Enhanced proteasomal degradation of mutant human thiopurine S-methyltransferase (TPMT) in mammalian cells: mechanism for TPMT protein deficiency inherited by TPMT*2, TPMT*3A, TPMT*3B or TPMT*3C

Inheritance of the TPMT*2, TPMT*3A and TPMT*3C mutant alleles is associated with deficiency of thiopurine S-methyltransferase (TPMT) activity in human s. However, unlike TPMT*2 and TPMT*3A, the catalytically active protein cod ed by TPMT*3C does not undergo enhanced proteolysis when heterologously exp ressed in yeast, making it unclear why this common mutant allele should be associated with inheritance of TPMT-deficiency. To further elucidate the me chanism for TPMT deficiency associated with these alleles, we characterized TPMT proteolysis following heterologous expression of wild-type and mutant proteins in mammalian cells. When expressed in COS-I cells, proteins encod ed by TPMT*2, TPMT*3A, and TPMT*3C cDNAs had significantly reduced steady-s tate levels and shorter degradation half-lives compared with the wild-type protein. Similarly, in rabbit reticulocyte lysate (RRL), these mutant TPMT proteins were degraded significantly faster than the wild-type protein. Thu s, enhanced proteolysis of TPMT*3C protein in mammalian cells is in contras t to its stability in yeast, but consistent with TPMT-deficiency in humans. Proteolysis was ATP-dependent and sensitive to proteasomal inhibitors MG11 5, MG132 and lactacystin, but not to calpain inhibitor II. We conclude that all of these mutant TPMT proteins undergo enhanced proteolysis in mammalia n cells, through an ATP-dependent proteasomal pathway, leading to low or un detectable levels of TPMT protein in humans who inherit these mutant allele s, Pharmacogenetics 9:641-650 (C) 1999 Lippincott Williams & Wilkins.