Human thiopurine S-methyltransferase (TPMT) pharmacogenetics: Variant allozyme aggresome formation

Abstract

Background/Aims TPMT*3A, the most common TPMT variant allozyme in Caucasians, has 2 alterations in amino acid sequence, resulting in striking decreases in TPMT protein levels as a result of rapid degradation. “Protein quality control” involves a dynamic balance among protein folding, degradation and aggregation with aggresome formation. We set out to test the hypothesis that, the presence of the proteasome inhibitor MG132, TPMT*3A can form aggresomes in cultured cells. Methods/Results TPMT*3A was highly ubiquinated and associated with hsp90 after transient expression in COS‐1 cells. In the presence of MG132, aggresomes formed in cells transfected with TPMT*3A, and ubiquinated TPMT*3A redistributed from the cytosol into the pellet ‐ compatible with aggresome formation. The aggresomes contained vimentin, dynein, γ‐tubulin, ubiquitin, hsp70, hsp90 and the deacetylating enzyme HDAC6 that co‐localized with TPMT*3A. Aggresome formation could be blocked by the microtubule destabilizing agent, vinblastine. Treatment with the HDAC inhibitor scriptaid inhibited aggresome formation and resulted in TPMT*3A microaggregate formation. Conclusions These results suggest that aggresome formation is one mechanism by which nonsynonymous cSNPs can alter function for proteins of pharmacogenetic importance such as TPMT. Clinical Pharmacology & Therapeutics (2005) 77, P95–P95; doi:

Thiopurine S-methyltransferase pharmacogenetics: variant allele functional and comparative genomics

“…That observation is in accordance with a growing body of evidence demonstrating that an alteration in the level of protein is a common mechanism responsible for the functional effects of genetic polymorphisms involving nonsynonymous cSNPs, most often as a result of accelerated protein degradation [30]. Recently, it has been reported that TPMT aggregation and aggresome formation may also contribute to decreased levels of TPMT*3A [31]. Multiple variant TPMT alleles with nonsynonymous cSNPs have been reported, making it possible to explore mechanisms responsible for the functional effects of naturally occurring alterations in amino acid sequences for this clinically important gene [11,[16][17][18][19][20][21][22][23][24][25].…”

Human Aromatase: Gene Resequencing and Functional Genomics

“…Functional characterization of the four variant allozymes encoded by alleles with nonsynonymous cSNPs showed a significant correlation between level of activity and immunoreactive protein (Fig. 3C), an observation compatible with a growing body of data that indicate that alteration in protein quantity is a common mechanism responsible for the functional effects of this type of polymorphism, most often as a result of accelerated protein degradation (37) but also, at times, involving intracellular protein aggregation (42). Obviously, our results must be confirmed in the future by in vivo genotypephenotype correlation studies.…”

“…One of the more striking observations made in the course of our studies was the significant correlation between levels of activity and protein for CYP19 variant allozymes-a phenomenon that confirms that a common, although certainly not the only, mechanism by which nonsynonymous cSNPs influence function is by altering levels of protein-most often as a result of accelerated degradation, at times with protein aggregation and aggresome formation (37,(40)(41)(42). Obviously, it would be ideal to know the structure of CYP19 to pursue our functional observations.…”

Human 3β-hydroxysteroid dehydrogenase types 1 and 2: Gene sequence variation and functional genomics

“…Several mechanisms might be responsible for these decreases in level of enzyme protein. However, the most common mechanism by which nonsynonymous cSNP affect protein quantity is accelerated degradation [23,24,39]. Therefore, in vitro degradation studies were performed with the HSD3B1 Phe96 variant allozyme that displayed a greater than 50% decrease in protein quantity.…”

“…Transcription and translation of HSD3B1 and HSD3B2 allozymes were performed with the TNT® coupled rabbit reticulocyte lysate (RRL) System (Promega), as described by Wang et al [23][24][25]. Specifically, 1 µg of expression construct DNA, together with 2 µL T7 buffer, 1 µL T7 polymerase, 1 µL of a mixture of amino acids that lacked methionine, 1 µL RNasin (Promega) and 2 µL of 35 S-methionine (1000 Ci/mM, 10 mCi/mL, 0.4 µM final concentration) (Amersham Pharmacia Biotech) were added to 25µL RRL that had been "treated" to inhibit protein degradation.…”