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Peter Reichard

Publications and source records attributed to Peter Reichard.

15 recordsLinked to original sources
PubMed2006

Mitochondrial deoxynucleotide pool sizes in mouse liver and evidence for a transport mechanism for thymidine monophosphate.

Dividing cultured cells contain much larger pools of the four dNTPs than resting cells. In both cases the sizes of the individual pools are only moderately different. The same applies to mitochondrial (mt) pools of cultured cells. Song et al. [Song S, Pursell ZF, Copeland WC, Longley MJ, Kunkel TA, Mathews CK (2005) Proc Natl Acad Sci USA 102:4990-4995] reported that mt pools of rat tissues instead are highly asymmetric, with the dGTP pool in some cases being several-hundred-fold larger than the dTTP pool, and suggested that the asymmetry contributes to increased mutagenesis during mt DNA replication. We have now investigated this discrepancy and determined the size of each dNTP pool in mouse liver mitochondria. We found large variations in pool sizes that closely followed variations in the ATP pool and depended on the length of time spent in the preparation of mitochondria. The proportion between dNTPs was in all cases without major asymmetries and similar to those found earlier in cultured resting cells. We also investigated the import and export of thymidine phosphates in mouse liver mitochondria and provide evidence for a rapid, highly selective, and saturable import of dTMP, not depending on a functional respiratory chain. At nM external dTMP the nucleotide is concentrated 100-fold inside the mt matrix. Export of thymidine phosphates was much slower and possibly occurred at the level of dTDP.

Animals↗
PubMed2006

Mitochondrial DNA depletion and thymidine phosphate pool dynamics in a cellular model of mitochondrial neurogastrointestinal encephalomyopathy.

Mitochondrial (mt) neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease associated with depletion, deletions, and point mutations of mtDNA. Patients lack a functional thymidine phosphorylase and their plasma contains high concentrations of thymidine and deoxyuridine; elevation of the corresponding triphosphates probably impairs normal mtDNA replication and repair. To study metabolic events leading to MNGIE we used as model systems skin and lung fibroblasts cultured in the presence of thymidine and/or deoxyuridine at concentrations close to those in the plasma of the patients, a more than 100-fold excess relative to controls. The two deoxynucleosides increased the mt and cytosolic dTTP pools of skin fibroblasts almost 2-fold in cycling cells and 8-fold in quiescent cells. During up to a two-month incubation of quiescent fibroblasts with thymidine (but not with deoxyuridine), mtDNA decreased to approximately 50% without showing deletions or point mutations. When we removed thymidine, but maintained the quiescent state, mtDNA recovered rapidly. With thymidine in the medium, the dTTP pool of quiescent cells turned over rapidly at a rate depending on the concentration of thymidine, due to increased degradation and resynthesis of dTMP in a substrate (=futile) cycle between thymidine kinase and 5'-deoxyribonucleotidase. The cycle limited the expansion of the dTTP pool at the expense of ATP hydrolysis. We propose that the substrate cycle represents a regulatory mechanism to protect cells from harmful increases of dTTP. Thus MNGIE patients may increase their consumption of ATP to counteract an unlimited expansion of the dTTP pool caused by circulating thymidine.

Adenosine Triphosphate↗
PubMed2006

Ribonucleotide reductases.

Ribonucleotide reductases (RNRs) transform RNA building blocks to DNA building blocks by catalyzing the substitution of the 2'OH-group of a ribonucleotide with a hydrogen by a mechanism involving protein radicals. Three classes of RNRs employ different mechanisms for the generation of the protein radical. Recent structural studies of members from each class have led to a deeper understanding of their catalytic mechanism and allosteric regulation by nucleoside triphosphates. The main emphasis of this review is on regulation of RNR at the molecular and cellular level. Conformational transitions induced by nucleotide binding determine the regulation of substrate specificity. An intricate interplay between gene activation, enzyme inhibition, and protein degradation regulates, together with the allosteric effects, enzyme activity and provides the appropriate amount of deoxynucleotides for DNA replication and repair. In spite of large differences in the amino acid sequences, basic structural features are remarkably similar and suggest a common evolutionary origin for the three classes.

Allosteric Regulation↗
PubMed2005

Euglena gracilis ribonucleotide reductase: the eukaryote class II enzyme and the possible antiquity of eukaryote B12 dependence.

Ribonucleotide reductases provide the building blocks for DNA synthesis. Three classes of enzymes are known, differing widely in amino acid sequence but with similar structural motives and allosteric regulation. Class I occurs in eukaryotes and aerobic prokaryotes, class II occurs in aerobic and anaerobic prokaryotes, and class III occurs in anaerobic prokaryotes. The eukaryote Euglena gracilis contains a class II enzyme (Gleason, F. K., and Hogenkamp, H. P. (1970) J. Biol. Chem. 245, 4894-4899) and, thus, forms an exception. Class II enzymes depend on vitamin B(12) for their activity. We purified the reductase from Euglena cells, determined partial peptide sequences, identified its cDNA, and purified the recombinant enzyme. Its amino acid sequence and general properties, including its allosteric behavior, were similar to the class II reductase from Lactobacillus leichmannii. Both enzymes belong to a distinct small group of reductases that unlike all other homodimeric reductases are monomeric. They compensate the loss of the second polypeptide of dimeric enzymes by a large insertion in the monomeric chain. Data base searching and sequence comparison revealed a homolog from the eukaryote Dictyostelium discoideum as the closest relative to the Euglena reductase, suggesting that the class II enzyme was present in a common, B(12)-dependent, eukaryote ancestor.

Algal Proteins↗
PubMed2005

Mitochondrial deoxynucleotide pools in quiescent fibroblasts: a possible model for mitochondrial neurogastrointestinal encephalomyopathy (MNGIE).

Mitochondrial (mt) DNA depletion syndromes can arise from genetic deficiencies for enzymes of dNTP metabolism, operating either inside or outside mitochondria. MNGIE is caused by the deficiency of cytosolic thymidine phosphorylase that degrades thymidine and deoxyuridine. The extracellular fluid of the patients contains 10-20 microM deoxynucleosides leading to changes in dTTP that may disturb mtDNA replication. In earlier work, we suggested that mt dTTP originates from two distinct pathways: (i) the reduction of ribonucleotides in the cytosol (in cycling cells) and (ii) intra-mt salvage of thymidine (in quiescent cells). In MNGIE and most other mtDNA depletion syndromes, quiescent cells are affected. Here, we demonstrate in quiescent fibroblasts (i) the existence of small mt dNTP pools, each usually 3-4% of the corresponding cytosolic pool; (ii) the rapid metabolic equilibrium between mt and cytosolic pools; and (iii) the intra-mt synthesis and rapid turnover of dTTP in the absence of DNA replication. Between 0.1 and 10 microM extracellular thymidine, intracellular thymidine rapidly approaches the extracellular concentration. We mimic the conditions of MNGIE by maintaining quiescent fibroblasts in 10-40 microM thymidine and/or deoxyuridine. Despite a large increase in intracellular thymidine concentration, cytosolic and mt dTTP increase at most 4-fold, maintaining their concentration for 41 days. Other dNTPs are marginally affected. Deoxyuridine does not increase the normal dNTP pools but gives rise to a small dUTP and a large dUMP pool, both turning over rapidly. We discuss these results in relation to MNGIE.

Cell Line↗
PubMed2004

Structural mechanism of allosteric substrate specificity regulation in a ribonucleotide reductase.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides into deoxyribonucleotides, which constitute the precursor pools used for DNA synthesis and repair. Imbalances in these pools increase mutational rates and are detrimental to the cell. Balanced precursor pools are maintained primarily through the regulation of the RNR substrate specificity. Here, the molecular mechanism of the allosteric substrate specificity regulation is revealed through the structures of a dimeric coenzyme B12-dependent RNR from Thermotoga maritima, both in complexes with four effector-substrate nucleotide pairs and in three complexes with only effector. The mechanism is based on the flexibility of loop 2, a key structural element, which forms a bridge between the specificity effector and substrate nucleotides. Substrate specificity is achieved as different effectors and their cognate substrates stabilize specific discrete loop 2 conformations. The mechanism of substrate specificity regulation is probably general for most class I and class II RNRs.

Adenosine Triphosphate↗
PubMed2004

Mitochondrial deoxyribonucleotides, pool sizes, synthesis, and regulation.

We quantify cytosolic and mitochondrial deoxyribonucleoside triphosphates (dNTPs) from four established cell lines using a recently described method for the separation of cytosolic and mitochondrial (mt) dNTPs from as little as 10 million cells in culture (Pontarin, G., Gallinaro, L., Ferraro, P., Reichard, P., and Bianchi, V. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 12159-12164). In cycling cells the concentrations of the phosphates of thymidine, deoxycytidine, and deoxyadenosine (combining mono-, di-, and triphosphates in each case) did not differ significantly between mitochondria and cytosol, whereas deoxyguanosine phosphates were concentrated to mitochondria. We study the source and regulation of the mt dTTP pool as an example of mt dNTPs. We suggest two pathways as sources for mt dTTP: (i) import from the cytosol of thymidine diphosphate by a deoxynucleotide transporter, predominantly in cells involved in DNA replication with an active synthesis of deoxynucleotides and (ii) import of thymidine followed by phosphorylation by the mt thymidine kinase, predominantly in resting cells. Here we demonstrate that the second pathway is regulated by a mt 5'-deoxyribonucleotidase (mdN). We modify the in situ activity of mdN and measure the transfer of radioactivity from [(3)H]thymidine to mt thymidine phosphates. In cycling cells lacking the cytosolic thymidine kinase, a 30-fold overproduction of mdN decreases the specific radioactivity of mt dTTP to 25%, and an 80% decrease of mdN by RNA interference increases the specific radioactivity 2-fold. These results suggest that mdN modulates the synthesis of mt dTTP by counteracting in a substrate cycle the phosphorylation of thymidine by the mt thymidine kinase.

5'-Nucleotidase↗
PubMed2004

Crystal structures of the mitochondrial deoxyribonucleotidase in complex with two specific inhibitors.

Monophosphate nucleotidases are enzymes that dephosphorylate nucleotides to their corresponding nucleosides. They play potentially important roles in controlling the activation of nucleotide-based drugs targeted against viral infections or cancer cells. The human mitochondrial deoxyribonucleotidase (dNT-2) dephosphorylates thymidine and deoxyuridine monophosphates. We describe the high resolution structures of the dNT-2 enzyme in complex with two potent nucleoside phosphonate inhibitors, (S)-1-[2'-deoxy-3',5'-O-(1-phosphono) benzylidene-beta-d-threo-pentofuranosyl]thymine (DPB-T) at 1.6-A resolution and (+/-)-1-trans-(2-phosphonomethoxycyclopentyl)uracil (PMcP-U) at 1.4-A resolution. The mixed competitive inhibitor DPB-T and the competitive inhibitor PMcP-U both bind in the active site of dNT-2 but in distinctly different binding modes, explaining their different kinetics of inhibition. The pyrimidine part of the inhibitors binds with very similar hydrogen bond interactions to the protein but with their phosphonate moieties in different binding sites compared with each other and to the previously determined position of phosphate bound to dNT-2. Together, these phosphate/phosphonate binding sites describe what might constitute a functionally relevant phosphate entrance tunnel to the active site. The structures of the inhibitors in complex with dNT-2, being the first such complexes of any nucleotidase, might provide important information for the design of more specific inhibitors to control the activation of nucleotide-based drugs.

5'-Nucleotidase↗
PubMed2003

Origins of mitochondrial thymidine triphosphate: dynamic relations to cytosolic pools.

Nuclear and mitochondrial (mt) DNA replication occur within two physically separated compartments and on different time scales. Both require a balanced supply of dNTPs. During S phase, dNTPs for nuclear DNA are synthesized de novo from ribonucleotides and by salvage of thymidine in the cytosol. Mitochondria contain specific kinases for salvage of deoxyribonucleosides that may provide a compartmentalized synthesis of dNTPs. Here we investigate the source of intra-mt thymidine phosphates and their relationship to cytosolic pools by isotope-flow experiments with [3H]thymidine in cultured human and mouse cells by using a rapid method for the clean separation of mt and cytosolic dNTPs. In the absence of the cytosolic thymidine kinase, the cells (i) phosphorylate labeled thymidine exclusively by the intra-mt kinase, (ii) export thymidine phosphates rapidly to the cytosol, and (iii) use the labeled dTTP for nuclear DNA synthesis. The specific radioactivity of dTTP is highly diluted, suggesting that cytosolic de novo synthesis is the major source of mt dTTP. In the presence of cytosolic thymidine kinase dilution is 100-fold less, and mitochondria contain dTTP with high specific radioactivity. The rapid mixing of the cytosolic and mt pools was not expected from earlier data. We propose that in proliferating cells dNTPs for mtDNA come largely from import of cytosolic nucleotides, whereas intra-mt salvage of deoxyribonucleosides provides dNTPs in resting cells. Our results are relevant for an understanding of certain genetic mitochondrial diseases.

Animals↗
PubMed2003

Cytosolic and mitochondrial deoxyribonucleotidases: activity with substrate analogs, inhibitors and implications for therapy.

Nucleoside analogs act as prodrugs that must be converted to 5'-phosphates by intracellular kinases to become active in the treatment of viral and oncological diseases. Activation may be reversed by dephosphorylation if the 5'-phosphates are substrates for 5'-nucleotidases. Dephosphorylation by cytosolic enzymes decreases the efficacy of the analogs, whereas dephosphorylation by mitochondrial enzymes may decrease mitochondrial toxicity. Both effects may influence the outcome of therapy. We investigated the dephosphorylation of the 5'-phosphates of commonly used nucleoside analogs by two cytosolic (cN-II and dNT-1) and one mitochondrial (dNT-2) nucleotidase. Most uracil/thymine nucleotide analogs were dephosphorylated by all three human enzymes but cytosine-containing nucleotide analogs were inactive. Only cN-II showed some activity with the monophosphates of the two purine analogs 2-chloro-2'-deoxyadenosine and 9-beta-D-arabinosylguanine. We conclude that overproduction of any of the three 5'-nucleotidases cannot explain development of resistance against cytosine analogs but that overproduction of cN-II could lead to resistance against purine analogs. Of the tested analogs, only (E)-5-(2-bromovinyl)-2'-deoxyuridine was preferentially dephosphorylated by mitochondrial dNT-2. We propose that in future developments of analogs this aspect be considered in order to reduce mitochondrial toxicity. We tested inhibition of dNT-1 and dNT-2 by a large variety of synthetic metabolically stable nucleoside phosphonate analogs and found one (PMcP-U) that inhibited dNT-1 and dNT-2 competitively and a second (DPB-T) that inhibited dNT-2 by mixed inhibition. Both inhibitors are useful for specific 5'-nucleotidase assays and structural studies and may open up possibilities for therapy.

5'-Nucleotidase↗
PubMed2002

Human mitochondrial 5'-deoxyribonucleotidase. Overproduction in cultured cells and functional aspects.

Deoxynucleoside triphosphates (dNTPs) used for mitochondrial DNA replication are mainly formed by phosphorylation of deoxynucleosides imported into mitochondria from the cytosol. We earlier obtained evidence for a mitochondrial 5'-nucleotidase (dNT2) with a pronounced specificity for dUMP and dTMP and suggested that the enzyme protects mitochondrial DNA replication from excess dTTP. In humans, accumulation of dTTP causes a mitochondrial genetic disease. We now establish that dNT2 in vivo indeed is located in mitochondria. The native enzyme shows the same substrate specificity and affinity for inhibitors as the recombinant dNT2. We constructed ponasterone-inducible cell lines overproducing dNT2 with and without the green fluorescent protein (GFP) linked to its C terminus. The fusion protein occurred in mitochondria mostly in an inactive truncated form, with only a short C-terminal fragment of dNT2 linked to GFP. No truncation occurred when dNT2 and GFP were not linked. The cell mitochondria then contained a large excess of active dNT2 with or without the mitochondrial presequence. After removal of ponasterone overproduced dNT2 disappeared only slowly from the cells, whereas dNT2-mRNA was lost rapidly. Overproduction of dNT2 did not lead to an increased excretion of pyrimidine deoxyribonucleosides, in contrast to overproduction of the corresponding cytosolic deoxynucleotidase, suggesting that the mitochondrial enzyme does not affect overall cellular deoxynucleotide turnover.

5'-Nucleotidase↗
PubMed2002

5'-Nucleotidases: specific assays for five different enzymes in cell extracts.

Several mammalian 5'-nucleotidases (5-NTs), attached to membranes or present in the cytosol or in mitochondria, remove the phosphate from ribo- and deoxyribonucleotides with different specificities for the sugar and base moieties. Some enzymes probably participate in signaling functions by producing adenosine from AMP. A more general function may be to prevent overproduction of deoxyribonucleotides. 5-NTs may affect the pharmacological activity of nucleoside analogs and also be involved in their mitochondrial toxicity. Here we describe for five cloned 5-NT specific assays that largely rely on new inhibitors for some of the enzymes. The assays can be used to quantitate each enzyme in crude cell extracts. To ascertain their validity we applied each assay to extracts from genetically modified cells that overproduce separately each of the five enzymes. The methodology should be useful in further studies of the physiological function of 5-NTs and their influence on the clinical use of nucleoside analogs.

5'-Nucleotidase↗
PubMed2002

Ribonucleotide reductases: the evolution of allosteric regulation.

Ribonucleotide reductases catalyze in all living organisms the production of the deoxyribonucleotides required for DNA replication and repair. Their appearance during evolution was a prerequisite for the transition from the "RNA world," where RNA sufficed for both catalysis and information transfer, to today's situation where life depends on the interplay among DNA, RNA, and protein. Three classes of ribonucleotide reductases exist today, widely differing in their primary and quaternary structures but all with a highly similar allosteric regulation of their substrate specificity. Here, I discuss the diversities between the three classes, describe their allosteric regulation, and discuss the evidence for their evolution. The appearance of oxygen on earth provided the likely driving force for enzyme diversification. From today's characteristics of the three classes, including their allosteric regulation, I propose that the anaerobic class III reductases with their iron-sulfur cluster and the requirement for S-adenosylmethionine for the generation of a glycyl protein free radical are the closest relatives to an ancestor ribonucleotide reductase.

Allosteric Regulation↗
PubMed2002

Crystal structure of a human mitochondrial deoxyribonucleotidase.

5' nucleotidases are ubiquitous enzymes that dephosphorylate nucleoside monophosphates and participate in the regulation of nucleotide pools. The mitochondrial 5'-(3') deoxyribonucleotidase (dNT-2) specifically dephosphorylates dUMP and dTMP, thereby protecting mitochondrial DNA replication from excess dTTP. We have solved the structure of dNT-2, the first of a mammalian 5' nucleotidase. The structure reveals a relationship to the HAD family, members of which use an aspartyl nucleophile as their common catalytic strategy, with a phosphoserine phosphatase as the most similar neighbor. A structure-based sequence alignment of dNT-2 with other 5' nucleotidases also suggests a common origin for these enzymes. Here we study the structures of dNT-2 in complex with bound phosphate and beryllium trifluoride plus thymidine as model for a phosphoenzyme-product complex. Based on these structures, determinants for substrate specificity recognition and the catalytic action of dNT-2 are outlined.

5'-Nucleotidase↗