Thiopurine S-methyltransferase pharmacogenetics: autophagy as a mechanism for variant allozyme degradation.
OBJECTIVE
Thiopurine S-methyltransferase (TPMT)*3A is degraded much more rapidly than is the 'wild-type' enzyme through a ubiquitin-proteasome-dependent process. It also forms aggresomes, suggesting a possible dynamic balance between degradation and aggregation. We set out to identify genes encoding proteins participating in these processes.


METHODS
Green fluorescent protein tagged TPMT*3A was expressed in a Saccharomyces cerevisiae gene deletion library, and flow cytometry was used to screen for cells with high fluorescence intensity, indicating the loss of a gene essential for TPMT*3A degradation.


RESULTS
Twenty-four yeast genes were identified in functional categories that included ubiquitin-dependent protein degradation, vesicle trafficking, and vacuolar degradation. The presence of genes encoding proteins involved in vesicular transport and vacuolar degradation suggested a possible role in TPMT*3A degradation for autophagy--a process not previously identified as a pharmacogenomic mechanism. In support of that hypothesis, TPMT*3A aggregates increased dramatically in mutants for vacuolar protease and autophagy-related genes. Furthermore, TPMT*3A expression in human cells induced autophagy, and small interfering RNA-mediated knockdown of ATG7, an autophagy-related human protein, enhanced TPMT*3A aggregation but not that of TPMT*3C or wild-type TPMT, indicating that autophagy contributes to TPMT*3A degradation in mammalian cells. We also demonstrated that UBE2G2, the human homologue of the E2 ubiquitin-conjugating enzyme identified during the yeast genetic screen, was involved in TPMT*3A degradation in human cells.


CONCLUSIONS
These results indicate that autophagy should be considered among mechanisms responsible for the effects of pharmacogenetically significant polymorphisms that alter encoded amino acids.
Li
Fang
F
Division of Clinical Pharmacology, Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic-Mayo Medical School, Rochester, Minnesota 55905, USA.
Wang
Liewei
L
Burgess
Rebecca J
RJ
Weinshilboum
Richard M
RM
D001343:Autophagy; D002460:Cell Line; D004398:Dyneins / Q000378:metabolism; D005434:Flow Cytometry; D017353:Gene Deletion; D005800:Genes, Fungal; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000235:genetics
D007527:Isoenzymes; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008780:Methyltransferases; C022745:thiopurine methyltransferase; D004398:Dyneins