Nonsynonymous SNPs are an important cause of inherited variation in drug response. Inherited alteration of only one or two amino acids often results in a decrease in the quantity of protein (see ) [4
]. When the mechanism responsible has been studied, protein degradation has been the most frequent cause [4
]. TPMT represents a striking example of both the clinical relevance of nonsynonymous SNPs for pharmacogenetics and the importance of protein degradation for these effects [1
]. TPMT genotyping studies have identified up to 30 variant alleles in different ethnic groups [12
] since the TPMT cDNA and gene were first cloned and characterized in 1993 and 1996, respectively [7
, 319 T>G, 107 Tyr>Asp, was recently reported in a renal transplantation recipient in Thailand although it was not functionally characterized [20
]. In the present study, we genotyped 220 healthy Thai subjects for TPMT
, but failed to identify any additional carriers of this novel allele, making it likely that, in this population, it represents a mutation rather than a polymorphism. However, the purpose of the present study was neither to determine the population frequency of this variant nor to determine its clinical implications, but rather to study mechanisms responsible for the decreased level of enzyme activity associated with this variant allele.
A chaperone protein-dependent, proteasome-medicated pathway [9
] and autophagy [8
], have both been shown to contribute to the degradation of TPMT*3A, the most common variant allele in Caucasian subjects. We tested the hypothesis that protein degradation might also contribute to the decrease in enzyme protein that we observed for TPMT*27. Transient expression of TPMT*27 in COS-1 cells, followed by quantitative Western blot analysis and assay of enzyme activity showed a dramatic decrease in levels of both immunoreactive protein and enzyme activity. Those results also fit well with the correlation between protein and activity that we reported previously for 13 TPMT allozymes () [6
]. Furthermore, TPMT*27 protein was rapidly degraded in a RRL system (). Immunoprecipitation demonstrated that two chaperone proteins which had been shown to be involved in targeting TPMT*3A for degradation, hsp70 and hsp 90 [11
], also interacted with TPMT*27. In addition, degradation of TPMT*27 was inhibited by the proteasome inhibitor MG132 (), and TPMT*27, like TPMT*3A [10
], formed aggresomes in COS-1 cells after proteasome inhibition (). We also observed that TPMT*27 protein levels increased after the inhibition of autophagy with 3-MA (), or after siRNA knockdown of ATG7 (). Therefore, autophagy appeared to be another route for TPMT*27 degradation just as it is for TPMT*3A [8
]. In summary, both proteasome-mediated degradation and autophagy contribute to TPMT*27 degradation. Finally, structural analysis and MD simulation suggested that substitution of Asp for Tyr at TPMT*27 position 107 might result in protein misfolding and resultant instability ().
It should be emphasized that not all TPMT variant allozymes with decreased activity can be explained by protein degradation. For example, TPMT*5 displays a moderately reduced level of immunoreactive protein together with nearly complete absence of enzyme activity [6
]. In the case of TPMT*5, an alteration at the active site of the enzyme rather than protein degradation appears to account for the loss of function [25
]. TPMT is not the only example for which inherited alteration in protein sequence results in reduced protein quantity. Other genes encoding enzymes with common nonsynonymous SNPs that are associated with functionally significant decreases in protein quantity include catechol O-methyltransferase (COMT), histamine N-methyltransferase (HNMT), a series of sulfotransferase (SULT) enzyme and quinone oxidoreductase 1 [1
]. Clearly, altered protein quantity is an important mechanism responsible for the functional effects of inherited variation in amino acid sequence, and this alteration, most often a decrease, frequently results from protein degradation.
In summary, we have studied mechanisms responsible for the decreased activity of TPMT*27, a novel variant allozyme for an important drug-metabolizing enzyme. We used this novel TPMT allele to test the hypothesis that protein clearance mechanisms might explain loss-of-function as a result of this genetic variation – perhaps as a result of protein misfolding. The mechanisms identified involved accelerated degradation mediated by both proteasome and autophagy pathways. Ultimately, it would be hoped that protein misfolding – and any related acceleration in degradation – might be predicted purely computationally, but that goal for pharmacogenetics still remains in the future.