Nicola Solaroli

Progressive loss of mitochondrial DNA in thymidine kinase 2-deficient mice

Deficient enzymatic activity of the mitochondrial deoxyribonucleoside kinases deoxyguanosine kinase (DGUOK) or thymidine kinase 2 (TK2) cause mitochondrial DNA (mtDNA)-depletion syndromes in humans. Here we report the generation of a Tk2-deficient mouse strain and show that the mice develop essentially normally for the first week but from then on exhibit growth retardation and die within 2-4 weeks of life. Several organs including skeletal muscle, heart, liver and spleen showed progressive loss of mtDNA without increased mtDNA mutations or structural alterations. There were no major histological changes in skeletal muscle, but heart muscle showed disorganized and damaged muscle fibers. Electron microscopy showed mitochondria with distorted cristae. The Tk2-deficient mice exhibited pronounced hypothermia and showed loss of hypodermal fat and abnormal brown adipose tissue. We conclude that Tk2 has a major role in supplying deoxyribonucleotides for mtDNA replication and that other pathways of deoxyribonucleotide synthesis cannot compensate for loss of this enzyme.

Two novel mutations in thymidine kinase-2 cause early onset fatal encephalomyopathy and severe mtDNA depletion

Deficiency of thymidine kinase-2 (TK2) has been described in children with early onset fatal skeletal myopathy. TK2 is a mitochondrial deoxyribonucleoside kinase required for the phosphorylation of deoxycytidine and deoxythymidine and hence is vital for the maintenance of a balanced mitochondrial dNTP pool in post-mitotic tissues. We describe a patient with two novel TK2 mutations, which caused disease onset shortly after birth and death at the age of three months. One mutation (219insCG) generated an early stop codon, thus preventing the synthesis of a functional protein. The second mutation (R130W) resulted in an amino acid substitution, which caused a severe reduction (<3%) of TK2 enzyme activity. These two novel TK2 mutations cause an extremely severe phenotype with overwhelming central nervous system symptoms not commonly seen in patients with TK2-deficiency. We conclude that the severe clinical presentation in this patient was due to a virtual lack of mitochondrial TK2 activity.

The many isoforms of human adenylate kinases.

Adenine nucleotides are involved in a variety of cellular metabolic processes, including nucleic acid synthesis and repair, formation of coenzymes, energy transfer, cell and ciliary motility, hormone secretion, gene expression regulation and ion-channel control. Adenylate kinases are abundant phosphotransferases that catalyze the interconversion of adenine nucleotides and thus regulate the adenine nucleotide ratios in different intracellular compartments. Nine different adenylate kinase isoenzymes have been identified and characterized so far in human tissues, named AK1 to AK9 according to their order of discovery. Adenylate kinases differ in molecular weight, tissue distribution, subcellular localization, substrate and phosphate donor specificity and kinetic properties. The preferred substrate and phosphate donor of all adenylate kinases are AMP and ATP respectively, but some members of the family can phosphorylate other substrates and use other phosphate donors. In addition to their nucleoside monophosphate kinase activity, adenylate kinases were found to possess nucleoside diphosphate kinase activity as they are able to phosphorylate both ribonucleoside and deoxyribonucleoside diphosphates to their corresponding triphosphates. Nucleoside analogues are structural analogues of natural nucleosides, used in the treatment of cancer and viral infections. They are inactive prodrugs that are dependent on intracellular phosphorylation to their pharmacologically active triphosphate form. Novel data presented in this review confirm the role of adenylate kinases in the activation of deoxyadenosine and deoxycytidine nucleoside analogues.

Engineering of a single conserved amino acid residue of herpes simplex virus type 1 thymidine kinase allows a predominant shift from pyrimidine to purine nucleoside phosphorylation.

Studies of herpes simplex virus type 1 (HSV-1) thymidine (dThd) kinase (TK) crystal structures show that purine and pyrimidine bases occupy distinct positions in the active site but approximately the same geometric plane. The presence of a bulky side chain, such as tyrosine at position 167, would not be sterically favorable for pyrimidine or pyrimidine nucleoside analogue binding, whereas purine nucleoside analogues would be less affected because they are located further away from the phenylalanine side chain. Site-directed mutagenesis of the conserved Ala-167 and Ala-168 residues in HSV-1 TK resulted in a wide variety of differential affinities and catalytic activities in the presence of the natural substrate dThd and the purine nucleoside analogue drug ganciclovir (GCV), depending on the nature of the amino acid mutation. A168H- and A167F-mutated HSV-1 TK enzymes turned out to have a virtually complete knock-out of dThd kinase activity (at least ∼4-5 orders of magnitude lower) presumably due to a steric clash between the mutated amino acid and the dThd ring. In contrast, a full preservation of the GCV (and other purine nucleoside analogues) kinase activity was achieved for A168H TK. The enzyme mutants also markedly lost their binding capacity for dThd and showed a substantially diminished feedback inhibition by thymidine 5′-triphosphate. The side chain size at position 168 seems to play a less important role regarding GCV or dThd selectivity than at position 167. Instead, the nitrogen-containing side chains from A168H and A168K seem necessary for efficient ligand discrimination. This explains why A168H-mutated HSV-1 TK fully preserves its GCV kinase activity (Vmax/Km 4-fold higher than wild-type HSV-1 TK), although still showing a severely compromised dThd kinase activity (Vmax/Km 3-4 orders of magnitude lower than wild-type HSV-1 TK).

The characterization of human adenylate kinases 7 and 8 demonstrates differences in kinetic parameters and structural organization among the family of adenylate kinase isoenzymes.

Differences in expression profiles, substrate specificities, kinetic properties and subcellular localization among the AK (adenylate kinase) isoenzymes have been shown to be important for maintaining a proper adenine nucleotide composition for many different cell functions. In the present study, human AK7 was characterized and its substrate specificity, kinetic properties and subcellular localization determined. In addition, a novel member of the human AK family, with two functional domains, was identified and characterized and assigned the name AK8. AK8 is the second known human AK with two complete and active AK domains within its polypeptide chain, a feature that has previously been shown for AK5. The full-length AK8, as well as its two domains AK8p1 and AK8p2, all showed similar AK enzyme activity. AK7, full-length AK8, AK8p1 and AK8p2 phosphorylated AMP, CMP, dAMP and dCMP with ATP as the phosphate donor, and also AMP, CMP and dCMP with GTP as the phosphate donor. Both AK7 and full-length AK8 showed highest affinity for AMP with ATP as the phosphate donor, and proved to be more efficient in AMP phosphorylation as compared with the major cytosolic isoform AK1. Expression of the proteins fused with green fluorescent protein demonstrated a cytosolic localization for both AK7 and AK8.

Enhanced toxicity of purine nucleoside analogs in cells expressing Drosophila melanogaster nucleoside kinase mutants

The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) is investigated for possible use as a suicide gene in combined gene/chemotherapy of cancer. The enzyme has broader substrate specificity and higher catalytic rate compared to herpes simplex type 1 thymidine kinase and other known dNKs. Although the enzyme has broad substrate specificity, it has a preference for pyrimidine nucleosides and nucleoside analogs. We have evaluated the substrate specificity and kinetic properties of Dm-dNK proteins containing M88R, V84A+M88R or V84A+M88R+A110D mutations in the amino-acid sequence. These engineered enzymes showed a relative increase in phosphorylation of purine nucleoside analogs such as ganciclovir, 9-β-D-arabinofuranosylguanine and 2′,2′-difluorodeoxyguanosine compared to the wild-type enzyme. The mutant enzymes were expressed in an osteosarcoma thymidine kinase-deficient cell line and the sensitivity of the cell line to nucleoside analogs was determined. The cells expressing the M88R mutant enzyme showed the highest increased sensitivity to purine nucleoside analogs with 8- to 80-fold decreased inhibition constant IC50 compared to untransduced control cells or cells expressing the wild-type nucleoside kinase. In summary, our data show that enzyme engineering can be used to shift the substrate specificity of the Dm-dNK to selectively increase the sensitivity of cells expressing the enzyme to purine nucleoside analogs.

Evidence of an intact N-terminal translocation sequence of human mitochondrial adenylate kinase 4.

Adenylate kinases are abundant nucleoside monophosphate kinases, which catalyze the phosphorylation of AMP by using ATP or GTP as phosphate donors. A previously cloned cDNA was named adenylate kinase 4 (AK4) based on its sequence similarity with known AKs but with no confirmed AK enzyme activity. In the present study the AK4 cDNA was expressed in Escherichia coli and the substrate specificity and kinetic properties of the recombinant protein were characterized. The enzyme catalyzed the phosphorylation of AMP, dAMP, CMP and dCMP with ATP or GTP as phosphate donors and AK4 also phosphorylated AMP with UTP as phosphate donor. The kinetic parameters of the enzyme were determined for AMP and dAMP with ATP as phosphate donor and for AMP with GTP as phosphate donor. AK4 showed its highest efficiency when phosphorylating AMP with GTP and a slightly lower efficiency for the phosphorylation of AMP with ATP. Among the three reactions for which kinetics were performed, dAMP was the poorest substrate. The AK4 mitochondrial localization was confirmed by expression of AK4 as a fusion protein with GFP in HeLa cells. The mitochondrial import sequence was shown to be located within the first N-terminal 11 amino acid residues, very close to the ATP-binding region of the enzyme. Import analysis suggested that the mitochondrial import sequence was not cleaved and thus the enzyme retained its activity upon entering the mitochondria. Site directed mutagenesis of amino acids Lys 4 and Arg 7 showed that these two residues were essential for mitochondrial import.

Late-onset respiratory failure due to TK2 mutations causing multiple mtDNA deletions

Mutations in nuclear genes involved in the maintenance of mitochondrial DNA (mtDNA) are associated with an extensive spectrum of clinical phenotypes, manifesting as either mtDNA depletion syndromes or multiple mtDNA deletion disorders.1

The human adenylate kinase 9 is a nucleoside mono- and diphosphate kinase.

Adenylate kinases regulate adenine nucleotide levels and are present in different intracellular compartments. These enzymes also participate in the activation of pharmacologically active nucleoside and nucleotide analogs. We have in the present study identified the ninth isoform of the adenylate kinase family of enzymes and accordingly named the protein adenylate kinase 9 (AK9). Initially a full-length cDNA of a hypothetical protein containing a predicted adenylate kinase domain was identified and subsequently cloned and expressed in Escherichia coli. The substrate specificity of the recombinant protein showed that the enzyme catalyzed the phosphorylation of AMP, dAMP, CMP and dCMP with ATP as phosphate donor, while only AMP and CMP were phosphorylated when GTP was the phosphate donor. The kinetic parameters of AK9 were determined for AMP, dAMP and CMP with ATP as phosphate donor. Interestingly, in addition to the diphosphate products, a nucleoside diphosphate kinase (NDPK) activity was also present with subsequent triphosphates formed. With ATP or GTP as phosphate donor it was possible to detect the production of ATP, CTP, GTP, UTP, dATP, dCTP, dGTP and TTP as enzymatic products from the corresponding diphosphate substrates. A number of previously characterized adenylate kinases were also tested and found to possess a broad phosphotransferase activity similar to AK9. These enzymes are accordingly suggested to be regarded as nucleoside mono- and diphosphate kinases with catalytic activities possibly determined by local substrate concentrations.

Identification of two active functional domains of human adenylate kinase 5

A full length cDNA that partially corresponded to human adenylate kinase 5 (AK5) was identified and shown to encode for two separate domains. The full length protein could be divided in two distinct functional domains, a previously unidentified domain of 338 amino acids and a second domain of 198 amino acids that corresponded to the protein characterized as AK5, now called AK5p2. The first domain, AK5p1, phosphorylated AMP, CMP, dAMP and dCMP with ATP or GTP as phosphate donors similarly to AK5p2. Our data demonstrate that human AK5 has two separate functional domains and that both have enzymatic activity.