Human thiopurine S-methyltransferase pharmacogenetics: variant allozyme misfolding and aggresome formation.
Thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs. TPMT genetic polymorphisms represent a striking example of the potential clinical value of pharmacogenetics. Subjects homozygous for TPMT*3A, the most common variant allele for low activity, an allele that encodes a protein with two changes in amino acid sequence, are at greatly increased risk for life-threatening toxicity when treated with standard doses of thiopurines. These subjects have virtually undetectable levels of TPMT protein. In this study, we tested the hypothesis that TPMT*3A might result in protein misfolding and aggregation. We observed that TPMT*3A forms aggresomes in cultured cells and that it aggregates in vitro, functional mechanisms not previously described in pharmacogenetics. Furthermore, there was a correlation among TPMT half-life values in rabbit reticulocyte lysate, aggresome formation in COS-1 cells, and protein aggregation in vitro for the three variant allozymes encoded by alleles that include the two TPMT*3A single-nucleotide polymorphisms. These observations were compatible with a common structural explanation for all of these effects, a conclusion supported by size-exclusion chromatography and CD spectroscopy. The results of these experiments provide insight into a unique pharmacogenetic mechanism by which common polymorphisms affect TPMT protein function and, as a result, therapeutic response to thiopurine drugs.
Wang
Liewei
L
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Nguyen
Tien V
TV
McLaughlin
Richard W
RW
Sikkink
Laura A
LA
Ramirez-Alvarado
Marina
M
Weinshilboum
Richard M
RM
D000483:Alleles; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D019556:COS Cells; D002845:Chromatography; D002942:Circular Dichroism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D014644:Genetic Variation; D000073864:Histone Deacetylase 6; D006655:Histone Deacetylases / Q000737:chemistry; D006720:Homozygote; D006801:Humans; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D008780:Methyltransferases / Q000235:genetics* / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism; D010597:Pharmacogenetics / Q000379:methods*; D011110:Polymorphism, Genetic; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D011687:Purines / Q000737:chemistry; D011804:Quinolines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature; D014162:Transfection; D025801:Ubiquitin / Q000737:chemistry; D014747:Vinblastine / Q000494:pharmacology
D001426:Bacterial Proteins; D015853:Cysteine Proteinase Inhibitors; D006898:Hydroxylamines; D007976:Leupeptins; D011506:Proteins; D011687:Purines; D011804:Quinolines; D011994:Recombinant Proteins; D025801:Ubiquitin; C410733:scriptaid; D014747:Vinblastine; D008780:Methyltransferases; C022745:thiopurine methyltransferase; C433811:HDAC6 protein, human; D000073864:Histone Deacetylase 6; D006655:Histone Deacetylases; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde