Three cytosolic and one plasma membrane-bound 5-nucleotidases have been cloned and characterized. Their various substrate specificities suggest widely different functions in nucleotide metabolism. We now describe a 5-nucleotidase in mitochondria. The enzyme, named dNT-2, dephosphorylates specifically the 5-and 2(3)-phosphates of uracil and thymine deoxyribonucleotides. The cDNA of human dNT-2 codes for a 25.9-kDa polypeptide with a typical mitochondrial leader peptide, providing the structural basis for two-step processing during import into the mitochondrial matrix. The deduced amino acid sequence is 52% identical to that of a recently described cytosolic deoxyribonucleotidase (dNT-1). The two enzymes share many catalytic properties, but dNT-2 shows a narrower substrate specificity. Mitochondrial localization of dNT-2 was demonstrated by the mitochondrial fluorescence of 293 cells expressing a dNT-2-green fluorescent protein (GFP) fusion protein. 293 cells expressing fusion proteins without leader peptide or with dNT-1 showed a cytosolic fluorescence. During in vitro import into mitochondria, the preprotein lost the leader peptide. We suggest that dNT-2 protects mitochondrial DNA replication from overproduction of dTTP, in particular in resting cells. Mitochondrial toxicity of dTTP can be inferred from a severe inborn error of metabolism in which the loss of thymidine phosphorylase led to dTTP accumulation and aberrant mitochondrial DNA replication. We localized the gene for dNT-2 on chromosome 17p11.2 in the Smith-Magenis syndrome-critical region, raising the possibility that dNT-2 is involved in the etiology of this genetic disease. Mitochondrial DNA synthesis occurs throughout the whole cell cycle, independent of nuclear DNA replication (1). It is catalyzed by a separate DNA polymerase that uses distinct 2Ј-deoxyribonucleoside 5Ј-triphosphate (dNTP) pools (2, 3), sequestered from cytosolic dNTPs by the mitochondrial membranes. What is the origin of mitochondrial dNTPs and how are pool sizes regulated? Despite considerable efforts, a mitochondrial ribonucleotide reductase has not been found, suggesting import of dNTPs or deoxyribonucleosides from the cytosol into mitochondria. dNTPs are synthesized by the cytosolic ribonucleotide reductase and can be imported directly by a permease of the mitochondrial membrane (4). Deoxyribonucleosides are derived from the extracellular fluid or by catabolism of dNTPs and, after import into mitochondria, phosphorylated by specific intramitochondrial deoxyribonucleoside kinases (5)(6)(7)(8).But what regulates the size of intramitochondrial dNTP pools? This is an important question, because it is well established for cytosolic dNTPs that pool imbalance is genotoxic (9) and can cause specific diseases. Thus, in some cases of hereditary severe immune deficiency, the accumulation of dATP (10, 11) or dGTP (12, 13) in the cytosol of blood cells results in the apoptotic destruction of B and͞or T cells. In a different autosomal recessive disease, neurogastrointestinal encephalomyopat...

In this account, we report the development of a series of substituted cinnamic anilides that represents a novel class of mitochondrial permeability transition pore (mPTP) inhibitors. Initial class expansion led to the establishment of the basic structural requirements for activity and to the identification of derivatives with inhibitory potency higher than that of the standard inhibitor cyclosporine-A (CsA). These compounds can inhibit mPTP opening in response to several stimuli including calcium overload, oxidative stress, and thiol cross-linkers. The activity of the cinnamic anilide mPTP inhibitors turned out to be additive with that of CsA, suggesting for these inhibitors a molecular target different from cyclophylin-D. In vitro and in vivo data are presented for (E)-3-(4-fluoro-3-hydroxy-phenyl)-N-naphthalen-1-yl-acrylamide 22, one of the most interesting compounds in this series, able to attenuate opening of the mPTP and limit reperfusion injury in a rabbit model of acute myocardial infarction.

Snake presynaptic neurotoxins with phospholipase A 2 activity are potent inducers of paralysis through inhibition of the neuromuscular junction. These neurotoxins were recently shown to induce exocytosis of synaptic vesicles following the production of lysophospholipids and fatty acids and a sustained influx of Ca 2؉ from the medium. Here, we show that these toxins are able to penetrate spinal cord motor neurons and cerebellar granule neurons and selectively bind to mitochondria. As a result of this interaction, mitochondria depolarize and undergo a profound shape change from elongated and spaghetti-like to round and swollen. We show that snake presynaptic phospholipase A 2 neurotoxins facilitate opening of the mitochondrial permeability transition pore, an inner membrane high-conductance channel. The relative potency of the snake neurotoxins was similar for the permeability transition pore opening and for the phospholipid hydrolysis activities, suggesting a causal relationship, which is also supported by the effect of phospholipid hydrolysis products, lysophospholipids and fatty acids, on mitochondrial pore opening. These findings contribute to define the cellular events that lead to intoxication of nerve terminals by these snake neurotoxins and suggest that mitochondrial impairment is an important determinant of their toxicity.Two classes of neurotoxins can paralyze the neuromuscular junction through their enzymatic activity: (i) the clostridial neurotoxins, metalloproteases acting specifically on SNARE (soluble NSF attachment protein receptor) proteins to cause tetanus and botulism, and (ii) the SPANs (1). SPANs 3 play a major role in envenomation and cause a botulism-like flaccid paralysis with autonomic symptoms (2, 3). The enzymatic activity and the neurospecificity make these toxins very effective; however, like botulinum neurotoxins, SPANs do not affect the cell body and axon of the motor neuron, allowing complete recovery in most patients (4). Impairment of neuromuscular transmission by SPANs is traditionally measured in nerve-muscle preparations isolated from the mouse hemidiaphragm or from the chicken biventer cervicis. A simpler and more sensitive assay, based on SPANinduced irreversible bulging of nerve terminals in culture, was recently described (5). It was also shown that an early consequence of the action of SPANs is the hydrolysis of phosphatidylcholine into lysophosphatidylcholine and fatty acids and that their equimolar mixture mimics the swelling response of nerve terminals to the toxin itself (6). The SPAN-induced nerve bulges accumulate Ca 2ϩ , and, this event is accompanied by mitochondrial rounding and depolarization (7). The cytosolic [Ca 2ϩ ] increase could also trigger the activity of many Ca 2ϩ -activated hydrolases of nucleic acids, proteins, and lipids, all factors that could account for the pronounced degeneration of nerve terminals poisoned by .Previous studies indicated that SPANs can gain access to the cell interior. Indeed, fluorescein-conjugated ␤-Btx was found to rapidly e...

Methylglyoxal and synthetic glyoxal derivatives react covalently with arginine residue(s) on the mitochondrial permeability transition pore (PTP). In this study, we have investigated how the binding of a panel of synthetic phenylglyoxal derivatives influences the opening and closing of the PTP. Using both isolated mitochondria and mammalian cells, we demonstrate that the resulting arginine-phenylglyoxal adduct can lead to either suppression or induction of permeability transition, depending on the net charge and hydrogen bonding capacity of the adduct. We report that phenylglyoxal derivatives that possess a net negative charge and/or are capable of forming hydrogen bonds induced permeability transition. Derivatives that were overall electroneutral and cannot form hydrogen bonds suppressed permeability transition. When mammalian cells were incubated with low concentrations of negatively charged phenylglyoxal derivatives, the addition of oligomycin caused a depolarization of the mitochondrial membrane potential. This depolarization was completely blocked by cyclosporin A, a PTP opening inhibitor, indicating that the depolarization was due to PTP opening. Collectively, these findings highlight that the target arginine(s) is functionally linked with the opening/closing mechanism of the PTP and that the electric charge and hydrogen bonding of the resulting arginine adduct influences the conformation of the PTP. These results are consistent with a model where the target arginine plays a role as a voltage sensor.

We have investigated the mitochondrial effects of BH3I-2, Chelerythrine, and HA14-1, small organic molecules that share the ability to bind the BH3 domain of BCL-2. All compounds displayed a biphasic effect on mitochondrial respiration with uncoupling at low concentrations and respiratory inhibition at higher concentrations, the relative uncoupling potency being BH3I-2 (half-maximal uncoupling at about 80 nM) > Chelerythrine (half-maximal uncoupling at about 2 M) > HA14-1 (half-maximal uncoupling at about 20 M). At concentrations lower than required for uncoupling all compounds sensitized the permeability transition pore (PTP) to opening both in isolated mitochondria and intact cells. To assess whether the effects on BCL-2 binding, PTP induction and respiration could be due to different structural determinants we have tested a set of HA14-1 analogs from the Hoffmann-La Roche chemical library. We have identified 5-(6-chloro-2,4-dioxo-1,3,4,10-tetrahydro-2H-9-oxa-1,3-diaza-anthracen-10-yl)-pyrimidine-2,4,6-trione (EM20-25) as a molecule devoid of effects on respiration that is able to induce PTP opening, to disrupt the BCL-2/BAX interactions in situ and to activate caspase-9 in BCL-2-overexpressing cells. EM20-25 neutralized the antiapoptotic activity of overexpressed BCL-2 toward staurosporine and sensitized BCL-2-expressing cells from leukemic patients to the killing effects of staurosporine, chlorambucil, and fludarabine. These results provide a proof of principle that the potentially toxic effects of BCL-2 ligands on mitochondrial respiration are not essential for their antiapoptotic activity and represent an important step forward in the development of tumor-selective drugs acting on BCL-2.

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.Mammalian 5Ј-nucleotidases form a heterogeneous group of enzymes differing in primary structure, cell localization, and tissue and substrate specificity. They dephosphorylate various nucleotides to nucleosides and inorganic phosphate. Originally they were distinguished from their enzyme activity, more recently via cloning of their cDNAs resulting in the clear definition of 7 different species (1-7). One enzyme is a ubiquitous ectonucleotidase anchored to the surface of the plasma membrane (1). Five enzymes occur in the cytosol (2-6), and one in mitochondria (7). The mitochondrial nucleotidase and one of the cytosolic enzymes are structurally related and show a preference for the hydrolysis of deoxyribonucleotides and were for this reason named deoxyribonucleotidases (dNTs), 1 with dNT1 in the cytosol (6) and dNT2 in mitochondria (7).We are investigating the regulation of deoxyribonucleoside triphosphate (dNTP) pools in relation to DNA synthesis and became first interested in the physiological function of dNT1 and cloned its cDNA (6). We obtained evidence that dNT1 participates in the regulation of pyrimidine but not purine dNTP pools via cytosolic substrate cycles (8). Subsequently we discovered the related dNT2, which in its cDNA contains a presumptive mitochondrial presequence, and obtained strong evidence that the enzyme was localized in mitochondria (7). We overproduced dNT2 in Escherichia coli, purified the recombinant protein close to homogeneity, and found that its substrate specificity was narrower than that of dNT1 and essentially limit...