Functional Study of the P32T ITPA Variant Associated with Drug Sensitivity in Humans

Elena I. Stepchenkova, Elena R. Tarakhovskaya, Kathryn Spitler, Christin Frahm, Miriam R. Menezes, Peter D. Simone, Carol Kolar, Luis A. Marky, Gloria E.O. Borgstahl, Youri I. Pavlov

Research output: Contribution to journalArticle

49 Scopus citations

Abstract

Sanitization of the cellular nucleotide pools from mutagenic base analogues is necessary for the accuracy of transcription and replication of genetic material and plays a substantial role in cancer prevention. The undesirable mutagenic, recombinogenic, and toxic incorporation of purine base analogues [i.e., ITP, dITP, XTP, dXTP, or 6-hydroxylaminopurine (HAP) deoxynucleoside triphosphate] into nucleic acids is prevented by inosine triphosphate pyrophosphatase (ITPA). The ITPA gene is a highly conserved, moderately expressed gene. Defects in ITPA orthologs in model organisms cause severe sensitivity to HAP and chromosome fragmentation. A human polymorphic allele, 94C→A, encodes for the enzyme with a P32T amino acid change and leads to accumulation of non-hydrolyzed ITP. ITPase activity is not detected in erythrocytes of these patients. The P32T polymorphism has also been associated with adverse sensitivity to purine base analogue drugs. We have found that the ITPA-P32T mutant is a dimer in solution, as is wild-type ITPA, and has normal ITPA activity in vitro, but the melting point of ITPA-P32T is 5 °C lower than that of wild-type. ITPA-P32T is also fully functional in vivo in model organisms as determined by a HAP mutagenesis assay and its complementation of a bacterial ITPA defect. The amount of ITPA protein detected by Western blot is severely diminished in a human fibroblast cell line with the 94C→A change. We propose that the P32T mutation exerts its effect in certain human tissues by cumulative effects of destabilization of transcripts, protein stability, and availability.

Original languageEnglish (US)
Pages (from-to)602-613
Number of pages12
JournalJournal of Molecular Biology
Volume392
Issue number3
DOIs
StatePublished - Sep 25 2009

Keywords

  • ITPA
  • base analogs
  • drug sensitivity
  • nucleotide pools

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

Fingerprint Dive into the research topics of 'Functional Study of the P32T ITPA Variant Associated with Drug Sensitivity in Humans'. Together they form a unique fingerprint.

  • Cite this

    Stepchenkova, E. I., Tarakhovskaya, E. R., Spitler, K., Frahm, C., Menezes, M. R., Simone, P. D., Kolar, C., Marky, L. A., Borgstahl, G. E. O., & Pavlov, Y. I. (2009). Functional Study of the P32T ITPA Variant Associated with Drug Sensitivity in Humans. Journal of Molecular Biology, 392(3), 602-613. https://doi.org/10.1016/j.jmb.2009.07.051