Gene variants associated with obstructive sleep apnea (OSA) in relation to sudden infant death syndrome (SIDS).

BACKGROUND: Both obstructive sleep apnea (OSA) and (at least a fraction of) sudden infant death syndrome (SIDS) are associated with impaired respiration. For OSA, an association with several gene variants was identified. Therefore, our hypothesis is that these polymorphisms might be of relevance in SIDS as well. METHODS: Twenty-four single nucleotide polymorphisms (SNPs) in 21 candidate genes connected to OSA, were genotyped in a total of 282 SIDS cases and 374 controls. Additionally, subgroups based on factors codetermining the SIDS risk (age, sex, season, and prone position) were established and compared as well. RESULTS: Two of the analyzed SNPs showed nominally significant differences between SIDS and control groups: rs1042714 in ADRB2 (adrenoceptor beta 2) and rs1800541 in EDN1 (endothelin 1). In the subgroup analyses, 10 further SNPs gave significant results. Nevertheless, these associations did not survive adjustment for multiple testing. CONCLUSIONS: Our results suggest that there might be a link between SIDS and OSA and its resulting respiratory and cardiovascular problems, albeit this predisposition might be dependent on the combination with other, hitherto unknown gene variants. These findings may encourage replication studies to get a better understanding of this connection.

Evaluation of variation in the phosphoinositide-3-kinase catalytic subunit alpha oncogene and breast cancer risk.

BACKGROUND: Somatic mutations in phosphoinositide-3-kinase catalytic subunit alpha (PIK3CA) are frequent in breast tumours and have been associated with oestrogen receptor (ER) expression, human epidermal growth factor receptor-2 overexpression, lymph node metastasis and poor survival. The goal of this study was to evaluate the association between inherited variation in this oncogene and risk of breast cancer. METHODS: A single-nucleotide polymorphism from the PIK3CA locus that was associated with breast cancer in a study of Caucasian breast cancer cases and controls from the Mayo Clinic (MCBCS) was genotyped in 5436 cases and 5280 controls from the Cancer Genetic Markers of Susceptibility (CGEMS) study and in 30 949 cases and 29 788 controls from the Breast Cancer Association Consortium (BCAC). RESULTS: Rs1607237 was significantly associated with a decreased risk of breast cancer in MCBCS, CGEMS and all studies of white Europeans combined (odds ratio (OR)=0.97, 95% confidence interval (CI) 0.95-0.99, P=4.6 × 10(-3)), but did not reach significance in the BCAC replication study alone (OR=0.98, 95% CI 0.96-1.01, P=0.139). CONCLUSION: Common germline variation in PIK3CA does not have a strong influence on the risk of breast cancer.

N-terminal truncation of the variable subunit stabilizes spinach ferredoxin:thioredoxin reductase.

The variable subunit of spinach ferredoxin:thioredoxin reductase (FTR) has an extended N-terminus compared to FTRs from other sources and this was proposed to contribute to the instability of the protein. We constructed two N-terminal truncation mutants of recombinant FTR by removing 16 or 24 residues from the variable subunit. The mutant proteins are readily expressed and show half-saturation values (S(0.5)) for ferredoxin and thioredoxin f comparable to WT. However, truncation increases significantly their stability. Using the stabilized FTR an exposed Cys on its thioredoxin contact surface could be substituted without altering its properties, whereas the replacement of an active site Cys by Ser completely destabilized the protein.

The ferredoxin/thioredoxin system: from discovery to molecular structures and beyond.

Experiments initiated in the early 1960s on fermentative bacteria led to the discovery of ferredoxin-dependent alpha-ketocarboxylation reactions that were later found to be key to a new cycle for the assimilation of carbon dioxide in photosynthetic bacteria (the reductive carboxylic acid or reverse citric cycle). The latter finding set the stage for the discovery of a regulatory system, the ferredoxin/thioredoxin system, functional in photosynthesis in chloroplasts and oxygen-evolving photosynthetic prokaryotes. The chloroplast research led to a description of the extraplastidic NADP/thioredoxin system that is now known to function in heterotrophic plant processes such as seed germination and self-incompatibility. Extensions of the fundamental research have begun to open doors to the broad application of thioredoxin in technology and medicine.

Oxidation-reduction and activation properties of chloroplast fructose 1,6-bisphosphatase with mutated regulatory site.

The concentration of Mg(2+) required for optimal activity of chloroplast fructose 1,6-bisphosphatase (FBPase) decreases when a disulfide, located on a flexible loop containing three conserved cysteines, is reduced by the ferredoxin/thioredoxin system. Mutation of either one of two regulatory cysteines in this loop (Cys155 and Cys174 in spinach FBPase) produces an enzyme with a S(0.5) for Mg(2+) (0.6 mM) identical to that observed for the reduced WT enzyme and significantly lower than the S(0.5) of 12.2 mM of oxidized WT enzyme. E(m) for the regulatory disulfide in WT spinach FBPase is -305 mV at pH 7.0, with an E(m) vs pH dependence of -59 mV/pH unit, from pH 5.5 to 8.5. Aerobic storage of the C174S mutant produces a nonphysiological Cys155/Cys179 disulfide, rendering the enzyme partially dependent on activation by thioredoxin. Circular dichroism spectra and thiol titrations provide supporting evidence for the formation of nonphysiological disulfide bonds. Mutation of Cys179, the third conserved cysteine, produces FBPase that behaves very much like WT enzyme but which is more rapidly activated by thioredoxin f, perhaps because the E(m) of the regulatory disulfide in the mutant has been increased to -290 mV (isopotential with thioredoxin f). Structural changes in the regulatory loop lower S(0.5) for Mg(2+) to 3.2 mM for the oxidized C179S mutant. These results indicate that opening the regulatory disulfide bridge, either through reduction or mutation, produces structural changes that greatly decrease S(0.5) for Mg(2+) and that only two of the conserved cysteines play a physiological role in regulation of FBPase.

Plant adenosine 5'-phosphosulfate reductase is a novel iron-sulfur protein.

Adenosine 5'-phosphosulfate reductase (APR) catalyzes the two-electron reduction of adenosine 5'-phosphosulfate to sulfite and AMP, which represents the key step of sulfate assimilation in higher plants. Recombinant APRs from both Lemna minor and Arabidopsis thaliana were overexpressed in Escherichia coli and isolated as yellow-brown proteins. UV-visible spectra of these recombinant proteins indicated the presence of iron-sulfur centers, whereas flavin was absent. This result was confirmed by quantitative analysis of iron and acid-labile sulfide, suggesting a [4Fe-4S] cluster as the cofactor. EPR spectroscopy of freshly purified enzyme showed, however, only a minor signal at g = 2.01. Therefore, Mössbauer spectra of (57)Fe-enriched APR were obtained at 4.2 K in magnetic fields of up to 7 tesla, which were assigned to a diamagnetic [4Fe-4S](2+) cluster. This cluster was unusual because only three of the iron sites exhibited the same Mössbauer parameters. The fourth iron site gave, because of the bistability of the fit, a significantly smaller isomer shift or larger quadrupole splitting than the other three sites. Thus, plant assimilatory APR represents a novel type of adenosine 5'-phosphosulfate reductase with a [4Fe-4S] center as the sole cofactor, which is clearly different from the dissimilatory adenosine 5'-phosphosulfate reductases found in sulfate reducing bacteria.

Heterodimer formation between thioredoxin f and fructose 1,6-bisphosphatase from spinach chloroplasts.

Chloroplast fructose 1,6-bisphosphatase (FBPase) is activated by reduction of a regulatory disulfide through thioredoxin f (Trx f). In the course of this reduction a transient mixed disulfide is formed linking covalently Trx f with FBPase, which possesses three Cys on a loop structure, two of them forming the redox-active disulfide bridge. The goal of this study was to identify the Cys involved in the transient mixed disulfide. To stabilize this reaction intermediate, mutant proteins with modified active sites were used. We identified Cys-155 of the FBPase as the one engaged in the formation of the mixed disulfide intermediate with Cys-46 of Trx f.

Characterization of the regulatory function of the 46-kDa isoform of Rubisco activase from Arabidopsis.

Arabidopsis Rubisco activase was recently shown to be regulated by redox changes in the larger (46-kDa) isoform specifically mediated by thioredoxin-f [Zhang and Portis (1999) Proc Natl Acad Sci USA 96: 9438-9443]. Reduction greatly increases the activity of the 46-kDa isoform and the native protein at physiological ATP/ADP ratios. In this study we conducted additional experiments to characterize the regulation of Rubisco activase by thioredoxin-f. The K(m) for both ATP hydrolysis and Rubisco activation by the 46-kDa isoform was lowered by 4 to 5-fold after reduction, but the maximum activity was increased by only 10%. Only 0.35 muM thioredoxin-f was required for a half-maximal activity change after a 10 min preincubation and activation with 1 muM was complete after 10 min. Equal amounts of 46-kDa and 43-kDa isoforms were required for a complete inhibition of the Rubisco activation activity after a reduction-oxidation cycle and assay at an ATP/ADP ratio of 3:1, whereas activity was only inhibited by 50% at a 2:1 ratio (43-/46-kDa) of the isoforms. This requirement is consistent with the fact that Arabidopsis normally contains about a 1:1 ratio of the two isoforms at both the mRNA and protein levels. Redox titrations indicated a midpoint potential of -344 mV for the 46-kDa isoform as compared to -342 mV for spinach fructose 1,6-bisphosphatase at pH 7.9, consistent with previous reports indicating that these proteins are co-regulated by light intensity in a similar manner.

C-MYC expression in medulloblastoma and its prognostic value.

To identify prognostic factors in medulloblastoma, a common malignant brain tumor of childhood, expression of the oncogene c-myc was examined at the mRNA level by in situ hybridization. c-myc mRNA expression was observed in 30 of 72 tumors (42%). The c-myc gene copy number was determined by quantitative PCR from genomic DNA of paraffin-embedded tumors. c-myc gene amplification was present in 5 of 62 cases (8.3%). Therefore, c-myc amplification was obviously not the cause of c-myc mRNA expression in most samples. Kaplan-Meier estimation revealed a significant correlation between c-myc mRNA expression and survival (total mean follow-up 4.6 +/- 3.6 years, log-rank p = 0.02). Multivariate logistic regression analysis including sex, age, histological type, degree of surgical resection and expression of synaptophysin, GFAP and c-myc, was carried out on 54 patients who received both radiotherapy and chemotherapy. The analysis identified expression of c-myc as an independent predictive factor of death from disease.

A dicistronic construct for the expression of functional spinach chloroplast ferredoxin:thioredoxin reductase in Escherichia coli.

Ferredoxin:thioredoxin reductase (FTR) is a heterodimeric Fe&z.sbnd;S containing disulfide reductase involved in the light-dependent activation of photosynthetic enzymes. We have designed a dicistronic construct for the heterologous expression of this nucleus encoded chloroplast protein in Escherichia coli. The coding sequences for the two mature subunits have been inserted in tandem into the expression vector pET-3d. This dicistronic construct is correctly translated yielding soluble, perfectly functional FTR. The recombinant enzyme is composed of both subunits, contains the correctly inserted Fe&z.sbnd;S cluster as evidenced by its spectral properties and is indistinguishable from the enzyme isolated from leaves in its capacity to activate chloroplast fructose-1,6-bisphosphatase, one of the well known light activated enzymes of the Calvin cycle.

Crystal structures of two functionally different thioredoxins in spinach chloroplasts.

Thioredoxins are small ubiquitous proteins which act as general protein disulfide reductases in living cells. Chloroplasts contain two distinct thioredoxins ( f and m) with different phylogenetic origin. Both act as enzyme regulatory proteins but have different specificities towards target enzymes. Thioredoxin f (Trx f), which shares only low sequence identity with thioredoxin m (Trx m) and with all other known thioredoxins, activates enzymes of the Calvin cycle and other photosynthetic processes. Trx m shows high sequence similarity with bacterial thioredoxins and activates other chloroplast enzymes. The here described structural studies of the two chloroplast thioredoxins were carried out in order to gain insight into the structure/function relationships of these proteins. Crystal structures were determined for oxidized, recombinant thioredoxin f (Trx f-L) and at the N terminus truncated form of it (Trx f-S), as well as for oxidized and reduced thioredoxin m (at 2.1 and 2.3 A resolution, respectively). Whereas thioredoxin f crystallized as a monomer, both truncated thioredoxin f and thioredoxin m crystallized as non-covalent dimers. The structures of thioredoxins f and m exhibit the typical thioredoxin fold consisting of a central twisted five-stranded beta-sheet surrounded by four alpha-helices. Thioredoxin f contains an additional alpha-helix at the N terminus and an exposed third cysteine close to the active site. The overall three-dimensional structures of the two chloroplast thioredoxins are quite similar. However, the two proteins have a significantly different surface topology and charge distribution around the active site. An interesting feature which might significantly contribute to the specificity of thioredoxin f is an inherent flexibility of its active site, which has expressed itself crystallographically in two different crystal forms.

Inhibition of Escherichia coli porphobilinogen synthase using analogs of postulated intermediates.

BACKGROUND: Porphobilinogen synthase is the second enzyme involved in the biosynthesis of natural tetrapyrrolic compounds, and condenses two molecules of 5-aminolevulinic acid (ALA) through a nonsymmetrical pathway to form porphobilinogen. Each substrate is recognized individually at two different active site positions to be regioselectively introduced into the product. According to pulse-labeling experiments, the substrate forming the propionic acid sidechain of porphobilinogen is recognized first. Two different mechanisms for the first bond-forming step between the two substrates have been proposed. The first involves carbon-carbon bond formation (an aldol-type reaction) and the second carbon-nitrogen bond formation, leading to an iminium ion. RESULTS: With the help of kinetic studies, we determined the Michaelis constants for each substrate recognition site. These results explain the Michaelis-Menten behavior of substrate analog inhibitors - they act as competitive inhibitors. Under standard conditions, however, another set of inhibitors demonstrates uncompetitive, mixed, pure irreversible, slow-binding or even quasi-irreversible inhibition behavior. CONCLUSIONS: Analysis of the different classes of inhibition behavior allowed us to make a correlation between the type of inhibition and a specific site of interaction. Analyzing the inhibition behavior of analogs of postulated intermediates strongly suggests that carbon-nitrogen bond formation occurs first.

Adenosine 5'-phosphosulfate sulfotransferase and adenosine 5'-phosphosulfate reductase are identical enzymes.

Adenosine 5'-phosphosulfate (APS) sulfotransferase and APS reductase have been described as key enzymes of assimilatory sulfate reduction of plants catalyzing the reduction of APS to bound and free sulfite, respectively. APS sulfotransferase was purified to homogeneity from Lemna minor and compared with APS reductase previously obtained by functional complementation of a mutant strain of Escherichia coli with an Arabidopsis thaliana cDNA library. APS sulfotransferase was a homodimer with a monomer M(r) of 43,000. Its amino acid sequence was 73% identical with APS reductase. APS sulfotransferase purified from Lemna as well as the recombinant enzyme were yellow proteins, indicating the presence of a cofactor. Like recombinant APS reductase, recombinant APS sulfotransferase used APS (K(m) = 6.5 microM) and not adenosine 3'-phosphate 5'-phosphosulfate as sulfonyl donor. The V(max) of recombinant Lemna APS sulfotransferase (40 micromol min(-1) mg protein(-1)) was about 10 times higher than the previously published V(max) of APS reductase. The product of APS sulfotransferase from APS and GSH was almost exclusively SO(3)(2-). Bound sulfite in the form of S-sulfoglutathione was only appreciably formed when oxidized glutathione was added to the incubation mixture. Because SO(3)(2-) was the first reaction product of APS sulfotransferase, this enzyme should be renamed APS reductase.

Activation of the potato tuber ADP-glucose pyrophosphorylase by thioredoxin.

The potato tuber (Solanum tuberosum L.) ADP-glucose pyrophosphorylase (ADP-GlcPPase) catalyzes the first committed step in starch biosynthesis. The main type of regulation of this enzyme is allosteric, and its activity is controlled by the ratio of activator, 3-phosphoglycerate to inhibitor, P(i). It was reported (Fu, Y., Ballicora, M. A., Leykam, J. F., and Preiss, J. (1998) J. Biol. Chem. 273, 25045-25052) that the enzyme was activated by reduction of the Cys(12) disulfide linkage present in the catalytic subunits. In this study, both reduced thioredoxin f and m from spinach (Spinacia oleracea) leaves reduced and activated the enzyme at low concentrations (10 microM) of activator (3-phosphoglycerate). Fifty percent activation was at 4.5 and 8.7 microM for reduced thioredoxin f and m, respectively, and 2 orders of magnitude lower than for dithiothreitol. The activation was reversed by oxidized thioredoxin. Cys(12) is conserved in the ADP-GlcPPases from plant leaves and other tissues except for the monocot endosperm enzymes. We postulate that in photosynthetic tissues, reduction could play a role in the fine regulation of the ADP-GlcPPase mediated by the ferredoxin-thioredoxin system. This is the first time that a covalent mechanism of regulation is postulated in the synthesis of starch.

Redox signaling in chloroplasts: cleavage of disulfides by an iron-sulfur cluster.

Light generates reducing equivalents in chloroplasts that are used not only for carbon reduction, but also for the regulation of the activity of chloroplast enzymes by reduction of regulatory disulfides via the ferredoxin:thioredoxin reductase (FTR) system. FTR, the key electron/thiol transducer enzyme in this pathway, is unique in that it can reduce disulfides by an iron-sulfur cluster, a property that is explained by the tight contact of its active-site disulfide and the iron-sulfur center. The thin, flat FTR molecule makes the two-electron reduction possible by forming on one side a mixed disulfide with thioredoxin and by providing on the opposite side access to ferredoxin for delivering electrons.

PLANT THIOREDOXIN SYSTEMS REVISITED.

Thioredoxins, the ubiquitous small proteins with a redox active disulfide bridge, are important regulatory elements in plant metabolism. Initially recognized as regulatory proteins in the reversible light activation of key photosynthetic enzymes, they have subsequently been found in the cytoplasm and in mitochondria. The various plant thioredoxins are different in structure and function. Depending on their intracellular location they are reduced enzymatically by an NADP-dependent or by a ferredoxin (light)-dependent reductase and transmit the regulatory signal to selected target enzymes through disulfide/dithiol interchange reactions. In this review we summarize recent developments that have provided new insights into the structures of several components and into the mechanism of action of the thioredoxin systems in plants.

Evidence of presynaptic location and function of the prion protein.

The prion protein (PrP(C)) is a copper-binding protein of unknown function that plays an important role in the etiology of transmissible spongiform encephalopathies. Using morphological techniques and synaptosomal fractionation methods, we show that PrP(C) is predominantly localized to synaptic membranes. Atomic absorption spectroscopy was used to identify PrP(C)-related changes in the synaptosomal copper concentration in transgenic mouse lines. The synaptic transmission in the presence of H(2)O(2), which is known to be decomposed to highly reactive hydroxyl radicals in the presence of iron or copper and to alter synaptic activity, was studied in these animals. The response of synaptic activity to H(2)O(2) was found to correlate with the amount of PrP(C) expression in the presynaptic neuron in cerebellar slice preparations from wild-type, Prnp(0/0), and PrP gene-reconstituted transgenic mice. Thus, our data gives strong evidence for the predominantly synaptic location of PrP(C), its involvement in the regulation of the presynaptic copper concentration, and synaptic activity in defined conditions.

Oxidation-reduction properties of chloroplast thioredoxins, ferredoxin:thioredoxin reductase, and thioredoxin f-regulated enzymes.

Oxidation-reduction midpoint potentials were determined, as a function of pH, for the disulfide/dithiol couples of spinach and pea thioredoxins f, for spinach and Chlamydomonas reinhardtii thioredoxins m, for spinach ferredoxin:thioredoxin reductase (FTR), and for two enzymes regulated by thioredoxin f, spinach phosphoribulokinase (PRK) and the fructose-1,6-bisphosphatases (FBPase) from pea and spinach. Midpoint oxidation-reduction potential (Em) values at pH 7.0 of -290 mV for both spinach and pea thioredoxin f, -300 mV for both C. reinhardtii and spinach thioredoxin m, -320 mV for spinach FTR, -290 mV for spinach PRK, -315 mV for pea FBPase, and -330 mV for spinach FBPase were obtained. With the exception of spinach FBPase, titrations showed a single two-electron component at all pH values tested. Spinach FBPase exhibited a more complicated behavior, with a single two-electron component being observed at pH values >/= 7.0, but with two components being present at pH values <7.0. The slopes of plots of Em versus pH were close to the -60 mV/pH unit value expected for a process that involves the uptake of two protons per two electrons (i. e., the reduction of a disulfide to two fully protonated thiols) for thioredoxins f and m, for FTR, and for pea FBPase. The slope of the Em versus pH profile for PRK shows three regions, consistent with the presence of pKa values for the two regulatory cysteines in the region between pH 7.5 and 9.0.