ABSTRACT
Lactoferrin (Lf) is a bioactive milk-derived protein with remarkable wide-spectrum antifungal activity. To deepen our understanding of the molecular mechanisms underlying Lf cytotoxicity, the role of plasma membrane ergosterol- and sphingolipid-rich lipid rafts and their association with the proton pump Pma1p was explored. Pma1p was previously identified as a Lf-binding protein. Results showed that bovine Lf (bLf) perturbs ergosterol-rich lipid rafts organization by inducing intracellular accumulation of ergosterol. Using yeast mutant strains lacking lipid rafts-associated proteins or enzymes involved in the synthesis of ergosterol and sphingolipids, we found that perturbations in the composition of these membrane domains increase resistance to bLf-induced yeast cell death. Also, when Pma1p-lipid rafts association is compromised in the Pma1-10 mutant and in the absence of the Pma1p-binding protein Ast1p, the bLf killing activity is impaired. Altogether, results showed that the perturbation of lipid rafts and the inhibition of both Pma1p and V-ATPase activities mediate the antifungal activity of bLf. Since it is suggested that the combination of conventional antifungals with lipid rafts-disrupting compounds is a powerful antifungal approach, our data will help to pave the way for the use of bLf alone or in combination for the treatment/eradication of clinically and agronomically relevant yeast pathogens/fungi.
Subject(s)
Antifungal Agents/pharmacology , Lactoferrin/pharmacology , Membrane Microdomains/drug effects , Proton-Translocating ATPases/physiology , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/drug effects , Adenosine Triphosphate/metabolism , Drug Resistance, Fungal , Ergosterol/metabolism , Filipin , Green Fluorescent Proteins/analysis , Membrane Microdomains/chemistry , Point Mutation , Proton-Translocating ATPases/biosynthesis , Proton-Translocating ATPases/genetics , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/biosynthesis , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/biosynthesis , Saccharomyces cerevisiae Proteins/genetics , Vacuoles/drug effects , Vacuoles/enzymology , beta-Cyclodextrins/pharmacologyABSTRACT
Glucose controls the phosphorylation of silent information regulator 2 (Sir2), a NAD+ -dependent protein deacetylase, which regulates the expression of the ATP-dependent proton pump Pma1 and replicative lifespan (RLS) in yeast. TORC1 signaling, which is a central regulator of cell growth and lifespan, is regulated by glucose as well as nitrogen sources. In this study, we demonstrate that TORC1 signaling controls Sir2 phosphorylation through casein kinase 2 (CK2) to regulate PMA1 expression and cytoplasmic pH (pHc) in yeast. Inhibition of TORC1 signaling by either TOR1 deletion or rapamycin treatment decreased PMA1 expression, pHc, and vacuolar pH, whereas activation of TORC1 signaling by expressing constitutively active GTR1 (GTR1Q65L) resulted in the opposite phenotypes. Deletion of SIR2 or expression of a phospho-mutant form of SIR2 increased PMA1 expression, pHc, and vacuolar pH in the tor1Δ mutant, suggesting a functional interaction between Sir2 and TORC1 signaling. Furthermore, deletion of TOR1 or KNS1 encoding a LAMMER kinase decreased the phosphorylation level of Sir2, suggesting that TORC1 signaling controls Sir2 phosphorylation. It was also found that Sit4, a protein phosphatase 2A (PP2A)-like phosphatase, and Kns1 are required for TORC1 signaling to regulate PMA1 expression and that TORC1 signaling and the cyclic AMP (cAMP)/protein kinase A (PKA) pathway converge on CK2 to regulate PMA1 expression through Sir2. Taken together, these findings suggest that TORC1 signaling regulates PMA1 expression and pHc through the CK2-Sir2 axis, which is also controlled by cAMP/PKA signaling in yeast.
Subject(s)
Mechanistic Target of Rapamycin Complex 1/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Sirtuins/metabolism , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cytoplasm/metabolism , Phosphorylation , Protein Phosphatase 2/metabolism , Protein Serine-Threonine Kinases/metabolism , Proton-Translocating ATPases/biosynthesis , Proton-Translocating ATPases/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/biosynthesis , Saccharomyces cerevisiae Proteins/genetics , Signal TransductionABSTRACT
BACKGROUND: This study aimed to study the osteo-preservative effects of captopril, an inhibitor on angiotensin-converting enzyme (ACE), on bone mass, micro-architecture and histomorphology as well as the modulation of captopril on skeletal renin-angiotensin system (RAS) and regulators for bone metabolism in mice with bilateral orchidectomy. METHODS: The orchidectomized (ORX) mice were orally administered with vehicle or captopril at low dose (10mg/kg) and high dose (50mg/kg) for six weeks. The distal femoral end, the proximal tibial head and the lumbar vertebra (LV) were stained by hematoxylin and eosin, Safranin O/Fast Green and masson-trichrome. Micro-computed tomography was performed to measure bone mineral density (BMD). RESULTS: Treatment with captopril increased trabecular bone area at distal metaphysis of femur, proximal metaphysis of tibia and LV-4, moreover, high dose of captopril significantly elevated trabecular BMD of LV-2 and LV-5. The mRNA expressions of renin receptor, angiotensinogen, carbonic anhydrase II, matrix metalloproteinase-9, and tumor necrosis factor-alpha were significantly decreased in tibia of ORX mice following treatment with captopril. The administration with captopril enhanced the ratio of OPG/RANKL mRNA expression, the mRNA expression of transforming growth factor-beta and the protein expression of bradykinin receptor-1. CONCLUSIONS: The inhibition on ACE by captopril exerts beneficial effects on trabecular bone of ORX mice. The therapeutic efficacy may be attributed to the regulation of captopril on local RAS and cytokines in bone.
Subject(s)
Bone Density/drug effects , Cancellous Bone/drug effects , Captopril/pharmacology , Femur/metabolism , Lumbar Vertebrae/metabolism , Tibia/metabolism , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Angiotensinogen/biosynthesis , Animals , Carbonic Anhydrase II/biosynthesis , Dose-Response Relationship, Drug , Male , Matrix Metalloproteinase 9/biosynthesis , Mice , Orchiectomy , Osteoprotegerin/biosynthesis , Proton-Translocating ATPases/biosynthesis , RANK Ligand/biosynthesis , Receptor, Bradykinin B1/biosynthesis , Receptors, Cell Surface/biosynthesis , Renin-Angiotensin System/drug effects , Transforming Growth Factor beta/biosynthesis , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
BACKGROUND/AIMS: High-density lipoproteins (HDL) exert multiple cardioprotective functions on the arterial wall, including the promotion of endothelial cell survival and proliferation. Among mechanism contributing to endothelial protection, it has been reported that apolipoprotein A-I (apoA-I), the major protein in HDL, binds and activates the endothelial ecto-F1-ATPase receptor. This generates extracellular ADP, which in turn promotes endothelial cell survival. In this study we aimed to further investigate the signaling pathway involved downstream of apoA-I-induced ecto-F1-ATPase activation. METHODS: In human umbilical vein endothelial cells (HUVECs), pharmacological and gene silencing approaches were used to study pathways involved downstream ecto-F1-ATPase activation by apoA-I. RESULTS: ApoA-I and HDL both induced Akt phosphorylation. F1-ATPase inhibitors such as inhibitory factor 1 and oligomycin completely blocked apoA-I-induced Akt phosphorylaton and significantly blocked HDL-induced phosphorylation, indicating that this signaling pathway is dependent on ecto-F1-ATPase activation by apoA-I. Further, we were able to specify roles for the P2Y1-ADPreceptor and the PI3Kß isoform in this pathway since pharmacological inhibition and silencing of these proteins dramatically inhibited apoA-I-induced Akt phosphorylation and cell proliferation. CONCLUSION: Altogether, these data highlight a key role of the P2Y1/PI3Kß axis in endothelial cell proliferation downstream of ecto-F1-ATPase activation by apoA-I. Pharmacological targeting of this pathway could represent a promising approach to enhance vascular endothelial protection.
Subject(s)
Apolipoprotein A-I/metabolism , Class II Phosphatidylinositol 3-Kinases/genetics , Endothelial Cells/metabolism , Proton-Translocating ATPases/genetics , Receptors, Purinergic P2Y1/genetics , Adenosine Diphosphate/metabolism , Apolipoprotein A-I/genetics , Arteries/metabolism , Arteries/pathology , Cell Proliferation/genetics , Cell Wall/metabolism , Cell Wall/pathology , Class II Phosphatidylinositol 3-Kinases/biosynthesis , Endothelial Cells/drug effects , Gene Expression Regulation/genetics , Gene Silencing , Human Umbilical Vein Endothelial Cells , Humans , Lipoproteins, HDL/metabolism , Oncogene Protein v-akt/genetics , Oncogene Protein v-akt/metabolism , Proton-Translocating ATPases/biosynthesis , Receptors, Purinergic P2Y1/metabolismABSTRACT
We investigated the influence of the quaternary ammonium salt (QAS) called IM (N-(dodecyloxycarboxymethyl)- N,N,N-trimethyl ammonium chloride) on yeast cells of the parental strain and the IM-resistant mutant (EO25 IMR) growth. The phenotype of this mutant was pleiotropic. The IMR mutant exhibited resistance to ethanol, osmotic shock and oxidative stress, as well as increased sensitivity to UV. Moreover, it was noted that mutant EO25 appears to have an increased resistance to clotrimazole, ketoconazole, fluconazole, nystatin and cycloheximide. It also tolerated growth in the presence of crystal violet, DTT and metals (selenium, tin, arsenic). It was shown that the presence of IM decreased ergosterol level in mutant plasma membrane and increased its unsaturation. These results indicate changes in the cell lipid composition. Western blot analysis showed the induction of Pma1 level by IM. RT-PCR revealed an increased PMA1 expression after IM treatment.
Subject(s)
Cell Membrane/drug effects , Ergosterol/biosynthesis , Proton-Translocating ATPases/biosynthesis , Saccharomyces cerevisiae Proteins/biosynthesis , Saccharomyces cerevisiae/genetics , Ammonium Chloride/pharmacology , Ergosterol/genetics , Ethanol/toxicity , Gene Expression Regulation, Fungal/drug effects , Mutation , Osmotic Pressure , Oxidative Stress/genetics , Proton-Translocating ATPases/genetics , Quaternary Ammonium Compounds/pharmacology , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
BACKGROUND: Kufor-Rakeb syndrome (KRS) is an autosomal recessive, juvenile-onset Parkinson's disease (PD) caused by loss-of-function mutations in ATP13A2 (PARK9). Impaired energy metabolism is considered a pathogenic mechanism in PD and mitochondrial dysfunction resulting from Zn(2+) dyshomeostasis has been found in KRS patient-derived cells. In addition to mitochondrial energy production, glycolysis plays an important role in cellular energy metabolism and glucose hypometabolism has been reported in PD. However, glycolytic status in KRS remains undetermined despite its potential importance. METHODS: We assessed glycolytic function in ATP13A2-deficient KRS patient-derived human olfactory neurosphere cells and fibroblasts and determined the effect of pyruvate supplementation on improving cellular energy production. RESULTS: We found impaired extracellular acidification, reduction in pyruvate production and a decrease in the NAD(+)/NADH ratio, indicative of glycolytic dysfunction. In addition, gene expression analysis revealed an altered expression profile for several glycolytic enzymes. Glycolytic dysfunction was aggravated when the intracellular Zn(2+) concentration was increased, while ATP13A2 overexpression and pyruvate supplementation blocked the observed Zn(2+)-mediated toxicity. Moreover, supplementation with pyruvate significantly increased basal mitochondrial ATP production and abolished Zn(2+)-induced cell death. CONCLUSIONS: These findings indicate that glycolytic dysfunction contributes to pathogenesis and pyruvate supplementation improves overall cellular bioenergetics in our KRS patient-derived cell model, highlighting a therapeutic potential.
Subject(s)
Glycolysis/physiology , Neurons/metabolism , Parkinsonian Disorders/metabolism , Proton-Translocating ATPases/deficiency , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Glycolysis/drug effects , Humans , Hydrogen Peroxide/toxicity , Neurons/drug effects , Neurons/pathology , Parkinson Disease/metabolism , Parkinson Disease/pathology , Parkinsonian Disorders/pathology , Proton-Translocating ATPases/biosynthesis , Pyruvic Acid/pharmacology , Zinc/metabolismABSTRACT
During the process of intra-uterine mammalian fetal development, the oxygen supply in growing fetus is low. A rapid switch from glycolysis-based metabolism to oxidative phosphorylation (OXPHOS) must proceed during early postnatal adaptation to extra-uterine conditions. Mitochondrial biogenesis and mammalian mitochondrial F(o)F(1)-ATP synthase assembly (complex V, EC 3.6.3.14, ATPase) are complex processes regulated by multiple transcription regulators and assembly factors. Using RNA expression analysis of rat liver and skeletal tissue (Rattus norvegicus, Berkenhout, 1769), we describe the expression profiles of 20 genes involved in mitochondrial maturation and ATP synthase biogenesis in detail between the 16th and 22nd day of gestation and the first 4 days of life. We observed that the most important expression shift occurred in the liver between the 20th and 22nd day of gestation, indicating that the fetus prepares for birth about two days before parturition. The detailed mechanism regulating the perinatal adaptation process is not yet known. Deeper insights in perinatal physiological development will help to assess mitochondrial dysfunction in the broader context of cell metabolism in preterm newborns or neonates with poor adaptation to extra-uterine life.
Subject(s)
Adaptation, Physiological , Animals, Newborn/metabolism , Liver/metabolism , Muscles/metabolism , Proton-Translocating ATPases/biosynthesis , Animals , Animals, Newborn/growth & development , Female , Gene Expression Profiling , Liver/embryology , Liver/growth & development , Muscle Development , Muscles/embryology , Organelle Biogenesis , Pilot Projects , Pregnancy , Rats, WistarABSTRACT
The activity of enzymes involved in active transport of matter across lipid bilayers can conveniently be assayed by measuring their consumption of energy, such as ATP hydrolysis, while it is more challenging to directly measure their transport activities as the transported substrate is not converted into a product and only moves a few nanometers in space. Here, we describe two methods for the measurement of active proton pumping across lipid bilayers and the concomitant formation of a membrane potential, applying the dyes 9-amino-6-chloro-2-methoxyacridine (ACMA) and oxonol VI. The methods are exemplified by assaying transport of the Arabidopsis thaliana plasma membrane H(+)-ATPase (proton pump), which after heterologous expression in Saccharomyces cerevisiae and subsequent purification has been reconstituted in proteoliposomes.
Subject(s)
Adenosine Triphosphate/chemistry , Lipid Bilayers/chemistry , Proton-Translocating ATPases/biosynthesis , Adenosine Triphosphate/biosynthesis , Aminoacridines/chemistry , Arabidopsis/enzymology , Biological Transport, Active , Gene Expression Regulation, Enzymologic , Hydrolysis , Isoxazoles/chemistry , Lipid Bilayers/metabolism , Membrane Potentials , Proton-Translocating ATPases/chemistry , Proton-Translocating ATPases/isolation & purification , Saccharomyces cerevisiae/geneticsABSTRACT
Our previous studies reported evidence for aerobic ATP synthesis by myelin from both bovine brainstem and rat sciatic nerve. Considering that the optic nerve displays a high oxygen demand, here we evaluated the expression and activity of the five Respiratory Complexes in myelin purified from either bovine or murine optic nerves. Western blot analyses on isolated myelin confirmed the expression of ND4L (subunit of Complex I), COX IV (subunit of Complex IV) and ß subunit of F1Fo-ATP synthase. Moreover, spectrophotometric and in-gel activity assays on isolated myelin, as well as histochemical activity assays on both bovine and murine transversal optic nerve sections showed that the respiratory Complexes are functional in myelin and are organized in a supercomplex. Expression of oxidative phosphorylation proteins was also evaluated on bovine optic nerve sections by confocal and transmission electron microscopy. Having excluded a mitochondrial contamination of isolated myelin and considering the results form in situ analyses, it is proposed that the oxidative phosphorylation machinery is truly resident in optic myelin sheath. Data may shed a new light on the unknown trophic role of myelin sheath. It may be energy supplier for the axon, explaining why in demyelinating diseases and neuropathies, myelin sheath loss is associated with axonal degeneration.
Subject(s)
Electron Transport Chain Complex Proteins/biosynthesis , Myelin Sheath/metabolism , Optic Nerve/metabolism , Proton-Translocating ATPases/biosynthesis , Adenosine Triphosphate/biosynthesis , Animals , Axons/metabolism , Cattle , Male , Mice , Mitochondria/metabolism , NADH Dehydrogenase/biosynthesis , Neuroglia/metabolism , Oxidative PhosphorylationABSTRACT
With the advent of nanotechnology, new functional modules (e.g., nanomotors, nanoprobes) have become essential in several medical fields. Generally, mechanical modulators systems are the principal components of most cutting-edge technologies in modern biomedical applications. However, the in vivo use of motile probes has raised many concerns due to their low sensitivity and non-biocompatibility. As an alternative, biological enzymatic engines have received increased attention. In particular, ATPases, which belong to a class of motile enzymes that catalyze chemical metabolic reactions, have emerged as a promising motor due to their improved biocompatibility and performance. However, ATPases usually suffer from lower functional activity and are difficult to express recombinantly in bacteria relative to their conventional and synthetic competitors. Here, we report a novel functional modified ATPase with both a simple purification protocol and enhanced motile activity. For this mutant ATPase, a new bacterial subcloning method was established. The ATPase-encoding sequence was redesigned so that the mutant ATPase could be easily produced in an Escherichia coli system. The modified thermophilic F1-ATPase (mTF1-ATPase) demonstrated 17.8unit/mg ATPase activity. We propose that derivatives of our ATPase may enable the development of novel in vitro and in vivo synthetic medical diagnostics, as well as therapeutics.
Subject(s)
Cloning, Molecular/methods , Proton-Translocating ATPases/genetics , Escherichia coli/genetics , Proton-Translocating ATPases/biosynthesis , Recombinant Proteins/biosynthesis , Recombinant Proteins/geneticsABSTRACT
Pseudomonas fluorescensâ PICF7 is an indigenous inhabitant of olive (Olea europaea L.) rhizosphere, able to display endophytic lifestyle in roots, to induce a wide range of defence responses upon colonization of this organ and to exert effective biological control against Verticillium wilt of olive (VWO) (Verticillium dahliae). We aimed to evaluate the involvement of specific PICF7 phenotypes in olive root colonization and VWO biocontrol effectiveness by generating mutants impaired in swimming motility (fliI) or siderophore pyoverdine production (pvdI). Besides, the performance of mutants with diminished in vitro growth in potato dextrose agar medium (gltA) and cysteine (Cys) auxotrophy was also assessed. Results showed that olive root colonization and VWO biocontrol ability of the fliI, pvdI and gltA mutants did not significantly differ from that displayed by the parental strain PICF7. Consequently, altered in vitro growth, swimming motility and pyoverdine production contribute neither to PICF7 VWO suppressive effect nor to its colonization ability. In contrast, the Cys auxotroph mutant showed reduced olive root colonization capacity and lost full biocontrol efficacy. Moreover, confocal laser scanning microscopy revealed that all mutants tested were able to endophytically colonize root tissue to the same extent as wild-type PICF7, discarding these traits as relevant for its endophytic lifestyle.
Subject(s)
Antibiosis , Biological Control Agents , Olea/microbiology , Oligopeptides/biosynthesis , Pseudomonas fluorescens/physiology , Verticillium/growth & development , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Cysteine/metabolism , Oligopeptides/genetics , Phenotype , Plant Diseases/microbiology , Plant Diseases/prevention & control , Plant Roots/microbiology , Proton-Translocating ATPases/biosynthesis , Proton-Translocating ATPases/genetics , Rhizosphere , Siderophores/biosynthesis , Siderophores/geneticsABSTRACT
The Arabidopsis thaliana plasma membrane proton ATPase genes, AHA1 and AHA2, are the two most highly expressed isoforms of an 11 gene family and are collectively essential for embryo development. We report the translational fusion of a tandem affinity-purification tag to the 5' end of the AHA1 open reading frame in a genomic clone. Stable expression of TAP-tagged AHA1 in Arabidopsis rescues the embryonic lethal phenotype of endogenous double aha1/aha2 knockdowns. Western blots of SDS-PAGE and Blue Native gels show enrichment of AHA1 in plasma membrane fractions and indicate a hexameric quaternary structure. TAP-tagged AHA1 rescue lines exhibited reduced vertical root growth. Analysis of the plasma membrane and soluble proteomes identified several plasma membrane-localized proteins with alterred abundance in TAP-tagged AHA1 rescue lines compared to wild type. Using affinity-purification mass spectrometry, we uniquely identified two additional AHA isoforms, AHA9 and AHA11, which copurified with TAP-tagged AHA1. In conclusion, we have generated transgenic Arabidopsis lines in which a TAP-tagged AHA1 transgene has complemented all essential endogenous AHA1 and AHA2 functions and have shown that these plants can be used to purify AHA1 protein and to identify in planta interacting proteins by mass spectrometry.
Subject(s)
Arabidopsis Proteins/biosynthesis , Arabidopsis/metabolism , Proton-Translocating ATPases/biosynthesis , Arabidopsis/genetics , Cell Membrane/metabolism , Gene Knockdown Techniques , Isoenzymes/biosynthesis , Isoenzymes/isolation & purification , Plants, Genetically Modified/metabolism , Transformation, Genetic , TransgenesABSTRACT
To address pivotal roles of cell surface F1/FO ATP synthase in the development of acidic microenvironment in tumor tissues, we investigated effects of shear stress stimulation on the cultured human breast cancer cells, MDA-MB-231 and MDA-MB-157, or human melanoma cells, SK-Mel-1. Shear stress stimulation (0.5-5.0 dyn/cm(2)), the levels of which are similar to those produced by the interstitial flow, induced strength-dependent corelease of ATP and H(+) from the cells, which triggered CO2 gas excretion. In contrast, the same level of shear stress stimulation did not induce significant ATP release and CO2 gas excretion from the control human mammary epithelial cells (HMEC). Marked immunocytochemical and mRNA expression of cell surface F1/FO ATP synthase, vacuolar-ATPase (V-ATPase), carbonic anhydrase type IX, and ectonucleoside triphosphate diphosphohydrolase (ENTPDase) 3 were detected in MDA-MB-231 cells, but little or no expression on the HMEC. Pretreatment with cell surface F1/FO ATP synthase inhibitors, but not cell surface V-ATPase inhibitors, caused a significant reduction of the shear stress stimulation-mediated ATP release and CO2 gas excretion from MDA-MB-231 cells. The ENTPDase activity in the shear stress-loaded MDA-MB-231 cell culture medium supernatant increased significantly in a time-dependent manner. In addition, MDA-MB-231 cells displayed strong staining for purinergic 2Y1 (P2Y1) receptors on their surfaces, and the receptors partially colocalized with ENTPDase 3. These findings suggest that cell surface F1/FO ATP synthase, but not V-ATPase, may play key roles in the development of interstitial flow-mediated acidic microenvironment in tumor tissues through the shear stress stimulation-induced ATP and H(+) corelease and CO2 gas production.
Subject(s)
Cell Membrane/enzymology , Extracellular Fluid/enzymology , Proton-Translocating ATPases/biosynthesis , Shear Strength/physiology , Tumor Microenvironment/physiology , Cell Culture Techniques/methods , Cell Line, Tumor , Cell Membrane/drug effects , Enzyme Inhibitors/pharmacology , Extracellular Fluid/drug effects , Humans , Hydrogen-Ion Concentration , Proton-Translocating ATPases/antagonists & inhibitors , Shear Strength/drug effectsABSTRACT
The ATP synthase (F(O)F1) of Escherichia coli couples the translocation of protons across the cytoplasmic membrane to the synthesis or hydrolysis of ATP. This nanomotor is composed of the rotor c10γε and the stator ab2α3ß3δ. To study the assembly of this multimeric enzyme complex consisting of membrane-integral as well as peripheral hydrophilic subunits, we combined nearest neighbor analyses by intermolecular disulfide bond formation or purification of partially assembled F(O)F1 complexes by affinity chromatography with the use of mutants synthesizing different sets of F(O)F1 subunits. Together with a time-delayed in vivo assembly system, the results demonstrate that F(O)F1 is assembled in a modular way via subcomplexes, thereby preventing the formation of a functional H(+)-translocating unit as intermediate product. Surprisingly, during the biogenesis of F(O)F1, F1 subunit δ is the key player in generating stable F(O). Subunit δ serves as clamp between ab2 and c10α3ß3γε and guarantees that the open H(+) channel is concomitantly assembled within coupled F(O)F1 to maintain the low membrane proton permeability essential for viability, a general prerequisite for the assembly of multimeric H(+)-translocating enzymes.
Subject(s)
Escherichia coli Proteins/biosynthesis , Escherichia coli/enzymology , Protein Subunits/biosynthesis , Proton-Translocating ATPases/biosynthesis , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Mutation , Protein Subunits/chemistry , Protein Subunits/genetics , Proton-Translocating ATPases/chemistry , Proton-Translocating ATPases/geneticsABSTRACT
The cellular morphology of the collecting duct is altered by chronic lithium treatment. We have previously shown that lithium increases the fraction of type-A intercalated cells and lowers the fraction of principal cells along the collecting duct. Moreover, type-A intercalated cells acquire a long-row distribution pattern along the tubules. In the present study, we show that these morphological changes reverse progressively after discontinuation of lithium and finally disappear after 19 days from lithium suspension. In this time frame we have identified for the first time, in vivo, a novel cellular type positive for both intercalated and principal cells functional markers, as recognized by colabeling with H(+)-ATPase/aquaporin-4 (AQP4) and anion exchanger-1 (AE-1)/AQP2 and Foxi1/AQP4. This cell type is mainly present after 6 days of lithium washout, and it disappears in parallel with the long-row pattern of the type-A intercalated cells. It usually localizes either in the middle or at the edge of the long-row pattern. Its ultrastructure resembles the intercalated cells as shown both by differential interference contrast and by electron microscopy. The time course of appearance, the localization along the collecting duct, and the ultrastructure suggest that the cells double labeled for principal and intercalated cells markers could represent a transition element driving the conversion of intercalated cells into principal cells.
Subject(s)
Diabetes Insipidus, Nephrogenic/chemically induced , Diabetes Insipidus, Nephrogenic/pathology , Kidney Tubules, Collecting/cytology , Lithium/adverse effects , Substance Withdrawal Syndrome , Animals , Aquaporin 2/biosynthesis , Aquaporin 4/biosynthesis , Cell Proliferation , Diabetes Insipidus, Nephrogenic/metabolism , Forkhead Transcription Factors/biosynthesis , Male , Proton-Translocating ATPases/biosynthesis , Rats , Substance Withdrawal Syndrome/pathologyABSTRACT
Cardiolipin, the main anionic phospholipid in the inner mitochondrial membrane, provides shape, charge and osmotic support to this membrane due to its biophysical properties. In addition, it helps form respiratory supercomplexes and provides functionality to mitochondrial proteins. Defects in the biosynthesis or remodeling of cardiolipin have been related to severe diseases, such as Barth syndrome. Opi1p, a transcriptional repressor for most enzymes in phospholipid biosynthesis found in Saccharomyces cerevisiae, has been demonstrated not to affect the biosynthesis of this mitochondrial phospholipid. However, we found that opi1 deletion compromises mitochondrial metabolism producing severe respiratory defects. The mechanism producing this phenotype was explored and found to be a mitochondrial cardiolipin depletion of almost 50%, resulting in low cytochrome content and high mitochondrial DNA instability. The origin of this low cardiolipin content strongly correlated with the overproduction of inositol, an intrinsic phenotype of this mutation. Overall, our results show that adequate regulation of phospholipid synthesis is essential for the maintenance of mitochondrial function.
Subject(s)
Cardiolipins/metabolism , Mitochondria/metabolism , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Cardiolipins/biosynthesis , DNA, Fungal/genetics , DNA, Mitochondrial/genetics , Electron Transport Complex IV/biosynthesis , Gene Dosage/genetics , Genomic Instability/genetics , Inositol/biosynthesis , Inositol/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proton-Translocating ATPases/biosynthesis , Proton-Translocating ATPases/biosynthesis , Repressor Proteins/deficiency , Repressor Proteins/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/biosynthesis , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
The Na(+) F(1)F(O) ATP synthase of the anaerobic, acetogenic bacterium Acetobacterium woodii has a unique F(O)V(O) hybrid rotor that contains nine copies of a F(O)-like c subunit and one copy of a V(O)-like c(1) subunit with one ion binding site in four transmembrane helices whose cellular function is obscure. Since a genetic system to address the role of different c subunits is not available for this bacterium, we aimed at a heterologous expression system. Therefore, we cloned and expressed its Na(+) F(1)F(O) ATP synthase operon in Escherichia coli. A Δatp mutant of E. coli produced a functional, membrane-bound Na(+) F(1)F(O) ATP synthase that was purified in a single step after inserting a His(6)-tag to its ß subunit. The purified enzyme was competent in Na(+) transport and contained the F(O)V(O) hybrid rotor in the same stoichiometry as in A. woodii. Deletion of the atpI gene from the A. woodii operon resulted in a loss of the c ring and a mis-assembled Na(+) F(1)F(O) ATP synthase. AtpI from E. coli could not substitute AtpI from A. woodii. These data demonstrate for the first time a functional production of a F(O)V(O) hybrid rotor in E. coli and revealed that the native AtpI is required for assembly of the hybrid rotor.
Subject(s)
Acetobacterium/enzymology , Bacterial Proteins/biosynthesis , Escherichia coli/enzymology , Proton-Translocating ATPases/biosynthesis , Sodium/metabolism , Acetobacterium/genetics , Bacterial Proteins/genetics , Escherichia coli/genetics , Ion Transport/physiology , Proton-Translocating ATPases/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/geneticsABSTRACT
Phosphatidic acid is a phospholipid second messenger implicated in various cellular processes in eukaryotes. In plants, production of phosphatidic acid is triggered in response to a number of biotic and abiotic stresses. Here, we show that phosphatidic acid binds to 14-3-3 proteins, a family of regulatory proteins which bind client proteins in a phosphorylation-dependent manner. Binding of phosphatidic acid involves the same 14-3-3 region engaged in protein target binding. Consequently, micromolar phosphatidic acid concentrations significantly hamper the interaction of 14-3-3 proteins with the plasma membrane H(+)-ATPase, a well characterized plant 14-3-3 target, thus inhibiting the phosphohydrolitic enzyme activity. Moreover, the proton pump is inhibited when endogenous PA production is triggered by phospholipase D and the G protein agonist mastoparan-7. Hence, our data propose a possible mechanism involving PA that regulates 14-3-3-mediated cellular processes in response to stress.
Subject(s)
14-3-3 Proteins/metabolism , Cell Membrane/enzymology , Phosphatidic Acids/metabolism , Plant Proteins/metabolism , Proton-Translocating ATPases/biosynthesis , Stress, Physiological , Catalytic Domain , Enzyme Activation , Intercellular Signaling Peptides and Proteins , Peptides/pharmacology , Phospholipase D/metabolism , Phosphorylation , Protein Binding , Zea mays/enzymology , Zea mays/physiologyABSTRACT
Various mechanisms exist to prevent a potentially deleterious maternal immune response that results in compromising survival of semiallogeneic fetus. In pregnancy, there is a necessary early preimplantation inflammatory stage followed by a postimplantation anti-inflammatory stage. Thus, there is a biphasic 'immune response' observed during the course of pregnancy. We provide the evidence that capacitation of sperm induced the expression of a2 isoform of V-ATPase (ATP6V0A2 referred to as a2V), leukemia inhibitory factor (Lif), Il1b, and Tnf in the sperm. Capacitated sperm also released cleaved N-terminal domain of a2V-ATPase (a2NTD), which upregulates the gene expression of Lif, Il1b, Tnf, and monocyte chemotactic protein-1 (Ccl2 (Mcp1)) in the uterus. Unfertilized eggs had low a2V expression, but after fertilization, the expression of a2V increased in zygotes. This increased level of a2V expression was maintained in preimplantation embryos. Seminal plasma was necessary for upregulation of a2V expression in preimplantation embryos, as mating with seminal vesicle-deficient males failed to elicit an increase in a2V expression in preimplantation embryos. The infiltration of macrophages into the uterus was significantly increased after insemination of both sperm and seminal plasma during the preimplantation period of pregnancy. This dynamic infiltration into the uterus corresponded with the uterine a2V expression through the induction of Ccl2 expression. Furthermore, the polarization ratio of M1:M2 (pro-inflammatory/anti-inflammatory) macrophages in the uterus fluctuated from a ratio of 1.60 (day 1) to 1.45 (day 4) when female mice were inseminated with both sperm and seminal plasma. These data provide evidence that exposure to semen may initiate an inflammatory milieu by inducing a2V and cytokine/chemokine expression, which triggers the influx of macrophages into the preimplantation uterus during the onset of pregnancy and ultimately leads to successful pregnancy outcome.
Subject(s)
Blastocyst/enzymology , Fertilization , Inflammation/enzymology , Proton-Translocating ATPases/biosynthesis , Sperm Capacitation , Spermatozoa/enzymology , Uterus/enzymology , Animals , Blastocyst/immunology , Chemokine CCL2/genetics , Chemokine CCL2/metabolism , Enzyme Induction , Female , Homeobox A10 Proteins , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Inflammation/genetics , Inflammation/immunology , Inflammation Mediators/metabolism , Insemination, Artificial , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Leukemia Inhibitory Factor/genetics , Leukemia Inhibitory Factor/metabolism , Ligation , Macrophages/immunology , Male , Mice , Mice, Inbred BALB C , Pregnancy , RNA, Messenger/metabolism , Seminal Vesicles/surgery , Spermatozoa/immunology , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , Up-Regulation , Uterus/immunology , Vas Deferens/surgery , VasectomyABSTRACT
P-ATPases are membrane transporters energized by ATP. The subfamily of P5-ATPases is the least studied P-ATPases and the ion substrate specificity of the P5 subfamily is not known. Mutations of the human P5ATPase gene ATP13A2 has been shown to underlie a form of Parkinson disease (PD). We investigated the link between ATP13A2 and environmental factors related to PD development. Increasing concentrations of the synthetic polyamine analog paraquat induced a greater cytotoxic effect over CHO cells expressing ATP13A2. Paraquat-toxicity was associated with increased production of cellular reactive oxygen species and this increment was reversed by the natural polyamine spermidine. Acridine orange fluorescence intensity suggested that ATP13A2 induced the expansion of acidic vesicles that become more alkaline upon external addition of spermidine. Polyamine uptake is proposed to be initiated by a plasma membrane carrier followed by sequestration into acidic vesicles of the late endocytic compartment through an unidentified active mechanism; because ATP13A2 is located in lysosomes and late endosomes, our results open the possibility that ATP13A2 could be one of those active transporters capable of transporting polyamines like spermidine as well as its toxic analog paraquat.