Quantitative in vitro and in vivo characterization of the human P32T mutant ITPase
Résumé
Human ITPase, encoded by the gene, and its orthologs (RdgB in and HAM1 in ) exclude noncanonical nucleoside triphosphates (NTPs) from NTP pools. Deoxyinosine triphosphate (dITP) and 2'-deoxy--6-hydroxylaminopurine triphosphate are both hydrolyzed by ITPase to yield the corresponding deoxynucleoside monophosphate and pyrophosphate. In addition, metabolites of thiopurine drugs such as azathioprine have been shown to be substrates for ITPase. The 94C>A [P32T] variant is one of two polymorphisms associated with decreased ITPase activity. Furthermore, the 94C>A [P32T] variant is associated with an increased risk of adverse drug reactions for patients treated with azathioprine. The nature of the observed phenotypes for 94C>A [P32T] variant individuals is currently unclear. Our biochemical assays indicate the P32T ITPase has 55% activity with dITP compared to wild-type ITPase. Complementation experiments at 37C show that -6-hydroxylaminopurine sensitivity of mutants is reduced with a plasmid bearing the 94C>A [P32T] gene approximately 50% less than with a plasmid bearing the wild-type gene. The reduction in sensitivity is less at 42C. Experiments with synthetic lethal (ts) mutants show that the 94C>A [P32T] gene also complements the (ts) growth deficiency at 42C approximately 40% lower than wild-type gene Western blot analysis indicates the expression level of P32T ITPase is reduced in these cells relative to wild-type. Our data support the idea that P32T ITPase is a functional protein, albeit with a reduced rate of noncanonical NTP pyrophosphohydrolase activity and reduced protein stability.
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