Functional study of the P32T ITPA variant associated with drug sensitivity in humans.
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 degrees 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.
Stepchenkova
Elena I
EI
Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, 68198-6805, USA.
Tarakhovskaya
Elena R
ER
Spitler
Kathryn
K
Frahm
Christin
C
Menezes
Miriam R
MR
Simone
Peter D
PD
Kolar
Carol
C
Marky
Luis A
LA
Borgstahl
Gloria E O
GE
Pavlov
Youri I
YI
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004351:Drug Resistance / Q000235:genetics*; D004795:Enzyme Stability; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011110:Polymorphism, Genetic; D011487:Protein Conformation; D011489:Protein Denaturation; D055503:Protein Multimerization; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000097630:Inosine Triphosphatase
D011755:Pyrophosphatases; C511829:ITPA protein, human; D000097630:Inosine Triphosphatase