Studies of enzymes in mitochondrial DNA precursor synthesis : regulatory mechanisms for human thymidine kinase 2 and deoxyguanosine kinase

Abstract: As important enzymes in mitochondrial nucleotide salvage pathway, thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) are expressed constitutively in almost all cells. These two enzymes catalyze the initial rate-limiting phosphorylation of pyrimidine and purine deoxynucleosides, respectively, providing DNA precursors for mitochondrial DNA (mtDNA) replication. Inherited genetic defects in TK2 have been associated with infantile myopathic form of mtDNA depletion syndrome (MDS). In study I, two mutations, R225W and a novel T230A, in TK2 are identified as a new genetic cause of adult-onset autosomal recessive progressive external ophthalmoplegia (arPEO) and the kinetic and structural effects of the two mutations on enzyme function have been characterized. Nucleoside analogs are widely used in anti-viral and anti-cancer chemotherapy, but they can cause severe side-effect such as mtDNA depletion. In study II, the potential mechanism underlying pyrimidine nucleoside analogs-associated mitochondrial toxicities was investigated, and showed that thymidine analogs had opposite effects on dThd and dCyd phosphorylation and thus can inhibit dThd salvage, leading to imbalanced dTTP and dCTP pools. It was found that the mechanism is most likely due to that TK2 normally exists in an inactive form with bound dTTP. The redox regulation of TK2 and dGK was studied in study III and IV. The activity of both enzymes was sensitive to the cellular redox status. Under oxidative stress, both TK2 and dGK can be reversibly S-glutathionylated by GSSG. The modification of the conserved Cys189 in TK2 was responsible for a partial inactivation and selective degradation of TK2 in mitochondria, most likely via the AAA⁺ Lon protease. The oxidative effect of nucleoside analogs was also evaluated. Treatment with 3’-azido- 2’,3’-dideoxythymidine (AZT) and 2’,3’-dideoxyinosine (ddI) led to degradation of mitochondrial TK2 and dGK, whereas uridine and guanosine supplementations to AZT respective ddI treatments prevented both proteins from degradation.