ENHANCED PROTEOLYSIS OF THIOPURINE S-METHYLTRANSFERASE (TPMT) ENCODEDBY MUTANT ALLELES IN HUMANS (TPMT-ASTERISK-3A, TPMT-ASTERISK-2) - MECHANISMS FOR THE GENETIC-POLYMORPHISM OF TPMT ACTIVITY

TPMT is a cytosolic enzyme that catalyzes the S-methylation of aromati c and heterocyclic sulfhydryl compounds, including medications such as mercaptopurine and thioguanine, TPMT activity exhibits autosomal codo minant genetic polymorphism, and patients inheriting TPMT deficiency a re at high risk of potentially fatal hematopoietic toxicity. The most prevalent mutant alleles associated with TPMT deficiency in humans hav e been cloned and characterized (TPMT2 and TPMT*3A), but the mechanis ms for loss of catalytic activity have not been elucidated, In the pre sent study, we established that erythrocyte TPMT activity was signific antly related to the amount of TPMT protein on Western blots of erythr ocytes from patients with TPMT activities of 0.4-23 units/ml pRBC (r(s ) = 0.99; P < 0.001), Similarly, heterologous expression of wild-type (TPMT1) and mutant (TPMT*2 and TPMT*3A) human cDNAs in yeast and COS- 1 cells demonstrated comparable levels of TPMT mRNA but significantly lower TPMT protein with the mutant cDNAs, Rates of protein synthesis w ere comparable for wild-type and mutant proteins expressed in yeast an d with in vitro translation in rabbit reticulocyte lysates, In contras t, pulse-chase experiments revealed significantly shorter degradation half-lives for TPMT2 and TPMT*3A (similar to 0.25 hr) compared with w ild-type TPMT1 (18 hr), The degradation of mutant proteins was impair ed by ATP depletion and in yeast with mutant proteasomes (pre-1 strain ) but unaffected by the lysosomal inhibitor chloroquine. These studies establish enhanced degradation of TPMT proteins encoded by TPMT2 and TPMT3A as mechanisms for lower TPMT protein and catalytic activity i nherited by the predominant mutant alleles at the human TPMT locus.