The protein that binds to DNA base J in trypanosomatids has features of a thymidine hydroxylase

Trypanosomatids contain an unusual DNA base J (beta-d-glucosylhydroxymethyluracil), which replaces a fraction of thymine in telomeric and other DNA repeats. To determine the function of base J, we have searched for enzymes that catalyze J biosynthesis. We present evidence that a protein that binds to J in DNA, the J-binding protein 1 (JBP1), may also catalyze the first step in J biosynthesis, the conversion of thymine in DNA into hydroxymethyluracil. We show that JBP1 belongs to the family of Fe(2+) and 2-oxoglutarate-dependent dioxygenases and that replacement of conserved residues putatively involved in Fe(2+) and 2-oxoglutarate-binding inactivates the ability of JBP1 to contribute to J synthesis without affecting its ability to bind to J-DNA. We propose that JBP1 is a thymidine hydroxylase responsible for the local amplification of J inserted by JBP2, another putative thymidine hydroxylase.     

Oxygen and iron regulation of iron regulatory protein 2

Iron regulatory protein 2 (IRP2) is a central regulator of cellular iron homeostasis due to its regulation of specific mRNAs encoding proteins of iron uptake and storage. Iron regulates IRP2 by mediating its rapid proteasomal degradation, where hypoxia and the hypoxia mimetics CoCl2 and desferrioxamine (DFO) stabilize it. Previous studies showed that iron-mediated degradation of IRP2 requires the presence of critical cysteines that reside within a 73-amino acid unique region. Here we show that a mutant IRP2 protein lacking this 73-amino acid region degraded at a rate similar to that of wild-type IRP2. In addition, DFO and hypoxia blocked the degradation of both the wild-type and mutant IRP2 proteins. Recently, members of the 2-oxoglutarate (2-OG)-dependent dioxygenase family have been shown to hydroxylate hypoxia-inducible factor-1 alpha (HIF-1 alpha), a modification required for its ubiquitination and proteasomal degradation. Since 2-OG-dependent dioxygenases require iron and oxygen, in addition to 2-OG, for substrate hydroxylation, we hypothesized that this activity may be involved in the regulation of IRP2 stability. To test this we used the 2-OG-dependent dioxygenase inhibitor dimethyloxalylglycine (DMOG) and showed that it blocked iron-mediated IRP2 degradation. In addition, hypoxia, DFO and DMOG blocked IRP2 ubiquitination. These data indicate that the region of IRP2 that is involved in IRP2 iron-mediated degradation lies outside of the 73-amino acid unique region and suggest a model whereby 2-OG-dependent dioxygenase activity may be involved in the oxygen and iron regulation of IRP2 protein stability.     

Regulation of trypanosome DNA glycosylation by a SWI2/SNF2-like protein

Synthesis of the modified thymine base beta-D-glucosyl-hydroxymethyluracil, or J, within telomeric DNA of Trypanosoma brucei correlates with the bloodstream-form-specific epigenetic silencing of telomeric variant surface glycoprotein genes involved in antigenic variation. The mechanism of developmental and telomeric-specific regulation of J synthesis is unknown. We have previously identified a J binding protein (JBP1) involved in propagating J synthesis. We have now identified a homolog of JBP1, JBP2, containing a domain related to the SWI2/SNF2 family of chromatin remodeling proteins that is upregulated in bloodstream form cells and interacts with nuclear chromatin. We show that expression of JBP2 in procyclic form cells leads to de novo J synthesis within telomeric regions of the chromosome and that this activity is inhibited after mutagenesis of conserved residues critical for SWI2/SNF2 function. We propose a model in which chromatin remodeling by JBP2 regulates the initial sites of J synthesis within bloodstream form trypanosome DNA, with further propagation and maintenance of J by JBP1.     

Site-specific interactions of JBP with base and sugar moieties in duplex J-DNA. Evidence for both major and minor groove contacts

Beta-D-Glucosyl-hydroxymethyluracil, also called base J, is an unusually modified DNA base conserved among Kinetoplastida. Base J is found predominantly in repetitive DNA and correlates with epigenetic silencing of telomeric variant surface glycoprotein genes. We have previously identified a J-binding protein (JBP) in Trypanosoma, Leishmania, and Crithidia, and we have shown that it is a structure-specific binding protein. Here we examine the molecular interactions that contribute to recognition of the glycosylated base in synthetic DNA substrates using modification interference, modification protection, DNA footprinting, and photocross-linking techniques. We find that the two primary requirements for J-DNA recognition include contacts at base J and a base immediately 5' of J (J-1). Methylation interference analysis indicates that the requirement of the base at position J-1 is due to a major groove contact independent of the sequence. DNA footprinting of the JBP.J-DNA complex with 1,10-phenanthroline-copper demonstrates that JBP contacts the minor groove at base J. Substitution of the thymine moiety of J with cytosine reduces the affinity for JBP approximately 15-fold. These data indicate that the sole sequence dependence for JBP binding may lie in the thymine moiety of base J and that recognition requires only two specific base contacts, base J and J-1, within both the major and minor groove of the J-DNA duplex.     

Identification of the Glucosyltransferase That Converts Hydroxymethyluracil to Base J in the Trypanosomatid Genome

O-linked glucosylation of thymine in DNA (base J) is an important regulatory epigenetic mark in trypanosomatids. β-d-glucopyranosyloxymethyluracil (base J) synthesis is initiated by the JBP1/2 enzymes that hydroxylate thymine, forming 5-hydroxymethyluracil (hmU). hmU is then glucosylated by a previously unknown glucosyltransferase. A recent computational screen identified a possible candidate for the base J-associated glucosyltransferase (JGT) in trypanosomatid genomes. We demonstrate that recombinant JGT utilizes uridine diphosphoglucose to transfer glucose to hmU in the context of dsDNA. Mutation of conserved residues typically involved in glucosyltransferase catalysis impairs DNA glucosylation in vitro. The deletion of both alleles of JGT from the genome of Trypanosoma brucei generates a cell line that completely lacks base J. Reintroduction of JGT in the JGT KO restores J synthesis. Ablation of JGT mRNA levels by RNAi leads to the sequential reduction in base J and increased levels of hmU that dissipate rapidly. The analysis of JGT function confirms the two-step J synthesis model and demonstrates that JGT is the only glucosyltransferase enzyme required for the second step of the pathway. Similar to the activity of the related Ten-Eleven Translocation (TET) family of dioxygenases on 5mC, our studies also suggest the ability of the base J-binding protein enzymes to catalyze iterative oxidation of thymine in trypanosome DNA. Here we discuss the regulation of hmU and base J formation in the trypanosome genome by JGT and base J-binding protein.     

Studies on the activity of the hypoxia-inducible-factor hydroxylases using an oxygen consumption assay

The activity and levels of the metazoan HIF (hypoxia-inducible factor) are regulated by its hydroxylation, catalysed by 2OG (2-oxoglutarate)- and Fe(II)-dependent dioxygenases. An oxygen consumption assay was developed and used to study the relationship between HIF hydroxylase activity and oxygen concentration for recombinant forms of two human HIF hydroxylases, PHD2 (prolyl hydroxylase domain-containing protein 2) and FIH (factor inhibiting HIF), and compared with two other 2OG-dependent dioxygenases. Although there are caveats on the absolute values, the apparent K(m) (oxygen) values for PHD2 and FIH were within the range observed for other 2OG oxygenases. Recombinant protein substrates were found to have lower apparent K(m) (oxygen) values compared with shorter synthetic peptides of HIF. The analyses also suggest that human PHD2 is selective for fragments of the C-terminal over the N-terminal oxygen-dependent degradation domain of HIF-1alpha. The present results, albeit obtained under non-physiological conditions, imply that the apparent K(m) (oxygen) values of the HIF hydroxylases enable them to act as oxygen sensors providing their in vivo capacity is appropriately matched to a hydroxylation-sensitive signalling pathway.     

Thymine 7-hydroxylase and pyrimidine deoxyribonucleoside 2' -hydroxylase activities in Rhodotorula glutinis

Cell-free preparations from Rhodotorula glutinis catalyzed the conversion of deoxyribonucleosides to ribonucleosides in a pyrimidine deoxyribonucleoside 2' -hydroxylase reaction. The reaction occurred with only thymidine or deoxyuridine, of the common deoxyribonucleosides, without detachment of the deoxyribose moiety, at the nucleoside level. The same enzyme preparations catalyzed the conversion of thymine to 5-hydroxymethyluracil in a thymine 7-hydroxylase reaction. Requirements for molecular oxygen, alpha-ketoglutarate, Fe2+, and ascorbate indicated that the 2' -hydroxylase and 7-hydroxylase reactions are of the alpha-keto-acid dioxygenases class. The requirements for alpha-ketoglutarate and Fe2+ were very stringent. During the course of the 2' -hydroxylase and 7-hydroxylase reactions, alpha-ketoglutarate was decarboxylated to form succinate and CO2 so that the ratio of hydroxylated nucleoside or pyrimidine to CO2 was 1:1.5-Hydroxymethyluracil and 5-formyluracil also stimulated the decarboxylation of alpha-ketoglutarate and thus appeared to undergo 7-hydroxylase reactions.     

Radiolytic hydroxylation of 1-substituted thymines promoted by transition metal salts in N2- and N2O-saturated aqueous solutions

The effect of high-valent transition metal salts on the radiation-induced hydroxylation of thymine (1a), 1-methylthymine (1b), and thymidine (1c) to the corresponding thymine glycol derivatives (2a-c) was studied at pH 7.0 in N2- and N2O-saturated aqueous solutions. The selectivities of (2a-c) based on converted (1a-c) increased to attain the maxima of 35-69% and then decreased, with increasing the one-electron reduction potential [E(M (n+1)+/Mn+)] of metal salts in the range of 0-1.0 V vs. NHE less than. Metal salts with E(M (n+1)+/Mn+) 0 or greater 1.0 V vs. NHE caused little change in the yields of (2a-c).     

An inner membrane dioxygenase that generates the 2-hydroxymyristate moiety of Salmonella lipid A

The lipid A residues of certain Gram-negative bacteria, including most strains of Salmonella and Pseudomonas, are esterified with one or two secondary S-2-hydroxyacyl chains. The S-2 hydroxylation process is O 2-dependent in vivo, but the relevant enzymatic pathways have not been fully characterized because in vitro assays have not been developed. We previously reported that expression of the Salmonella lpxO gene confers upon Escherichia coli K-12 the ability to synthesize 2-hydroxymyristate modified lipid A ( J. Biol. Chem. (2000) 275, 32940-32949). We now demonstrate that inactivation of lpxO, which encodes a putative Fe (2+)/O 2/alpha-ketoglutarate-dependent dioxygenase, abolishes S-2-hydroxymyristate formation in S. typhimurium. Membranes of E. coli strains expressing lpxO are able to hydroxylate Kdo 2-[4'- (32)P]-lipid A in vitro in the presence of Fe (2+), O 2, alpha-ketoglutarate, ascorbate, and Triton X-100. The Fe (2+) chelator 2,2'-bipyridyl inhibits the reaction. The product generated in vitro is a monohydroxylated Kdo 2-lipid A derivative. The [4'- (32)P]-lipid A released by mild acid hydrolysis from the in vitro product migrates with authentic S-2-hydroxlyated lipid A isolated from (32)P-labeled S. typhimurium cells. Electrospray ionization mass spectrometry and gas chromatography/mass spectrometry of the in vitro product are consistent with the 2-hydroxylation of the 3'-secondary myristoyl chain of Kdo 2-lipid A. LpxO contains two predicted trans-membrane helices (one at each end of the protein), and its active site likely faces the cytoplasm. LpxO is an unusual example of an integral membrane protein that is a member of the Fe (2+)/O 2/alpha-ketoglutarate-dependent dioxygenase family.     

Kinetic studies of iron deposition in horse spleen ferritin using O2 as oxidant

An optical flow cell provided a means to conveniently measure the rate of successive Fe(2+) oxidation reactions catalyzed by horse spleen ferritin (HoSF) to determine if both ferroxidase and mineral core Fe(2+) oxidation reactions occur. The oxygen concentration and pH were held constant and multiple additions of Fe(2+)/HoSF ratios of 1, 10, 100, 150, 250 and 400 were conducted, creating core sizes ranging from 12 to 2800. During these oxidations, the absence of nonspecific Fe(OH)(3) formation and the presence (>95%) of Fe(OH)(3) deposited within the core of HoSF demonstrated the validity of monitoring iron deposition into HoSF by this procedure. Initial rates for oxidation of 5-50 Fe(2+)/HoSF established that the reaction is overall first order in Fe(2+) concentration. However, when full progress curves were analyzed at a variety of Fe(2+)/HoSF ratios, two first-order reactions (k(1) approximately 0.035 s(-1) and k(2) approximately 0.007 s(-1)) were found to contribute to the overall Fe(2+) oxidation reaction. The proportion of the fast reaction increased with increasing Fe(2+)/HoSF ratio until at approximately 400, it was the dominant reaction. For the Fe(2+)/HoSF ratios examined, the overall rate of iron deposition is independent of the size of the mineral core, a result suggesting that an increasing mineral core size does not enhance the rate of Fe(2+) oxidation. Comparison of successive additions of 1.0 Fe(2+)/HoSF showed that oxidation of the first 8-10 Fe(2+) produced a Fe(III) species with a lower molar absorptivity per Fe(III) than that of the bulk core. Measurement of the H(+)/Fe(2+) ratio confirmed this difference in behavior by giving an H(+)/Fe(2+) ratio of approximately 1.0 below and 2.0 for ratios >30 Fe(2+)/HoSF. The faster reaction was attributed to ferroxidase catalysis and the slow reaction to nonspecific ferroxidase activity of the HoSF protein shell.     

A novel mechanism for Ca(2+)-dependent regulation of hepatic gluconeogenesis: stimulation of mitochondrial phosphoenolpyruvate synthesis by Ca2+.

1. Isolated guinea pig liver mitochondria were used to assess a possible effect of Ca2+ on the rate of phosphoenolpyruvate (PEP) synthesis. 2. PEP synthesis from 2-oxoglutarate (2-OG), but not from malate, was stimulated by [Ca2+] between 200 and 1200 nM. The effect was more pronounced at low [2-OG] (i.e. 0.1 and 0.3 mM) and it reached 58 and 22%, respectively, at 1200 nM as compared to 200 nM [Ca2+]. 3. Ruthenium red (1.8 microM) totally suppressed the stimulatory effect of Ca2+. 4. Malonate (5 mM) abolished PEP formation with 2-OG alone but inhibited only slightly the process with 2-OG + malate. 5. The results suggest that the stimulation by Ca2+ of 2-OG dehydrogenase and, therefore, of GTP synthesis, provides a mechanism for an enhanced PEP synthesis and for regulation of hepatic gluconeogenesis by Ca(2+)-mobilizing hormones.     

J-binding protein increases the level and retention of the unusual base J in trypanosome DNA

The nuclear DNA of Trypanosoma brucei and other kinetoplastid flagellates contains the unusual base beta-d-glucosyl-hydroxymethyluracil, called J, replacing part of the thymine in repetitive sequences. We have described a 100 kDa protein that specifically binds to J in duplex DNA. We have now disrupted the genes for this J-binding protein (JBP) in T. brucei. The disruption does not affect growth, gene expression or the stability of some repetitive DNA sequences. Unexpectedly, however, the JBP KO trypanosomes contain only about 5% of the wild-type level of J in their DNA. Excess J, randomly introduced into T. brucei DNA by growing the cells in the presence of the J precursor 5-hydroxymethyldeoxyuridine, is lost by simple dilution as the KO trypanosomes multiply, showing that JBP does not protect J against removal. In contrast, cells containing JBP lose excess J only sluggishly. We conclude that JBP is able to activate the thymine modification enzymes to introduce additional J in regions of DNA already containing a basal level of J. We propose that JBP is a novel DNA modification maintenance protein.     

Effect of JBP485 on obstructive jaundice is related to regulation of renal Oat1, Oat3 and Mrp2 expression in ANIT-treated rats

The objective was to determine whether protective effects of JBP485 on biliary obstruction induced by alpha-naphthylisothiocyanate (ANIT) are mediated by the organic anion transporters Oat1, Oat3 and the multidrug resistance-associated protein Mrp2. The ANIT-induced increases in bilirubin (BIL), alanine aminotransferase (ALT) and aspartate transaminase (AST) in rat serum were inhibited significantly by oral administration of JBP485. The plasma concentration of JBP485 which is the substrate of Oat1 and Oat3 determined by LC–MS/MS was markedly increased after intravenous administration in ANIT-treated rats, whereas cumulative urinary excretion of JBP485 in vivo and the uptake of JBP485 in kidney slices were decreased remarkably. RT-PCR and Western blot showed the decreased expression of Oat1 and Oat3, increased expression of Mrp2 in ANIT-induced rats, meanwhile, the expression levels of Mrp2 and Oat1 were up-regulated after administration of JBP485. The up-regulation of Mrp2 and Oat1 was associated with a concomitant increase in urinary BIL after treatment with JBP485 in ANIT-treated rats. The mechanism for JBP485 to restore liver function might be related to improvement of the expression and function for Oat1 and Mrp2 as well as facilitation of urinary excretion for hepatoxic substance.     

Inhibition of Na+/K(+)-ATPase and Mg(2+)-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na(+)/K(+)-ATPase and Mg(2+)-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu(2+), Zn(2+), Fe(2+) and Co(2+)) and heavy metals (Hg(2+) and Pb(2+)) ions were studied. All investigated metals produced a larger maximum inhibition of Na(+)/K(+)-ATPase than Mg(2+)-ATPase activity. The free concentrations of the key species (inhibitor, MgATP(2-), MeATP(2-)) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu(2+)/Fe(2+) or Hg(2+)/Pb(2+) caused additive inhibition, while Cu(2+)/Zn(2+) or Fe(2+)/Zn(2+) inhibited Na(+)/K(+)-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu(2+)/Fe(2+) or Cu(2+)/Zn(2+) inhibited Mg(2+)-ATPase activity synergistically, while Hg(2+)/Pb(2+) or Fe(2+)/Zn(2+) induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na(+)/K(+)-ATPase activity by reducing the maximum velocities (V(max)) rather than the apparent affinity (Km) for substrate MgATP(2-), implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg(2+)-ATPase activity by Zn(2+), Fe(2+) and Co(2+) as well as kinetic analysis indicated two distinct Mg(2+)-ATPase subtypes activated in the presence of low and high MgATP(2-) concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na(+)/K(+)-ATPase with various potencies. Furthermore, these ligands also reversed Na(+)/K(+)-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg(2+)-induced inhibition was not obtained.