the salvage of FOH and GGOH. The enzyme was expressed in Saccharomyces cerevisiae, and its FOH/GGOH kinase activities were experimentally validated. Furthermore, conditional knockout parasites (Δ-PolK) were created to investigate the biological importance of the FOH/GGOH salvage pathway. Δ-PolK parasites were viable but displayed increased susceptibility to fosmidomycin. Their sensitivity to MEP inhibitors could not be rescued by adding prenols. Additionally, Δ-PolK parasites lost their capability to utilize prenols for protein prenylation. Experiments using culture medium supplemented with whole/delipidated human plasma in transgenic parasites revealed that human plasma has components that can diminish the effectiveness of fosmidomycin. Mass spectrometry tests indicated that both bovine supplements used in culture and human plasma contain GGOH. These findings suggest that the FOH/GGOH salvage pathway might offer an alternate source of isoprenoids for malaria parasites when de novo biosynthesis is inhibited. This study also identifies a novel kind of enzyme related to isoprenoid metabolism.
Assuntos
Diterpenos , Fosfomicina/análogos & derivados , Hemiterpenos , Parasitos , Pentanóis , Humanos , Animais , Bovinos , Parasitos/metabolismo , Fosfatos , Terpenos/farmacologia , Terpenos/metabolismoRESUMO
Enzyme behaviour is characterised in the laboratory using diluted solutions of enzyme. However, in vivo processes usually occur at [S T ] ≈ [E T ] ≈ K m . Furthermore, the study of enzyme action involves characterisation of inhibitors and their mechanisms. However, to date, there have been no reports proposing mathematical expressions that can be used to describe enzyme activity at high enzyme concentration apart from the simplest single substrate, irreversible case. Using a continued fraction approach, equations can be easily derived for the most common cases in monosubstrate reactions, such as irreversible or reversible reactions and effector (inhibitor or activator) kinetic interactions. These expressions are an extension of the classical Michaelis-Menten equations. A first analysis using these expressions permits to deduce some differences at high versus low enzyme concentration, such as the greater effectiveness of allosteric inhibitors compared to catalytic ones. Also, they can be used to understand catalyst saturation in a reaction. Although they can be linearised, these equations also show differences that need to be taken into account. For example, the different meaning of line intersection points in Dixon plots. All in all, these expressions may be useful tools for modelling in vivo and biotechnological processes.
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Cinética , CatáliseRESUMO
Isoprenoids are the output of the polymerization of five-carbon, branched isoprenic chains derived from isopentenyl pyrophosphate (IPP) and its isomer, dimethylallyl pyrophosphate (DMAPP). Isoprene units are consecutively condensed to form longer structures such as farnesyl and geranylgeranyl pyrophosphate (FPP and GGPP, respectively), necessary for the biosynthesis of several metabolites. Polyprenyl transferases and synthases use polyprenyl pyrophosphates as their natural substrates; however, it is known that free polyprenols, such as farnesol (FOH), and geranylgeraniol (GGOH) can be incorporated into prenylated proteins, ubiquinone, cholesterol, and dolichols. Furthermore, FOH and GGOH have been shown to block the effects of isoprenoid biosynthesis inhibitors such as fosmidomycin, bisphosphonates, or statins in several organisms. This phenomenon is the consequence of a short pathway, which was observed for the first time more than 25 years ago: the polyprenol salvage pathway, which works via the phosphorylation of FOH and GGOH. Biochemical studies in bacteria, animals, and plants suggest that this pathway can be carried out by two enzymes: a polyprenol kinase and a polyprenyl-phosphate kinase. However, to date, only a few genes have been unequivocally identified to encode these enzymes in photosynthetic organisms. Nevertheless, pieces of evidence for the importance of this pathway abound in studies related to infectious diseases, cancer, dyslipidemias, and nutrition, and to the mitigation of the secondary effects of several drugs. Furthermore, nowadays it is known that both FOH and GGOH can be incorporated via dietary sources that produce various biological effects. This review presents, in a simplified but comprehensive manner, the most important data on the FOH and GGOH salvage pathway, stressing its biomedical importance The main objective of this review is to bring to light the need to discover and characterize the kinases associated with the isoprenoid salvage pathway in animals and pathogens.
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Diterpenos , Inibidores de Hidroximetilglutaril-CoA Redutases , Animais , Farneseno Álcool/farmacologia , Diterpenos/farmacologia , Diterpenos/metabolismo , Terpenos/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologiaRESUMO
Abnormal sterols disrupt cellular functions through yet unclear mechanisms. In Saccharomyces cerevisiae, accumulation of Δ8-sterols, the same type of sterols observed in patients of Conradi-Hünermann-Happle syndrome or in fungi after amine fungicide treatment, leads to cell wall weakness. We have studied the influence of Δ8-sterols on the activity of glucan synthase I, the protein synthetizing the main polymer in fungal cell walls, its regulation by the Cell Wall Integrity (CWI) pathway, and its transport from the endoplasmic reticulum to the plasma membrane. We ascertained that the catalytic characteristics were mostly unaffected by the presence of abnormal sterols but the enzyme was partially retained in the endoplasmic reticulum, leading to glucan deficit at the cell wall. Furthermore, we observed that glucan synthase I traveled through an unconventional exocytic route to the plasma membrane that is associated with low density intracellular membranes. Also, we found out that the CWI pathway remained inactive despite low glucan levels at the cell wall. Taken together, these data suggest that Δ8-sterols affect cell walls by inhibiting unconventional secretion of proteins leading to retention and degradation of glucan synthase I, while the compensatory CWI pathway is unable to activate. These results could be instrumental to understand defects of bone development in cholesterol biosynthesis disorders and fungicide mechanisms of action.
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The sarcoplasmic/endoplasmic reticulum (SR/ER) is the main intracellular calcium (Ca2+) pool in muscle and non-muscle eukaryotic cells, respectively. The reticulum accumulates Ca2+ against its electrochemical gradient by the action of sarco/endoplasmic reticulum calcium ATPases (SERCA pumps), and the capacity of this Ca2+ store is increased by the presence of Ca2+ binding proteins in the lumen of the reticulum. A diversity of physical and chemical signals, activate the main Ca2+ release channels, i.e. ryanodine receptors (RyRs) and inositol (1, 4, 5) trisphosphate receptors (IP3Rs), to produce transient elevations of the cytoplasmic calcium concentration ([Ca2+]i) while the reticulum is being depleted of Ca2+. This picture is incomplete because it implies that the elements involved in the Ca2+ release process are acting alone and independently of each other. However, it appears that the Ca2+ released by RyRs and IP3Rs is trapped in luminal Ca2+ binding proteins (Ca2+ lattice), which are associated with these release channels, and the activation of these channels appears to facilitate that the trapped Ca2+ ions become available for release. This situation makes the initial stage of the Ca2+ release process a highly efficient one; accordingly, there is a large increase in the [Ca2+]i with minimal reductions in the bulk of the free luminal SR/ER [Ca2+] ([Ca2+]SR/ER). Additionally, it has been shown that active SERCA pumps are required for attaining this highly efficient Ca2+ release process. All these data indicate that Ca2+ release by the SR/ER is a highly regulated event and not just Ca2+ coming down its electrochemical gradient via the open release channels. One obvious advantage of this sophisticated Ca2+ release process is to avoid depletion of the ER Ca2+ store and accordingly, to prevent the activation of ER stress during each Ca2+ release event.
Assuntos
Cálcio , Retículo Endoplasmático , Retículo Sarcoplasmático , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Retículo Endoplasmático/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismoRESUMO
PURPOSE: The EPIC-26 (Expanded Prostate Cancer Index Composite-Short Form) is a validated questionnaire for measuring health related quality of life. However, the relationship between domain scores and functional outcomes remains unclear, leading to potential confusion about expectations after treatment. For instance, does a sexual function domain score of 80 mean that a patient can achieve erection sufficient for intercourse? Consequently we sought to determine the relationship between the domain score and the response to obtaining the best possible outcome for each question. MATERIALS AND METHODS: Using data from the CEASAR (Comparative Effectiveness Analysis of Surgery and Radiation) study, a multicenter, prospective study of men diagnosed with localized prostate cancer, we analyzed 11,464 EPIC-26 questionnaires from a total of 2,563 men at baseline through 60 months of followup who were treated with robotic prostatectomy, radiotherapy or active surveillance. We dichotomized every item into its best possible outcome and assessed the percent of men at each domain score who achieved the best result. RESULTS: For every EPIC-26 item the frequency of the best possible outcome was reported by domain score category. For example, a score of 80 to 100 on sexual function corresponded to 97% of men reporting erections sufficient for intercourse while at a score of 40 to 60 only 28% reported adequate erections. Also, at a score of 80 to 100 on the urinary incontinence domain 93% of men reported rarely or never leaking vs 6% at a score of 61 to 80. CONCLUSIONS: Our findings indicate a novel way to interpret EPIC-26 domain scores, demonstrating large variations in the percent of respondents reporting the best possible outcomes over narrow domain score differences. This information may be valuable when counseling men on treatment options.
Assuntos
Medidas de Resultados Relatados pelo Paciente , Neoplasias da Próstata/terapia , Qualidade de Vida , Recuperação de Função Fisiológica , Adulto , Idoso , Idoso de 80 Anos ou mais , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Prostatectomia , Radioterapia , Fatores de RiscoRESUMO
Reported experimental results, in which transient elevations of sarcoplasmic calcium levels are induced by caffeine in smooth muscle cells, apparently contradict the principle of mass conservation. The commonly accepted model assumes that the total number of Ca2+ binding sites is fixed. A former work dealing with this problem proved that assuming the presence within the reticulum of calcium sequestering proteins whose total number of calcium binding sites increases as the existent sites get occupied, is enough to explain the above referred counter-intuitive experimental results. However, no chemical explanation was given to account for this binding-site count increase. In the present work, we propose a chemical-kinetics scheme for the binding of calcium to calsequestrin (a protein found within the reticulum) and the polymerization of this protein. On the one hand, this scheme is in agreement with reported results on calsequestrin binding kinetics, but it is also fully capable of explaining the observed intriguing performance of the sarcoplasmic reticulum. We further explore the behavior of the resulting nonlinear dynamic system and discuss possible physiological implications of the proposed scheme.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Multimerização Proteica/fisiologia , Retículo Sarcoplasmático/metabolismo , Animais , Cafeína/farmacologia , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Calsequestrina/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Cinética , Modelos Teóricos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Polimerização/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Retículo Sarcoplasmático/efeitos dos fármacosRESUMO
In stably-transfected human neuroblastoma SH-SY5Y cells, we have compared the effect of activating two isoforms of 445 and 365 amino acids of the human histamine H3 receptor (hH3R445 and hH3R365) on [35S]-GTPγS binding, forskolin-induced cAMP formation, depolarization-induced increase in the intracellular concentration of Ca2+ ions ([Ca2+]i) and depolarization-evoked [3 H]-dopamine release. Maximal specific binding (Bmax) of [3 H]-N-methyl-histamine to cell membranes was 953 ± 204 and 555 ± 140 fmol/mg protein for SH-SY5Y-hH3R445 and SH-SY5Y-hH3R365 cells, respectively, with similar dissociation constants (Kd, 0.86 nM and 0.81 nM). The mRNA of the hH3R365 isoform was 40.9 ± 7.9% of the hH3R445 isoform. No differences in receptor affinity were found for the H3R ligands histamine, immepip, (R)(-)-α-methylhistamine (RAMH), A-331440, clobenpropit and ciproxifan. Both the stimulation of [35S]-GTPγS binding and the inhibition of forskolin-stimulated cAMP accumulation by the agonist RAMH were significantly larger in SH-SY5Y-hH3R445 cells ([35S]-GTPγS binding, 158.1 ± 7.5% versus 136.5 ± 3.6% for SH-SY5Y-hH3R365 cells; cAMP accumulation, -74.0 ± 4.9% versus -43.5 ± 5.3%), with no significant effect on agonist potency. In contrast, there were no differences in the efficacy and potency of RAMH to inhibit [3 H]-dopamine release evoked by 100 mM K+ (-18.9 ± 3.0% and -20.5 ± 3.3%, for SH-SY5Y-hH3R445 and SH-SY5Y-hH3R365 cells), or the inhibition of depolarization-induced increase in [Ca2+]i (S2/S1 ratios: parental cells 0.967 ± 0.069, SH-SY5Y-hH3R445 cells 0.639 ± 0.049, SH-SY5Y-hH3R365 cells 0.737 ± 0.045). These results indicate that in SH-SY5Y cells, hH3R445 and hH3R365 isoforms regulate in a differential manner the signaling pathways triggered by receptor activation.
Assuntos
Aminoácidos/metabolismo , Neuroblastoma/metabolismo , Receptores Histamínicos H3/metabolismo , Transdução de Sinais , Cálcio/metabolismo , Linhagem Celular Tumoral , Colforsina/farmacologia , AMP Cíclico/metabolismo , Dopamina/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Agonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores Histamínicos/farmacologia , Humanos , Cinética , Ligantes , Potenciais da Membrana/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trítio/metabolismoRESUMO
8-Dehydrosterols are present in a wide range of biologically relevant situations, from human rare diseases to amine fungicide-treated fungi and crops. However, the molecular bases of their toxicity are still obscure. We show here that 8-dehydrosterols, but not other sterols, affect yeast vacuole acidification through V-ATPases. Moreover, erg2Δ cells display reductions in proton pumping rates consistent with ion-transport uncoupling in vitro. Concomitantly, subunit Vph1p shows conformational changes in the presence of 8-dehydrosterols. Expression of a plant vacuolar H(+)-pumping pyrophosphatase as an alternative H(+)-pump relieves Vma(-)-like phenotypes in erg2Δ-derived mutant cells. As a consequence of these acidification defects, endo- and exo-cytic traffic deficiencies that can be alleviated with a H(+)-pumping pyrophosphatase are also observed. Despite their effect on membrane traffic, 8-dehydrosterols do not induce endoplasmic reticulum stress or assembly defects on the V-ATPase. Autophagy is a V-ATPase dependent process and erg2Δ mutants accumulate autophagic bodies under nitrogen starvation similar to Vma(-) mutants. In contrast to classical Atg(-) mutants, this defect is not accompanied by impairment of traffic through the CVT pathway, processing of Pho8Δ60p, GFP-Atg8p localisation or difficulties to survive under nitrogen starvation conditions, but it is concomitant to reduced vacuolar protease activity. All in all, erg2Δ cells are autophagy mutants albeit some of their phenotypic features differ from classical Atg(-) defective cells. These results may pave the way to understand the aetiology of sterol-related diseases, the cytotoxic effect of amine fungicides, and may explain the tolerance to these compounds observed in plants.
Assuntos
Autofagia/efeitos dos fármacos , Membrana Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Esteróis/farmacologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Autofagia/genética , Membrana Celular/genética , Humanos , Mutação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
Inorganic pyrophosphatases are required for anabolism to take place in all living organisms. Defects in genes encoding these hydrolytic enzymes are considered inviable, although their exact nature has not been studied at the cellular and molecular physiology levels. Using a conditional mutant in IPP1, the Saccharomyces cerevisiae gene encoding the cytosolic soluble pyrophosphatase, we show that respiring cells arrest in S phase upon Ipp1p deficiency, but they remain viable and resume growth if accumulated pyrophosphate is removed. However, fermenting cells arrest in G1/G0 phase and suffer massive vacuolization and eventual cell death by autophagy. Impaired NAD(+) metabolism is a major determinant of cell death in this scenario because demise can be avoided under conditions favoring accumulation of the oxidized pyridine coenzyme. These results posit that the mechanisms related to excess pyrophosphate toxicity in eukaryotes are dependent on the energy metabolism of the cell.
Assuntos
Autofagia/fisiologia , Metabolismo Energético/fisiologia , Pirofosfatase Inorgânica/metabolismo , NAD/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Difosfatos/metabolismo , Pirofosfatase Inorgânica/genética , NAD/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
BACKGROUND AND PURPOSE: Gastrointestinal tumours overexpress voltage-gated calcium (CaV3) channels (CaV3.1, 3.2 and 3.3). CaV3 channels regulate cell growth and apoptosis colorectal cancer. Gossypol, a polyphenolic aldehyde found in the cotton plant, has anti-tumour properties and inhibits CaV3 currents. A systematic study was performed on gossypol blocking mechanism on CaV3 channels and its potential anticancer effects in colon cancer cells, which express CaV3 isoforms. EXPERIMENTAL APPROACH: Transcripts for CaV3 proteins were analysed in gastrointestinal cancers using public repositories and in human colorectal cancer cell lines HCT116, SW480 and SW620. The gossypol blocking mechanism on CaV3 channels was investigated by combining heterologous expression systems and patch-clamp experiments. The anti-tumoural properties of gossypol were estimated by cell proliferation, viability and cell cycle assays. Ca2+ dynamics were evaluated with cytosolic and endoplasmic reticulum (ER) Ca2+ indicators. KEY RESULTS: High levels of CaV3 transcripts correlate with poor prognosis in gastrointestinal cancers. Gossypol blockade of CaV3 isoforms is concentration- and use-dependent interacting with the closed, activated and inactivated conformations of CaV3 channels. Gossypol and CaV3 channels down-regulation inhibit colorectal cancer cell proliferation by arresting cell cycles at the G0/G1 and G2/M phases, respectively. CaV3 channels underlie the vectorial Ca2+ uptake by endoplasmic reticulum in colorectal cancer cells. CONCLUSION AND IMPLICATIONS: Gossypol differentially blocked CaV3 channel and its anticancer activity was correlated with high levels of CaV3.1 and CaV3.2 in colorectal cancer cells. The CaV3 regulates cell proliferation and Ca2+ dynamics in colorectal cancer cells. Understanding this blocking mechanism maybe improve cancer therapies.
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Ubiquinone (UQ) is a fundamental mitochondrial electron transport chain component. This compound is synthesized as the condensation of a p-substituted benzoic acid and a polyisoprenic moiety catalyzed by the enzyme 4-hydroxybenzoate polyprenyltransferase (EC 2.5.1.39). In Plasmodium spp., this enzyme is still uncharacterized. In this work, we expressed the sequence of the Plasmodium falciparum PF3D7_0607500 gene (abbreviated as PfCOQ2) in a coq2Δ mutant strain of Saccharomyces cerevisiae, and studied the functionality of its gene product. This open reading frame could complement S. cerevisiae coq2Δ mutant growth defect on media with glycerol as a carbon source. Further, UQ was unequivocally identified in lipid extracts from this coq2Δ mutant when expressing PfCOQ2. Remarkably, UQ was detected under those conditions when S. cerevisiae cells were metabolically labeled with either [ring-14C(U)]-p-aminobenzoic acid or [ring-14C(U)]-4-hydroxybenzoic acid. However, no UQ was detected in P. falciparum if labeled with p-aminobenzoic acid. These results indicate that PfCOQ2 is a 4-hydroxybenzoate polyprenyltransferase. Further, its substrate profile seems not dissimilar to that of S. cerevisiae, but, as in other organisms, p-aminobenzoic acid does not act as an aromatic precursor in UQ biosynthesis in P. falciparum. The reason for this last feature remains to be established, but may lie upstream of PfCOQ2.
Assuntos
Plasmodium falciparum , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Plasmodium falciparum/genética , Ácido 4-AminobenzoicoRESUMO
Contact sites between the endoplasmic reticulum (ER) and mitochondria play a pivotal role in cell signaling, and the interaction between these organelles is dynamic and finely regulated. We have studied the role of ER Ca2+ concentration ([Ca2+]ER) in modulating this association in HeLa and HEK293 cells and human fibroblasts. According to Manders' coefficient, ER-mitochondria colocalization varied depending on the ER marker; it was the highest with ER-Tracker and the lowest with ER Ca2+ indicators (Mag-Fluo-4, erGAP3, and G-CEPIA1er) in both HeLa cells and human fibroblasts. Only GEM-CEPIA1er displayed a high colocalization with elongated mitochondria in HeLa cells, this ER Ca2+ indicator reveals low Ca2+ regions because this ion quenches its fluorescence. On the contrary, the typical rounded and fragmented mitochondria of HEK293 cells colocalized with Mag-Fluo-4 and, to a lesser extent, with GEM-CEPIA1er. The ablation of the three IP3R isoforms in HEK293 cells increased mitochondria-GEM-CEPIA1er colocalization. This pattern of colocalization was inversely correlated with the rate of ER Ca2+ leak evoked by thapsigargin (Tg). Moreover, Tg and Histamine in the absence of external Ca2+ increased mitochondria-ER colocalization. On the contrary, in the presence of external Ca2+, both Bafilomycin A1 and Tg reduced the mitochondria-ER interaction. Notably, knocking down MCU decreased mitochondria-ER colocalization. Overall, our data suggest that the [Ca2+] is not homogenous within the ER lumen and that mitochondria-ER interaction is modulated by the ER Ca2+ leak and the [Ca2+]i.
Assuntos
Retículo Endoplasmático , Mitocôndrias , Humanos , Células HeLa , Células HEK293 , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Tapsigargina/farmacologia , Cálcio/metabolismo , Sinalização do CálcioRESUMO
V-ATPases (vacuolar H+-ATPases) are a specific class of multi-subunit pumps that play an essential role in the generation of proton gradients across eukaryotic endomembranes. Another simpler proton pump that co-localizes with the V-ATPase occurs in plants and many protists: the single-subunit H+-PPase [H+-translocating PPase (inorganic pyrophosphatase)]. Little is known about the relative contribution of these two proteins to the acidification of intracellular compartments. In the present study, we show that the expression of a chimaeric derivative of the Arabidopsis thaliana H+-PPase AVP1, which is preferentially targeted to internal membranes of yeast, alleviates the phenotypes associated with V-ATPase deficiency. Phenotypic complementation was achieved both with a yeast strain with its V-ATPase specifically inhibited by bafilomycin A1 and with a vma1-null mutant lacking a catalytic V-ATPase subunit. Cell staining with vital fluorescent dyes showed that AVP1 recovered vacuole acidification and normalized the endocytic pathway of the vma mutant. Biochemical and immunochemical studies further demonstrated that a significant fraction of heterologous H+-PPase is located at the vacuolar membrane. These results raise the question of the occurrence of distinct proton pumps in certain single-membrane organelles, such as plant vacuoles, by proving yeast V-ATPase activity dispensability and the capability of H+-PPase to generate, by itself, physiologically suitable internal pH gradients. Also, they suggest new ways of engineering macrolide drug tolerance and outline an experimental system for testing alternative roles for fungal and animal V-ATPases, other than the mere acidification of subcellular organelles.
Assuntos
Pirofosfatase Inorgânica/metabolismo , Macrolídeos/farmacologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Proteínas de Arabidopsis , Farmacorresistência Fúngica , Bombas de Próton/metabolismo , Força Próton-Motriz , Saccharomyces cerevisiae/metabolismoRESUMO
PKC inhibitors stimulate Ca2+ release from internal stores in diverse cell types. Our data indicate that this action cannot be explained by an increased agonist-induced IP3 production or an overloaded SR Ca2+ pool in smooth muscle cells from guinea pig urinary bladder. The incubation of these cells with three different PKC inhibitors, such as Go6976, Go6983, and BIM 1, resulted in a higher SR Ca2+ leak revealed by inhibition of the SERCA pump with thapsigargin. This SR Ca2+ leakage was sensitive to protein translocation inhibitors such as emetine and anisomycin. Since this increased SR Ca2+ leak did not result in a depleted SR Ca2+ store, we have inferred there was a compensatory increase in SERCA pump activity, resulting in a higher steady-state. This new steady-state increased the frequency of Spontaneous Transient Outward Currents (STOCs), which reflect the activation of high conductance, Ca2+-sensitive potassium channels in response to RyR-mediated Ca2+ sparks. This increased STOC frequency triggered by PKC inhibition was restored to normal by inhibiting translocon-mediated Ca2+ leak with emetine. These results suggest a critical role of PKC-mediated translocon phosphorylation in regulating SR Ca2+ steady-state, which, in turn, alters SR Ca2+ releasing activity.
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In mesenteric arteries (MAs), aldosterone (ALDO) binds to the endogenous mineralocorticoid receptor (MR) and increases the expression of the voltage-gated L-type Cav1.2 channel, an essential ion channel for vascular contraction, sarcoplasmic reticulum (SR) Ca2+ store refilling, and Ca2+ spark generation. In mesenteric artery smooth muscle cells (MASMCs), Ca2+ influx through Cav1.2 is the indirect mechanism for triggering Ca2+ sparks. This process is facilitated by plasma membrane-sarcoplasmic reticulum (PM-SR) nanojunctions that drive Ca2+ from the extracellular space into the SR via Sarco/Endoplasmic Reticulum Ca2+ (SERCA) pump. Ca2+ sparks produced by clusters of Ryanodine receptors (RyRs) at PM-SR nanodomains, decrease contractility by activating large-conductance Ca2+-activated K+ channels (BKCa channels), which generate spontaneous transient outward currents (STOCs). Altogether, Cav1.2, SERCA pump, RyRs, and BKCa channels work as a functional unit at the PM-SR nanodomain, regulating intracellular Ca2+ and vascular function. However, the effect of the ALDO/MR signaling pathway on this functional unit has not been completely explored. Our results show that short-term exposure to ALDO (10 nM, 24 h) increased the expression of Cav1.2 in rat MAs. The depolarization-induced Ca2+ entry increased SR Ca2+ load, and the frequencies of both Ca2+ sparks and STOCs, while [Ca2+]cyt and vasoconstriction remained unaltered in Aldo-treated MAs. ALDO treatment significantly increased the mRNA and protein expression levels of the SERCA pump, which counterbalanced the augmented Cav1.2-mediated Ca2+ influx at the PM-SR nanodomain, increasing SR Ca2+ content, Ca2+ spark and STOC frequencies, and opposing to hyperpolarization-induced vasoconstriction while enhancing Acetylcholine-mediated vasorelaxation. This work provides novel evidence for short-term ALDO-induced upregulation of the functional unit comprising Cav1.2, SERCA2 pump, RyRs, and BKCa channels; in which the SERCA pump buffers ALDO-induced upregulation of Ca2+ entry at the superficial SR-PM nanodomain of MASMCs, preventing ALDO-triggered depolarization-induced vasoconstriction and enhancing vasodilation. Pathological conditions that lead to SERCA pump downregulation, for instance, chronic exposure to ALDO, might favor the development of ALDO/MR-mediated augmented vasoconstriction of mesenteric arteries.
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The overexpression of the Orai1 channel inhibits SOCE when using the Ca2+ readdition protocol. However, we found that HeLa cells overexpressing the Orai1 channel displayed enhanced Ca2+ entry and a limited ER depletion in response to the combination of ATP and thapsigargin (TG) in the presence of external Ca2+. As these effects require the combination of an agonist and TG, we decided to study whether the phosphorylation of Orai1 S27/S30 residues had any role using two different mutants: Orai1-S27/30A (O1-AA, phosphorylation-resistant) and Orai1-S27/30D (O1-DD, phosphomimetic). Both O1-wt and O1-AA supported enhanced Ca2+ entry, but this was not the case with O1-E106A (dead-pore mutant), O1-DD, and O1-AA-E106A, while O1-wt, O1-E106A, and O1-DD inhibited the ATP and TG-induced reduction of ER [Ca2+], suggesting that the phosphorylation of O1 S27/30 interferes with the IP3R activity. O1-wt and O1-DD displayed an increased interaction with IP3R in response to ATP and TG; however, the O1-AA channel decreased this interaction. The expression of mCherry-O1-AA increased the frequency of ATP-induced sinusoidal [Ca2+]i oscillations, while mCherry-O1-wt and mCherry-O1-DD decreased this frequency. These data suggest that the combination of ATP and TG stimulates Ca2+ entry, and the phosphorylation of Orai1 S27/30 residues by PKC reduces IP3R-mediated Ca2+ release.
Assuntos
Canais de Cálcio , Cálcio , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Células HeLa , Humanos , Proteína ORAI1/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Tapsigargina/farmacologiaRESUMO
Experimental evidence in mice models has demonstrated that a high regulator of G-protein signaling 2 (RSG2) protein levels precede an insulin resistance state. In the same context, a diet rich in saturated fatty acids induces an increase in RGS2 protein expression, which has been associated with decreased basal metabolism in mice; however, the above has not yet been analyzed in humans. For this reason, in the present study, we examined the association between RGS2 expression and insulin resistance state. The incubation with palmitic acid (PA), which inhibits insulin-mediated Akt Ser473 phosphorylation, resulted in the increased RGS2 expression in human umbilical vein endothelial-CS (HUVEC-CS) cells. The RGS2 overexpression without PA was enough to inhibit insulin-mediated Akt Ser473 phosphorylation in HUVEC-CS cells. Remarkably, the platelet RGS2 expression levels were higher in type 2 diabetes mellitus (T2DM) patients than in healthy donors. Moreover, an unbiased principal component analysis (PCA) revealed that RGS2 expression level positively correlated with glycated hemoglobin (HbA1c) and negatively with age and high-density lipoprotein cholesterol (HDL) in T2DM patients. Furthermore, PCA showed that healthy subjects segregated from T2DM patients by having lower levels of HbA1c and RGS2. These results demonstrate that RGS2 overexpression leads to decreased insulin signaling in a human endothelial cell line and is associated with poorly controlled diabetes.
RESUMO
We have previously shown that rapid inhibition of sarcoplasmic reticulum (SR) ATPase (SERCA pumps) decreases the amplitude and rate of rise (synchronization) of caffeine induced-Ca(2+) release without producing a reduction of free luminal SR Ca(2+) level in smooth muscle cells (Gómez-Viquez L, Guerrero-Serna G, García U, Guerrero-Hernández A. Biophys J 85: 370-380, 2003). Our aim was to investigate the role of luminal SR Ca(2+) content in the communication between ryanodine receptors (RyRs) and SERCA pumps. To this end, we studied the effect of SERCA pump inhibition on RyR-mediated Ca(2+) release in smooth muscle cells with overloaded SR Ca(2+) stores. Under this condition, the amplitude of RyR-mediated Ca(2+) release was not affected but the rate of rise was still decreased. In addition, the caffeine-induced Ca(2+)-dependent K(+) outward currents revealed individual events, suggesting that SERCA pump inhibition reduces the coordinated activation of RyRs. Collectively, our results indicate that SERCA pumps facilitate the activation of RyRs by a mechanism that does not involve the regulation of SR Ca(2+) content. Importantly, SERCA pumps and RyRs colocalize in smooth muscle cells, suggesting a possible local communication between these two proteins.
Assuntos
Cálcio/metabolismo , Miócitos de Músculo Liso/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Cafeína/farmacologia , Agonistas dos Canais de Cálcio/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Cobaias , Miócitos de Músculo Liso/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Tapsigargina/farmacologiaRESUMO
AIM: To evaluate the effect of supplementing a hypocaloric diet with mandarin juice, a food with a high content of antioxidants (vitamin C, flavonoids and carotenoids), on biomarkers of oxidant/antioxidant status of severe obese children. METHODS: Forty obese children were randomized into two groups pair-wise in a 4-week controlled intervention study. Both groups followed a hypocaloric diet. One group received additionally a supplementation of 500mL of 100% mandarin juice daily. Clinical data, anthropometry, dietary intake and fasting blood samples were collected at baseline and after the intervention. Lipid peroxidation was assessed by circulating levels of malondialdehyde, and protein oxidation was determined by the concentration of plasma carbonyl groups. The antioxidant defence was evaluated by red cell-reduced glutathione and plasma levels of α-tocopherol and vitamin C. RESULTS: The supplemented group experienced a decrease in the levels of malondialdehyde (-9.6%, p =0.014) and carbonyl groups (-36.1%, p =0.006) and an increase in antioxidants (α-tocopherol +16.1%, p=0.006, glutathione +36.1%, p < 0.0001, and vitamin C + 94.6%, p < 0.0001). CONCLUSION: The mandarin juice consumption with a reduced calorie diet positively affects the antioxidant defence and produces a decrease in biomarkers of oxidative stress in obese children.