Search

Contains fulltext : 184340.pdf (publisher's version ) (Open Access), The extracellular pH (pHe) is a key determinant of the cellular (micro)environment and needs to be maintained within strict boundaries to allow normal cell function. Here we used HEK293 cells to study the effects of pHe acidification (24h), induced by mitochondrial inhibitors (rotenone, antimycin A) and/or extracellular HCl addition. Lowering pHe from 7.2 to 5.8 reduced cell viability by 70% and was paralleled by a decrease in cytosolic pH (pHc), hyperpolarization of the mitochondrial membrane potential (Deltapsi), increased levels of hydroethidine-oxidizing ROS and stimulation of protein carbonylation. Co-treatment with the antioxidant alpha-tocopherol, the mitochondrial permeability transition pore (mPTP) desensitizer cyclosporin A and Necrostatin-1, a combined inhibitor of Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and Indoleamine 2,3-dioxygenase (IDO), prevented acidification-induced cell death. In contrast, the caspase inhibitor zVAD.fmk and the ferroptosis inhibitor Ferrostatin-1 were ineffective. We conclude that extracellular acidification induces necroptotic cell death in HEK293 cells and that the latter involves intracellular acidification, mitochondrial functional impairment, increased ROS levels, mPTP opening and protein carbonylation. These findings suggest that acidosis of the extracellular environment (as observed in mitochondrial disorders, ischemia, acute inflammation and cancer) can induce cell death via a ROS- and mPTP opening-mediated pathogenic mechanism.

Contains fulltext : 155167.pdf (publisher's version ) (Closed access), Hereditary sensorineural hearing loss is an extremely clinical and genetic heterogeneous disorder in humans. Especially, syndromic hearing loss is subdivided by combinations of various phenotypes, and each subtype is related to different genes. We present a new form of progressive hearing loss with migraine found to be associated with a variant in the ATP1A2 gene. The ATP1A2 gene has been reported as the major genetic cause of familial migraine by several previous studies. A Korean family presenting progressive hearing loss with migraine was ascertained. The affected members did not show any aura or other neurologic symptoms during migraine attacks, indicating on a novel phenotype of syndromic hearing loss. To identify the causative gene, linkage analysis and whole-exome sequencing were performed. A novel missense variant, c.571G>A (p.(Val191Met)), was identified in the ATP1A2 gene that showed co-segregation with the phenotype in the family. In silico studies suggest that this variant causes a change in hydrophobic interactions and thereby slightly destabilize the A-domain of Na(+)/K(+)-ATPase. However, functional studies failed to show any effect of the p.(Val191Met) substitution on the catalytic rate of this enzyme. We describe a new phenotype of progressive hearing loss with migraine associated with a variant in the ATP1A2 gene. This study suggests that a variant in Na(+)/K(+)-ATPase can be involved in both migraine and hearing loss.

Contains fulltext : 135976.pdf (publisher's version ) (Closed access), Digitalis-like compounds (DLCs) comprise a diverse group of molecules characterized by a cis-trans-cis ring-fused steroid core linked to a lactone. They have been used in the treatment of different medical problems including heart failure, where their inotropic effect on heart muscle is attributed to potent Na(+),K(+)-ATPase inhibition. Their application as drugs, however, has declined in recent past years due to their small safety margin. Since human Na(+),K(+)-ATPase is represented by four different isoforms expressed in a tissue-specific manner, one of the possibilities to improve the therapeutic index of DLCs is to exploit and amend their isoform selectivity. Here, we aimed to reveal the determinants of selectivity of the ubiquitously expressed alpha1 isoform and the more restricted alpha2 isoform toward several well-known DLCs and their hydrogenated forms. Using baculovirus to express various mutants of the alpha2 isoform, we were able to link residues Met(119) and Ser(124) to differences in affinity between the alpha1 and alpha2 isoforms to ouabain, dihydro-ouabain, digoxin, and dihydro-digoxin. We speculate that the interactions between these amino acids and DLCs affect the initial binding of these DLCs. Also, we observed isoform selectivity for DLCs containing no sugar groups.

Contains fulltext : 133904.pdf (publisher's version ) (Closed access), Sporadic hemiplegic migraine type 2 (SHM2) and familial hemiplegic migraine type 2 (FHM2) are rare forms of hemiplegic migraine caused by mutations in the Na(+),K(+)-ATPase alpha2 gene. Today, more than 70 different mutations have been linked to SHM2/FHM2, randomly dispersed over the gene. For many of these mutations, functional studies have not been performed. Here, we report the functional characterization of nine SHM2/FHM2 linked mutants that were produced in Spodoptera frugiperda (Sf)9 insect cells. We determined ouabain binding characteristics, apparent Na(+) and K(+) affinities, and maximum ATPase activity. Whereas membranes containing T345A, R834Q or R879W possessed ATPase activity significantly higher than control membranes, P796S, M829R, R834X, del 935-940 ins Ile, R937P and D999H membranes showed significant loss of ATPase activity compared to wild type enzyme. Further analysis revealed that T345A and R879W showed no changes for any of the parameters tested, whereas mutant R834Q possessed significantly decreased Na(+) and increased K(+) apparent affinities as well as decreased ATPase activity and ouabain binding. We hypothesize that the majority of the mutations studied here influence interdomain interactions by affecting formation of hydrogen bond networks or interference with the C-terminal ion pathway necessary for catalytic activity of Na(+),K(+)-ATPase, resulting in decreased functionality of astrocytes at the synaptic cleft expressing these mutants.

Contains fulltext : 128411.pdf (publisher's version ) (Closed access), De novo mutations in ATP1A3, the gene encoding the alpha3-subunit of Na(+),K(+)-ATPase, are associated with the neurodevelopmental disorder Alternating Hemiplegia of Childhood (AHC). The aim of this study was to determine the functional consequences of six ATP1A3 mutations (S137Y, D220N, I274N, D801N, E815K, and G947R) associated with AHC. Wild type and mutant Na(+),K(+)-ATPases were expressed in Sf9 insect cells using the baculovirus expression system. Ouabain binding, ATPase activity, and phosphorylation were absent in mutants I274N, E815K and G947R. Mutants S137Y and D801N were able to bind ouabain, although these mutants lacked ATPase activity, phosphorylation, and the K(+)/ouabain antagonism indicative of modifications in the cation binding site. Mutant D220N showed similar ouabain binding, ATPase activity, and phosphorylation to wild type Na(+),K(+)-ATPase. Functional impairment of Na(+),K(+)-ATPase in mutants S137Y, I274N, D801N, E815K, and G947R might explain why patients having these mutations suffer from AHC. Moreover, mutant D801N is able to bind ouabain, whereas mutant E815K shows a complete loss of function, possibly explaining the different phenotypes for these mutations.

Contains fulltext : 125451.pdf (publisher's version ) (Closed access), Familial hemiplegic migraine (FHM) is a monogenic variant of migraine with aura. One of the three known causative genes, ATP1A2, which encodes the alpha2 isoform of Na,K-ATPase, causes FHM type 2 (FHM2). Over 50 FHM2 mutations have been reported, but most have not been characterized functionally. Here we study the molecular mechanism of Na,K-ATPase alpha2 missense mutations. Mutants E700K and P786L inactivate or strongly reduce enzyme activity. Glutamic acid 700 is located in the phosphorylation (P) domain and the mutation most likely disrupts the salt bridge with Lysine 35, thereby destabilizing the interaction with the actuator (A) domain. Mutants G900R and E902K are present in the extracellular loop at the interface of the alpha and beta subunit. Both mutants likely hamper the interaction between these subunits and thereby decrease enzyme activity. Mutants E174K, R548C and R548H reduce the Na(+) and increase the K(+) affinity. Glutamic acid 174 is present in the A domain and might form a salt bridge with Lysine 432 in the nucleotide binding (N) domain, whereas Arginine 548, which is located in the N domain, forms a salt bridge with Glutamine 219 in the A domain. In the catalytic cycle, the interactions of the A and N domains affect the K(+) and Na(+) affinities, as observed with these mutants. Functional consequences were not observed for ATP1A2 mutations found in two sporadic hemiplegic migraine cases (Y9N and R879Q) and in migraine without aura (R51H and C702Y).

Item does not contain fulltext, Mutagenesis analyses and a recent crystal structure of the mammalian Na,K-ATPase have identified amino acids which are responsible for high affinity binding of cardenolides (such as ouabain) which at higher doses block the enzyme in the phosphorylated state. Genetic analysis of the Na,K-ATPase of insects adapted to cardenolides in their food plants revealed that some species possess substitutions which confer strongly increased resistance to ouabain in the mammalian enzyme such as the substitution T797A or combined substitutions at positions 111 and 122. To test for the effect of these mutations against the background of insect Na,K-ATPase, we here expressed the ouabain sensitive Na,K-ATPase alpha-subunit of Drosophila melanogaster together with the beta-subunit Nrv3 in baculovirus-infected Sf9 cells and introduced the substitutions N122H, T797A, Q111T-N122H, Q111V-N122H, all of which have been observed in cardenolide-adapted insects. While all constructs showed similar expression levels, ouabain affinity of mutated Na,K-ATPases was reduced compared to the wild-type fly enzyme. Ouabain sensitivity of the ATPase activity in inhibition assays was significantly decreased by all mutations, yet whereas the IC50 for the single mutations of N122H (61.0 muM) or T797A (63.3 muM) was increased roughly 250-fold relative to the wild-type (0.24 muM), the double mutations of Q111V-N122H (IC50 550 muM) and Q111T-N122H (IC50 583 muM) proved to be still more effective yielding a 2.250-fold increased resistance to ouabain. The double mutations identified in cardenolide-adapted insects are more effective in reducing ouabain sensitivity of the enzyme than those found naturally in the rat Na,K-ATPase (Q111R-N122D) or in mutagenesis screens of the mammalian enzyme. Obviously, the intense selection pressure on cardenolide exposed insects has resulted in very efficient substitutions that decrease cardenolide sensitivity extremely.

Item does not contain fulltext, AIMS: The BolA protein family is widespread among eukaryotes and bacteria. In Escherichia coli, BolA causes a spherical cell shape and is overexpressed during oxidative stress. Here we aim to elucidate the possible role of its human homolog BOLA1 in mitochondrial morphology and thiol redox potential regulation. RESULTS: We show that BOLA1 is a mitochondrial protein that counterbalances the effect of L-buthionine-(S,R)-sulfoximine (BSO)-induced glutathione (GSH) depletion on the mitochondrial thiol redox potential. Furthermore, overexpression of BOLA1 nullifies the effect of BSO and S-nitrosocysteine on mitochondrial morphology. Conversely, knockdown of the BOLA1 gene increases the oxidation of mitochondrial thiol groups. Supporting a role of BOLA1 in controlling the mitochondrial thiol redox potential is that BOLA1 orthologs only occur in aerobic eukaryotes. A measured interaction of BOLA1 with the mitochondrial monothiol glutaredoxin GLRX5 provides hints for potential mechanisms behind BOLA1's effect on mitochondrial redox potential. Nevertheless, we have no direct evidence for a role of GLRX5 in BOLA1's function. INNOVATION: We implicate a new protein, BOLA1, in the regulation of the mitochondrial thiol redox potential. CONCLUSION: BOLA1 is an aerobic, mitochondrial protein that prevents mitochondrial morphology aberrations induced by GSH depletion and reduces the associated oxidative shift of the mitochondrial thiol redox potential.

Contains fulltext : 110064.pdf (publisher's version ) (Closed access), AIMS: Cell regulation by signaling reactive oxygen species (sROS) is often incorrectly studied through extracellular oxidant addition. Here, we used the membrane-permeable antioxidant Trolox to examine the role of sROS in mitochondrial morphology, oxidative phosphorylation (OXPHOS), and cytosolic calcium (Ca(2+)) handling in healthy human skin fibroblasts. RESULTS AND INNOVATION: Trolox treatment reduced the levels of 5-(and-6)-chloromethyl-2',7'-dichlorodihydro-fluorescein (CM-H(2)DCF) oxidizing ROS, lowered cellular lipid peroxidation, and induced a less oxidized mitochondrial thiol redox state. This was paralleled by increased glutathione- and mitofusin-dependent mitochondrial filamentation, increased expression of fully assembled mitochondrial complex I, elevated activity of citrate synthase and OXPHOS enzymes, and a higher cellular O(2) consumption. In contrast, Trolox did not alter hydroethidium oxidation, cytosolic thiol redox state, mitochondrial NAD(P)H levels, or mitochondrial membrane potential. Whole genome expression profiling revealed that Trolox did not trigger significant changes in gene expression, suggesting that Trolox acts downstream of this process. Cytosolic Ca(2+) transients, induced by the hormone bradykinin, were of a higher amplitude and decayed faster in Trolox-treated cells. These effects were dose-dependently antagonized by hydrogen peroxide. CONCLUSIONS: Our findings suggest that Trolox-sensitive sROS are upstream regulators of mitochondrial mitofusin levels, morphology, and function in healthy human skin fibroblasts. This information not only facilitates the interpretation of antioxidant effects in cell models (of oxidative-stress), but also contributes to a better understanding of ROS-related human pathologies, including mitochondrial disorders.

1 oktober 2012, Item does not contain fulltext, Human mitochondrial complex I (CI) deficiency is associated with progressive neurological disorders. To better understand the CI pathomechanism, we here studied how deletion of the CI gene NDUFS4 affects cell metabolism. To this end we compared immortalized mouse embryonic fibroblasts (MEFs) derived from wildtype (wt) and whole-body NDUFS4 knockout (KO) mice. Mitochondria from KO cells lacked the NDUFS4 protein and mitoplasts displayed virtually no CI activity, moderately reduced CII, CIII and CIV activities and normal citrate synthase and CV (F(o)F(1)-ATPase) activity. Native electrophoresis of KO cell mitochondrial fractions revealed two distinct CI subcomplexes of ~830kDa (enzymatically inactive) and ~200kDa (active). The level of fully-assembled CII-CV was not affected by NDUFS4 gene deletion. KO cells exhibited a moderately reduced maximal and routine O(2) consumption, which was fully inhibited by acute application of the CI inhibitor rotenone. The aberrant CI assembly and reduced O(2) consumption in KO cells were fully normalized by NDUFS4 gene complementation. Cellular [NAD(+)]/[NADH] ratio, lactate production and mitochondrial tetramethyl rhodamine methyl ester (TMRM) accumulation were slightly increased in KO cells. In contrast, NDUFS4 gene deletion did not detectably alter [NADP(+)]/[NADPH] ratio, cellular glucose consumption, the protein levels of hexokinases (I and II) and phosphorylated pyruvate dehydrogenase (P-PDH), total cellular adenosine triphosphate (ATP) level, free cytosolic [ATP], cell growth rate, and reactive oxygen species (ROS) levels. We conclude that the NDUFS4 subunit is of key importance in CI stabilization and that, due to the metabolic properties of the immortalized MEFs, NDUFS4 gene deletion has only modest effects at the live cell level. This article is part of a special issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

1 mei 2012, Contains fulltext : 108799.pdf (publisher's version ) (Closed access), Digitalis-like compounds (DLCs), specific inhibitors of Na,K-ATPase, are implicated in cellular signaling. Exposure of cell cultures to ouabain, a well-known DLC, leads to up- or down regulation of various processes and involves activation of Src kinase. Since Na,K-ATPase is the only known target for DLC binding an in vitro experimental setup using highly purified Na,K-ATPase from pig kidney and commercially available recombinant Src was used to investigate the mechanism of coupling between the Na,K-ATPase and Src. Digoxin was used as a representative DLC for inhibition of Na,K-ATPase. The activation of Src kinase was measured as the degree of its autophosphorylation. It was observed that in addition to digoxin, Src activation was dependent on concentrations of other specific ligands of Na,K-ATPase: Na(+), K(+), vanadate, ATP and ADP. The magnitude of the steady-state ATPase activity therefore seemed to affect Src activation. Further experiments with an ATP regenerating system showed that the ATP/ADP ratio determined the extent of Src activation. Thus, our model system which represents the proposed very proximal part of the Na,K-ATPase-Src signaling cascade, shows that Src kinase activity is regulated by both ATP and ADP concentrations and provides no evidence for a direct interaction between Na,K-ATPase and Src.

Contains fulltext : 109628.pdf (Publisher’s version ) (Open Access), Studies employing native PAGE suggest that most nDNA-encoded CI subunits form subassemblies before assembling into holo-CI. In addition, in vitro evidence suggests that some subunits can directly exchange in holo-CI. Presently, data on the kinetics of these two incorporation modes for individual CI subunits during CI maintenance are sparse. Here, we used inducible HEK293 cell lines stably expressing AcGFP1-tagged CI subunits and quantified the amount of tagged subunit in mitoplasts and holo-CI by non-native and native PAGE, respectively, to determine their CI incorporation efficiency. Analysis of time courses of induction revealed three subunit-specific patterns. A first pattern, represented by NDUFS1, showed overlapping time courses, indicating that imported subunits predominantly incorporate into holo-CI. A second pattern, represented by NDUFV1, consisted of parallel time courses, which were, however, not quantitatively overlapping, suggesting that imported subunits incorporate at similar rates into holo-CI and CI assembly intermediates. The third pattern, represented by NDUFS3 and NDUFA2, revealed a delayed incorporation into holo-CI, suggesting their prior appearance in CI assembly intermediates and/or as free monomers. Our analysis showed the same maximum incorporation into holo-CI for NDUFV1, NDUFV2, NDUFS1, NDUFS3, NDUFS4, NDUFA2, and NDUFA12 with nearly complete loss of endogenous subunit at 24 h of induction, indicative of an equimolar stoichiometry and unexpectedly rapid turnover. In conclusion, the results presented demonstrate that newly formed nDNA-encoded CI subunits rapidly incorporate into holo-CI in a subunit-specific manner.

Contains fulltext : 98278.pdf (publisher's version ) (Closed access), The functioning and survival of mammalian cells requires an active energy metabolism. Metabolic dysfunction plays an important role in many human diseases, including diabetes, cancer, inherited mitochondrial disorders, and metabolic syndrome. The monosaccharide glucose constitutes a key source of cellular energy. Following its import across the plasma membrane, glucose is converted into pyruvate by the glycolysis pathway. Pyruvate oxidation supplies substrates for the ATP-generating mitochondrial oxidative phosphorylation (OXPHOS) system. To gain cell-biochemical knowledge about the operation and regulation of the cellular energy metabolism in the healthy and diseased state, quantitative knowledge is required about (changes in) metabolite concentrations under (non) steady-state conditions. This information can, for instance, be used to construct more realistic in silico models of cell metabolism, which facilitates understanding the consequences of metabolic dysfunction as well as on- and off-target effects of mitochondrial drugs. Here we review the current state-of-the-art live-cell quantification of two key cellular metabolites, glucose and ATP, using protein-based sensors. The latter apply the principle of FRET (fluorescence resonance energy transfer) and allow measurements in different cell compartments by fluorescence microscopy. We further summarize the properties and applications of the FRET-based sensors, their calibration, pitfalls, and future perspectives.

Contains fulltext : 96788.pdf (publisher's version ) (Closed access), Complex I (CI) of the oxidative phosphorylation system is assembled from 45 subunits encoded by both the mitochondrial and nuclear DNA. Defective mitochondrial translation is a major cause of mitochondrial disorders and proper understanding of its mechanisms and consequences is fundamental to rational treatment design. Here, we used a live cell approach to assess its consequences on CI assembly. The approach consisted of fluorescence recovery after photobleaching (FRAP) imaging of the effect of mitochondrial translation inhibition by chloramphenicol (CAP) on the dynamics of AcGFP1-tagged CI subunits NDUFV1, NDUFS3, NDUFA2 and NDUFB6 and assembly factor NDUFAF4. CAP increased the mobile fraction of the subunits, but not NDUFAF4, and decreased the amount of CI, demonstrating that CI is relatively immobile and does not associate with NDUFAF4. CAP increased the recovery kinetics of NDUFV1-AcGFP1 to the same value as obtained with AcGFP1 alone, indicative of the removal of unbound NDUFV1 from the mitochondrial matrix. Conversely, CAP decreased the mobility of NDUFS3-AcGFP1 and, to a lesser extent, NDUFB6-AcGFP1, suggestive of their enrichment in less mobile subassemblies. Little, if any, change in mobility of NDUFA2-AcGFP1 could be detected, suggesting that the dynamics of this accessory subunit of the matrix arm remains unaltered. Finally, CAP increased the mobility of NDUFAF4-AcGFP1, indicative of interaction with a more mobile membrane-bound subassembly. Our results show that the protein interactions of CI subunits and assembly factors are differently altered when mitochondrial translation is defective.

Contains fulltext : 92348.pdf (publisher's version ) (Closed access)

Contains fulltext : 81222.pdf (publisher's version ) (Closed access), Based on studies with chimeras between (non-)gastric H,K-ATPase and Na,K-ATPase, a model for the ouabain binding site has recently been presented (Qiu et al. J.Biol.Chem. 280 (2005) 32349). In this model, hydrogen bonds between specific amino acid residues of Na,K-ATPase and hydroxyl groups of ouabain play a crucial role. In the present study, a series of ouabain analogues were tested on baculovirus-expressed Na,K-ATPase and an ouabain-sensitive mutant of non-gastric H,K-ATPase (D312E/ S319G/ A778P/ I795L/ F802C). For each analogue, the results obtained by measuring ATPase inhibition and [(3)H]ouabain replacement agreed rather well. In Na,K-ATPase, strophanthidin had a 7-10 times higher and digoxin a 4-12 times lower affinity than ouabain. The results of the non-gastric H,K-ATPase mutant were rather similar to that of Na,K-ATPase with exception of dihydro-ouabain that showed a much lower affinity with the non-gastric H,K-ATPase mutant. Docking studies showed that all analogues bind to the same pocket in Na,K-ATPase. However, the amino acids to which hydrogen bonds were formed differed and depended on the availability of hydroxyl or keto groups in the ouabain analogues.

Contains fulltext : 80715.pdf (publisher's version ) (Closed access), Autosomal dominant renal hypomagnesemia (OMIM 154020), associated with hypocalciuria, has been linked to a 121G to A mutation in the FXYD2 gene. To gain insight into the molecular mechanisms linking this mutation to the clinical phenotype, we studied isolated proximal tubular cells from urine of a patient and a healthy subject. Cells were immortalized and used to assess the effects of hypertonicity-induced overexpression of FXYD2 on amount, activity and apparent affinities for Na(+), K(+) and ATP of Na,K-ATPase. Both cell lines expressed mRNA for FXYD2a and FXYD2b, and patient cells contained both the wild-type and mutated codons. FXYD2 protein expression was lower in patient cells and could be increased in both cell lines upon culturing in hyperosmotic medium but to a lesser extent in patient cells. Similarly, hyperosmotic culturing increased Na,K-ATPase protein expression and ATP hydrolyzing activity but, again, to a lesser extent in patient cells. Apparent affinities of Na,K-ATPase for Na(+), K(+) and ATP did not differ between patient and control cells or after hyperosmotic induction. We conclude that human proximal tubular cells respond to a hyperosmotic challenge with an increase in FXYD2 and Na,K-ATPase protein expression, though to a smaller absolute extent in patient cells.

Contains fulltext : 81145.pdf (publisher's version ) (Closed access), Ubiquitination plays a major role in many aspects of hematopoiesis. Alterations in ubiquitination have been implicated in hematological cancer. The ubiquitin ligase Triad1 controls the proliferation of myeloid cells. Here, we show that two RING (really interesting new gene) domains in Triad1 differentially bind ubiquitin-conjugating enzymes, UbcH7 and Ubc13. UbcH7 and Ubc13 are known to catalyze the formation of different poly-ubiquitin chains. These chains mark proteins for proteasomal degradation or serve crucial non-proteolytic functions, respectively. In line with the dual Ubc interactions, we observed that Triad1 catalyzes the formation of both types of ubiquitin chains. The biological relevance of this finding was studied by testing Triad1 mutants in myeloid clonogenic assays. Full-length Triad1 and three mutants lacking conserved domains inhibited myeloid colony formation by over 50%. Strikingly, deletion of either RING finger completely abrogated the inhibitory effect of Triad1 in clonogenic growth. We conclude that Triad1 exhibits dual ubiquitin ligase activity and that both of its RING domains are crucial to inhibit myeloid cell proliferation. The differential interaction of the RINGs with Ubcs strongly suggests that the ubiquitination mediated through UbcH7 as well as Ubc13 plays a major role in myelopoiesis.

Contains fulltext : 70919.pdf (Publisher’s version ) (Open Access), Mitochondrial complex I (CI) is a large assembly of 45 different subunits, and defects in its biogenesis are the most frequent cause of mitochondrial disorders. In vitro evidence suggests a stepwise assembly process involving pre-assembled modules. However, whether these modules also exist in vivo is as yet unresolved. To answer this question, we here applied submitochondrial fluorescence recovery after photobleaching to HEK293 cells expressing 6 GFP-tagged subunits selected on the basis of current CI assembly models. We established that each subunit was partially present in a virtually immobile fraction, possibly representing the holo-enzyme. Four subunits (NDUFV1, NDUFV2, NDUFA2, and NDUFA12) were also present as highly mobile matrix-soluble monomers, whereas, in sharp contrast, the other two subunits (NDUFB6 and NDUFS3) were additionally present in a slowly mobile fraction. In the case of the integral membrane protein NDUFB6, this fraction most likely represented one or more membrane-bound subassemblies, whereas biochemical evidence suggested that for the NDUFS3 protein this fraction most probably corresponded to a matrix-soluble subassembly. Our results provide first time evidence for the existence of CI subassemblies in mitochondria of living cells.

Contains fulltext : 70882.pdf (publisher's version ) (Closed access), Mitochondrial isolated complex I deficiency is the most frequently encountered OXPHOS defect. We report a patient with an isolated complex I deficiency expressed in skin fibroblasts as well as muscle tissue. Because the parents were consanguineous, we performed homozygosity mapping to identify homozygous regions containing candidate genes such as NDUFA2 on chromosome 5. Screening of this gene on genomic DNA revealed a mutation that interferes with correct splicing and results in the skipping of exon 2. Exon skipping was confirmed on the mRNA level. The mutation in this accessory subunit causes reduced activity and disturbed assembly of complex I. Furthermore, the mutation is associated with a mitochondrial depolarization. The expression and activity of complex I and the depolarization was (partially) rescued with a baculovirus system expressing the NDUFA2 gene.

Contains fulltext : 69840.pdf (publisher's version ) (Closed access), Autosomal dominant renal hypomagnesemia, associated with hypocalciurea, has been linked to a G to A mutation at nucleotide position 121 in the FXYD2 gene, resulting in the substitution of Gly with Arg at residue 41 of the protein. FXYD2, also called the Na,K-ATPase gamma-subunit, binds to Na,K-ATPase and influences its cation affinities. In this paper, we provide evidence for the molecular mechanism underlying the dominant character of the disorder. Co-immunoprecipitation experiments using tagged FXYD2 proteins demonstrated that wild type FXYD2 proteins oligomerise. Moreover, FXYD2-G41R also shows oligomerisation with itself and with the wild type protein. In the case of FXYD2-G41R, however, formation of homo-oligomers was prevented by addition of DTT or introduction of the C52A mutation. Finally, we demonstrated that artificial glycosylation of the wild type FXYD2 is reduced when co-expressed with FXYD2-G41R. These data indicate that binding of FXYD2-G41R to wild type FXYD2 subunit might abrogate the routing of wild type FXYD2 to the plasma membrane thus causing the dominant nature of this mutation.

Contains fulltext : 70443.pdf (publisher's version ) (Closed access), Structural biology is increasingly reliant on elevated throughput methods for protein production. In particular, development of efficient methods of heterologous production of membrane proteins is essential. Here, we describe the heterologous overproduction of 24 membrane proteins from the human pathogen Legionella pneumophila in Escherichia coli. Protein production was performed in 0.5 ml cultures in standard 24-well plates, allowing increased throughput with minimal effort. The effect of the location of a histidine purification tag was analyzed, and the effect of decreasing the length of the N- and C-terminal extensions introduced by the Gateway cloning strategy is presented. We observed that the location and length of the purification tag significantly affected protein production levels. In addition, an auto-induction protocol for membrane protein expression was designed to enhance the overproduction efficiency such that, regardless of the construct used, much higher expression was achieved when compared with standard induction approaches such as isopropyl-beta-d-thiogalactopyranoside (IPTG). All 24 targets were produced at levels exceeding 2mg/l, with 18 targets producing at levels of 5mg/l or higher. In summary, we have designed a fast and efficient process for the production of medically relevant membrane proteins with a minimum number of screening parameters.

Contains fulltext : 51463.pdf (publisher's version ) (Closed access), The primary sequence of non-gastric H,K-ATPase differs much more between species than that of Na,K-ATPase or gastric H,K-ATPase. To investigate whether this causes species-dependent differences in enzymatic properties, we co-expressed the catalytic subunit of human non-gastric H,K-ATPase in Sf9 cells with the beta(1) subunit of rat Na,K-ATPase and compared its properties with those of the rat enzyme (Swarts et al., J. Biol. Chem. 280, 33115-33122, 2005). Maximal ATPase activity was obtained with NH(4)(+) as activating cation. The enzyme was also stimulated by Na(+), but in contrast to the rat enzyme, hardly by K(+). SCH 28080 inhibited the NH(4)(+)-stimulated activity of the human enzyme much more potently than that of the rat enzyme. The steady-state phosphorylation level of the human enzyme decreased with increasing pH, [K(+)], and [Na(+)] and nearly doubled in the presence of oligomycin. Oligomycin increased the sensitivity of the phosphorylated intermediate to ADP, demonstrating that it inhibited the conversion of E(1)P to E(2)P. All three cations stimulated the dephosphorylation rate dose-dependently. Our studies support a role of the human enzyme in H(+)/Na(+) and/or H(+)/NH(4)(+) transport but not in Na(+)/K(+) transport.

Contains fulltext : 50988.pdf (Publisher’s version ) (Open Access), Coxsackievirus infection leads to a rapid reduction of the filling state of the endoplasmic reticulum (ER) and Golgi Ca2+ stores. The coxsackievirus 2B protein, a small membrane protein that localizes to the Golgi and to a lesser extent to the ER, has been proposed to play an important role in this effect by forming membrane-integral pores, thereby increasing the efflux of Ca2+ from the stores. Here, evidence is presented that supports this idea and that excludes the possibility that 2B reduces the uptake of Ca2+ into the stores. Measurement of intra-organelle-free Ca2+ in permeabilized cells revealed that the ability of 2B to reduce the Ca2+ filling state of the stores was preserved at steady ATP. Biochemical analysis in a cell-free system further showed that 2B had no adverse effect on the activity of the sarco/endoplasmic reticulum calcium ATPase, the Ca2+-ATPase that transports Ca2+ from the cytosol into the stores. To investigate whether 2B specifically affects Ca2+ homeostasis or other ion gradients, we measured the lumenal Golgi pH. Expression of 2B resulted in an increased Golgi pH, indicative for the efflux of H+ from the Golgi lumen. Together, these data support a model that 2B increases the efflux of ions from the ER and Golgi by forming membrane-integral pores. We have demonstrated that a major consequence of this activity is the inhibition of protein trafficking through the Golgi complex.

Contains fulltext : 49286.pdf (Publisher’s version ) (Open Access), P-type ATPases of the IIC subfamily exhibit large differences in sensitivity toward ouabain. This allows a strategy in which ouabain-insensitive members of this subfamily are used as template for mutational elucidation of the ouabain-binding site. With this strategy, we recently identified seven amino acids in Na,K-ATPase that conferred high affinity ouabain binding to gastric H,K-ATPase (Qiu, L. Y., Krieger, E., Schaftenaar, G., Swarts, H. G. P., Willems, P. H. G. M., De Pont, J. J. H. H. M., and Koenderink, J. B. (2005) J. Biol. Chem. 280, 32349-32355). Because important, but identical, amino acids were not recognized in that study, here we used the non-gastric H,K-ATPase, which is rather ouabain-insensitive, as template. The catalytic subunit of this enzyme, in which several amino acids from Na,K-ATPase were incorporated, was expressed with the Na,K-ATPase beta1 subunit in Xenopus laevis oocytes. A chimera containing 14 amino acids, located in M4, M5, and M6, which are unique to Na,K-ATPase, displayed high affinity ouabain binding. Four of these residues, all located in M5, appeared dispensable for high affinity binding. Individual mutation of the remaining 10 residues to their non-gastric H,K-ATPase counterparts yielded five amino acids (Glu312,Gly319, Pro778, Leu795, and Cys802) whose mutation resulted in a loss of ouabain binding. In a final gain-of-function experiment, we introduced these five amino acids in different combinations in non-gastric H,K-ATPase and demonstrated that all five were essential for high affinity ouabain binding. The non-gastric H,K-ATPase with these five mutations had a similar apparent affinity for ouabain as the wild type Na,K-ATPase and showed a 2000 times increased affinity for ouabain in the NH4+-stimulated ATPase activity in membranes of transfected Sf9 cells.

Contains fulltext : 48560.pdf (Publisher’s version ) (Open Access), We used the baculovirus/Sf9 expression system to gain new information on the mechanistic properties of the rat non-gastric H,K-ATPase, an enzyme that is implicated in potassium homeostasis. The alpha2-subunit of this enzyme (HKalpha2) required a beta-subunit for ATPase activity thereby showing a clear preference for NaKbeta1 over NaKbeta3 and gastric HKbeta. NH4(+), K+, and Na+ maximally increased the activity of HKalpha2-NaKbeta1 to 24.0, 14.2, and 5.0 micromol P(i) x mg(-1) protein x h(-1), respectively. The enzyme was inhibited by relatively high concentrations of ouabain and SCH 28080, whereas it was potently inhibited by oligomycin. From the phosphorylation level in the presence of oligomycin and the maximal NH4(+)-stimulated ATPase activity, a turnover number of 20,000 min(-1) was determined. All three cations decreased the steady-state phosphorylation level and enhanced the dephosphorylation rate, disfavoring the hypothesis that Na+ can replace H+ as the activating cation. The potency with which vanadate inhibited the cation-activated enzyme decreased in the order K+ > NH4(+) > Na+, indicating that K+ is a stronger E2 promoter than NH4(+), whereas in the presence of Na+ the enzyme is in the E1 form. For K+ and NH4(+), the E2 to E1 conformational equilibrium correlated with their efficacy in the ATPase reaction, indicating that here the transition from E2 to E1 is rate-limiting. Conversely, the low maximal ATPase activity with Na+ is explained by a poor stimulatory effect on the dephosphorylation rate. These data show that NH4(+) can replace K+ with similar affinity but higher efficacy as an extracellular activating cation in rat nongastric H,K-ATPase.

Contains fulltext : 32913.pdf (Publisher’s version ) (Open Access), Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na, K-ATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we started from the fact that a closely related enzyme, the gastric H, K-ATPase, does not bind glycosides like ouabain. Previously, we showed that a chimera of these two enzymes, in which only the M3-M4 and M5-M6 hairpins were of Na, K- ATPase, bound ouabain with high affinity (Koenderink, J. B., Hermsen, H. P. H., Swarts, H. G. P., Willems, P. H. G. M., and De Pont, J. J. H. H. M. ( 2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11209 11214). We also demonstrated that only three amino acids (Phe(783), Thr(797), and Asp(804)) present in the M5-M6 hairpin of Na,K-ATPase were sufficient to confer high affinity ouabain binding to a chimera which contained in addition the M3-M4 hairpin of Na, K-ATPase (Qiu, L. Y., Koenderink, J. B., Swarts, H. G., Willems, P. H., and De Pont, J. J. H. H. M. ( 2003) J. Biol. Chem. 278, 47240 - 47244). To further pinpoint the ouabain-binding site here we used a chimerabased loss-of-function strategy and identified four amino acids (Glu(312), Val(314), Ile(315), Gly(319)), all present in M4, as being important for ouabain binding. In a final gain-of-function study we showed that a gastric H, K- ATPase that contained Glu312, Val314, Ile315, Gly319, Phe783, Thr797, and Asp804 of Na, K- ATPase bound ouabain with the same affinity as the native enzyme. Based on the E2P crystal structure of Ca2+ -ATPase we constructed a homology model for the ouabain-binding site of Na, K- ATPase involving all seven amino acids as well as several earlier postulated amino acids.

Contains fulltext : 48060.pdf (Publisher’s version ) (Open Access), Asn792 present in M5 of gastric H,K-ATPase is highly conserved within the P-type ATPase family. A direct role in K+ binding was postulated for Na,K-ATPase but was not found in a recent model for gastric H,K-ATPase (Koenderink, J. B., Swarts, H. G. P., Willems, P. H. G. M., Krieger, E., and De Pont, J. J. H. H. M. (2004) J. Biol. Chem. 279, 16417-16424). Therefore, its role in K+ binding and E1/E2 conformational equilibrium in gastric H,K-ATPase was studied by site-directed mutagenesis and expression in Sf9 cells. N792Q and N792A, but not N792D and N792E, had a markedly reduced K+ affinity in both the ATPase and dephosphorylation reactions. In addition, N792A shifted the conformational equilibrium to the E1 form. In double mutants, the effect of N792A on K+ sensitivity was overruled by either E820Q (K(+)-independent activity) or E343D (no dephosphorylation activity). Models were made for the mutants based on the E2 structure of Ca(2+)-ATPase. In the wild-type model the acid amide group of Asn792 has hydrogen bridges to Lys791, Ala339, and Val341. Comparison of the effects of the various mutants suggests that the hydrogen bridge between the carbonyl oxygen of Asn792 and the amino group of Lys791 is essential for the K+ sensitivity and the E2 preference of wild-type enzyme. Moreover, there was a high positive correlation (r = 0.98) between the in silico calculated energy difference of the E2 form (mutants versus wild type) and the experimentally measured IC50 values for vanadate, which reflects the direction of the E2<-->E1 conformational equilibrium. These data strongly support the validity of the model in which Asn792 participates in the hydrogen bond network around the K(+)-binding pocket.

Item does not contain fulltext

Contains fulltext : 57308.pdf (Publisher’s version ) (Open Access), Homology modeling of gastric H, K-ATPase based on the E-2 model of sarcoplasmic reticulum Ca2+-ATPase (Toyoshima, C., and Nomura, H. (2002) Nature 392, 835-839) revealed the presence of a single high-affinity binding site for K+ and an E-2 form-specific salt bridge between Glu(820) (M6) and Lys(791) (M5). In the E820Q mutant this salt bridge is no longer possible, and the head group of Lys791, together with a water molecule, fills the position of the K+ ion and apparently mimics the K+-filled cation binding pocket. This gives an explanation for the K+-independent ATPase activity and dephosphorylation step of the E820Q mutant (Swarts, H. G. P., Hermsen, H. P. H., Koenderink, J. B., Schuurmans Stekhoven, F. M. A. H., and De Pont, J. J. H. H. M. ( 1998) EMBO J. 17, 3029 - 3035) and, indirectly, for its E-1 preference. The model is strongly supported by a series of reported mutagenesis studies on charged and polar amino acid residues in the membrane domain. To further test this model, Lys(791) was mutated alone and in combination with other crucial residues. In the K791A mutant, the K+ affinity was markedly reduced without altering the E-2 preference of the enzyme. The K791A mutation prevented, in contrast to the K791R mutation, the spontaneous dephosphorylation of the E820Q mutant as well as its conformational equilibrium change toward E-1. This indicates that the salt bridge is essential for high-affinity K+ binding and the E-2 preference of H,K-ATPase. Moreover, its breakage (E820Q) can generate a K+-insensitive activity and an E-1 preference. In addition, the study gives a molecular explanation for the electroneutrality of H, K-ATPases.

Item does not contain fulltext

Contains fulltext : 142680.pdf (Publisher’s version ) (Open Access), Ouabain is a glycoside that binds to and inhibits the action of Na+,K+-ATPase. Little is known, however, about the specific requirements of the protein surface for glycoside binding. Using chimeras of gastric H+,K+-ATPase and Na+,K+-ATPase, we demonstrated previously that the combined presence of transmembrane hairpins M3-M4 and M5-M6 of Na+,K+-ATPase in a backbone of H+,K+-ATPase (HN34/56) is both required and sufficient for high affinity ouabain binding. Since replacement of transmembrane hairpin M3-M4 by the N terminus up to transmembrane segment 3 (HNN3/56) resulted in a low affinity ouabain binding, hairpin M5-M6 seems to be essential for ouabain binding. To assess which residues of M5-M6 are required for ouabain action, we divided this transmembrane hairpin in seven parts and individually replaced these parts by the corresponding sequences of H+,K+-ATPase in chimera HN34/56. Three of these chimeras failed to bind ouabain following expression in Xenopus laevis oocytes. Altogether, these three chimeras contained 7 amino acids that were specific for Na+,K+-ATPase. Individual replacement of these 7 amino acids by the corresponding amino acids in H+,K+-ATPase revealed a dramatic loss of ouabain binding for F783Y, T797C, and D804E. As a proof of principle, the Na+,K+-ATPase equivalents of these 3 amino acids were introduced in different combinations in chimera HN34. The presence of all 3 amino acids appeared to be required for ouabain action. Docking of ouabain onto a three-dimensional-model of Na+,K+-ATPase suggests that Asp804, in contrast to Phe783 and Thr797, does not actually form part of the ouabain-binding pocket. Most likely, the presence of this amino acid is required for adopting of the proper conformation for ouabain binding.

Item does not contain fulltext

Item does not contain fulltext, Six double mutants of Glu(795) and Glu(820) present in transmembrane domains 5 and 6 of the alpha-subunit of rat gastric H(+),K(+)-ATPase were generated and expressed with the baculovirus expression system. Five of the six mutants exhibited an SCH 28080-sensitive ATPase activity in the absence of K(+). The activity levels decreased in the following order: E795Q/E820A > E795Q/E820Q > E795Q/E820D congruent with E795A/E820A > E795L/E820Q. The E795L/E820D mutant possessed no constitutive activity. The relative low ATPase activity of the E795L/E820Q mutant is due to its low phosphorylation rate so that the dephosphorylation step was no longer rate-limiting. The constitutively active mutants showed a much lower vanadate sensitivity than the wild-type enzyme and K(+)-sensitive mutants, indicating that these mutants have a preference for the E(1) conformation. In contrast to the constitutively active single mutants generated previously, the double mutants exhibited a high spontaneous dephosphorylation rate at 0 degrees C compared to that of the wild-type enzyme. In addition, the H(+),K(+)-ATPase inhibitor SCH 28080 increased the steady-state phosphorylation level of the constitutively active mutants, due to the formation of a stable complex with the E(2)-P form. These studies further substantiate the idea that the empty ion binding pockets of some mutants apparently mimic the K(+)-filled binding pocket of the native enzyme.

Item does not contain fulltext

Item does not contain fulltext

Item does not contain fulltext

Item does not contain fulltext

Contains fulltext : 216410.pdf (Publisher’s version ) (Open Access)

Item does not contain fulltext

Item does not contain fulltext

Item does not contain fulltext