The human ITPA polymorphic variant P32T is destabilized by the unpacking of the hydrophobic core

Abstract

Inosine triphosphate pyrophosphatase (ITPA), a key enzyme involved in maintaining the purity of cellular nucleoside triphosphate pools, specifically recognizes inosine triphosphate and xanthosine triphosphate (including the deoxyribose forms) and detoxifies them by catalyzing the hydrolysis of a phosphoanhydride bond, releasing pyrophosphate. This prevents their inappropriate use as substrates in enzymatic reactions utilizing (d)ATP or (d)GTP. A human genetic polymorphism leads to the substitution of Thr for Pro32 (P32T) and causes ITPA deficiency in erythrocytes, with heterozygotes having on average 22.5% residual activity, and homozygotes having undetectable activity. This polymorphism has been implicated in modulating patients’ response to mercaptopurines and ribavirin. Human fibroblasts containing this variant have elevated genomic instability upon treatment with base analogs. We find that the wild-type and P32T forms are dimeric in solution and in the crystal structure. This abolishes the previous speculation that the P32T change disrupts dimerization as a mechanism of inactivation. The only difference in structure from the wild-type protein is that the area surrounding Thr32 is disrupted. Phe31 is flipped from the hydrophobic core out into the solvent, leaving a hole in the hydrophobic core of the protein which likely accounts for the reduced thermal stability of P32T ITPA and ultimately leads to its susceptibility to degradation in human cells. Circular dichroism and thermal denaturation studies confirm these structural results. We propose that the dimer of P32T variant subunit with wild-type subunit is degraded in cells similarly to the P32T homodimer explaining the level of loss of ITPA activity in heterozygotes.

Recurrent activating mutations of CD28 in peripheral T-cell lymphomas

“…Briefly, to analyze the effects of the D124V mutation in CD28, PDB files 1YJD (CD28) 28 , 1I85 (CD86 and CTLA4) 29 , 1I8L (CD80 and CTLA4) 30 , and 3WA4 (GRB2 and CD28) 31 were used and analyzed as previously described 32–34 .…”

Effect of nitric oxide on conformational changes of ovalbumin accompanying self-assembly into non-disease-associated fibrils

“…This region is located close to helix A (hA; 1 GSIGAASMEFCFDVFKELKVHH 22 ) and helix B (hB; 32 IAIMSALAMVYL 43 ), which are in an amphiphilic region and an amyloidogenic core region of the N-terminal, respectively. Several proteins showed structurally unstable through enhanced polarity by substitution of charged residues in hydrophobic core [28,29]. Cysteine residues susceptible to S-nitrosylation are found mainly in peptides within hydrophobic pockets.…”

Inosine Triphosphate Pyrophosphatase Gene Polymorphisms and Ribavirin-Induced Anemia in HCV Patients

“…The ITPase catalyses the conversion of inosine triphosphate (ITP) to inosine monophosphate (IMP), and pyrophosphate, so the ITP does not accumulate in normal cells. There are two single nucleotide polymorphisms (SNPs) in ITPA gene, located on chromosome 20 [12]. The first polymorphism concerns a missense variant in exon 2; a variant of ITPA 94C>A (rs1127354), the second concerns a splicing-altering single nucleotide polymorphism (SNP) in intron 2; IVS2+21 A>C (rs7270101) [7,12].…”

“…There are two single nucleotide polymorphisms (SNPs) in ITPA gene, located on chromosome 20 [12]. The first polymorphism concerns a missense variant in exon 2; a variant of ITPA 94C>A (rs1127354), the second concerns a splicing-altering single nucleotide polymorphism (SNP) in intron 2; IVS2+21 A>C (rs7270101) [7,12]. These two ITPA polymorphisms are associated with reduced ITPase activity resulting in accumulation of ITP in red blood cells (RBCs) and subsequent hemolysis [13,14].…”