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  • br Results and Discussion br Materials and


    Results and Discussion
    Materials and Methods
    Introduction Inorganic pyrophosphate is generated as a by-product in several enzymatic reactions in cells. These reactions include DNA and RNA synthesis, activation of fatty acids and amino acids, as well as synthesis of the cyclic nucleotides cAMP and cGMP [1], [2]. Pyrophosphate cannot be exported efficiently across the cell membrane and rapid intracellular hydrolysis of pyrophosphate is important for the progression and thermodynamic pull of the reactions. Pyrophosphatases (PPases)1 catalyze the intracellular hydrolysis of pyrophosphate to inorganic phosphate. A family of soluble PPases has been identified and members of this enzyme family are present in both prokaryotic and eukaryotic cells [1], [3]. Escherichia coli contain one major soluble PPase, which has been shown to be essential for cell growth [4], [5]. The yeast Saccharomyces cerevisiae contains two sequence-related PPase genes: PPA1 and PPA2 [6], [7]. The PPA1 gene encodes a cytosolic PPase that, similar to the E. coli PPase, has been shown to be required for cell growth [7]. The yeast PPA2 gene contains an N-terminal mitochondrial import signal sequence and the enzyme is localized in the mitochondrial matrix. The PPA2 gene product has been shown to be required for mitochondrial DNA (mtDNA) maintenance. Yeast cells deficient in expression of the mitochondrial PPase exhibit a ρ0 phenotype, with no detectable mtDNA, and require growth medium that supports Wiskostatin metabolism [7]. In mammalian cells, both a soluble cytosolic and a mitochondrial PPase have Wiskostatin been detected in biochemical studies [8], [9], [10], [11], [12]. Yang and co-workers purified a cytosolic PPase from bovine retina and cloned the cDNA encoding the enzyme [12], [13]. The enzyme was shown to be sequence related to the family of soluble PPase\'s of yeast and bacteria. Recently, the cloning and recombinant expression of the human homologue of the bovine cytosolic PPase were reported [14], [15]. Less is known about the molecular properties of the mammalian mitochondrial PPase. The enzyme is located in the mitochondrial matrix and biochemical studies suggest that the enzyme may be associated with mitochondrial inner membrane proteins such as components of the respiratory chain [2], [10], [11]. Several different PPase-containing protein complexes with different molecular masses have been purified from mammalian mitochondria, but it appears that the catalytically active subunit in each complex has a size similar to that of the yeast mitochondrial PPase [10], [11]. The ρ0 phenotype of yeast cells lacking the mitochondrial PPase suggests that deficiency in the expression of the human mitochondrial PPase could result in mtDNA depletion. Inherited mtDNA depletion syndromes (MDS) are a heterogeneous group of diseases that usually present with severe symptoms of mitochondrial dysfunction in early childhood [16], [17]. For many of these patients the genetic defect causing MDS remains undetermined. We cloned the cDNA and gene encoding the human mitochondrial PPase to investigate the role of this enzyme in mitochondrial pyrophosphate metabolism and mtDNA maintenance. To investigate if PPase-2 is involved in MDS in humans, we sequenced the PPA2 gene from patients with MDS of unknown cause. In the present study, we report the molecular characterization of the human mitochondrial PPase and compare its properties to those of the human cytosolic PPase. We also report the sequencing of the PPA2 gene from 13 patients with MDS.
    Discussion Inherited MDS are rare and usually severe diseases that often present in infancy or early childhood [16], [17]. The symptoms of these disorders are related to mitochondrial dysfunction and decreased oxidative phosphorylation. The organs most affected are highly dependent on aerobic metabolism, such as muscle, liver, and brain. However, there is wide tissue specificity among MDS patients, likely associated with different genetic etiologies. Recently, mutations in the mitochondrial deoxyribonucleoside kinases, deoxyguanosine kinase, and thymidine kinase 2 have been identified in some patients with mtDNA depletion [20], [21]. However, subsequent studies suggest that mutations in these genes account for only a minority of MDS cases and that other genetic defects are responsible for the syndrome in the majority of patients [22]. The importance of the yeast mitochondrial PPase in mtDNA maintenance suggests that the human PPase-2 also may be required for normal mtDNA integrity and patients with mtDNA depletion syndrome of unknown cause were therefore screened for mutations in the PPA2 gene. However, no pathogenic mutations were identified in the PPA2 gene of the 13 MDS patients studied. Although we found no evidence in the investigated patients that PPA2 gene mutations are likely to be a common cause of MDS in humans, additional patients with MDS are being investigated for PPA2 gene mutations. Accordingly, identification of further genes needed for mtDNA maintenance might pinpoint a major gene causing MDS.