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. CONCLUSION: These results indicate that autophagy should be considered among mechanisms responsible for the effects of pharmacogenetically significant polymorphisms that alter encoded amino acids.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|