Rapid polyubiquitination and proteasomal degradation of a mutant form of NAD(P)H : Quinone oxidoreductase 1

The NAD(P) H: quinone oxidoreductase 1 (NQO1)*2 polymorphism is characteriz ed by a single proline-to-serine amino acid substitution. Cell lines and ti ssues from organisms genotyped as homozygous for the NQO1*2 polymorphism ar e deficient in NQO1 activity. In studies with cells homozygous for the wild type allele and cells homozygous for the mutant NQO1*2 allele, no differenc e in the half-life of NQO1 mRNA transcripts was observed. Similarly, in vit ro transcription/translation studies showed that both wild-type and mutant NQO1 coding regions were transcribed and translated into full-length protei n with equal efficiency. Protein turnover studies in NQO1 wild-type and mut ant cell lines demonstrated that the half-life of wild-type NQO1 was greate r than 18 h, whereas the half-life of mutant NQO1 was 1.2 h. Incubation of NQO1 mutant cell lines with proteasome inhibitors increased the amount of i mmunoreactive NQO1 protein, suggesting that mutant protein may be degraded via the proteasome pathway. Additional studies were performed using purifie d recombinant NQO1 wild-type and mutant proteins incubated in a rabbit reti culocyte lysate system. In these studies, no degradation of wild-type NQO1 protein was observed; however, mutant NQO1 protein was completely degraded in 2 h. Degradation of mutant NQO1 was inhibited by proteasome inhibitors a nd was ATP-dependent. Mutant NQO1 incubated in rabbit reticulocyte lysate w ith MG132 resulted in the accumulation of proteins with increased molecular masses that were immunoreactive for both NQO1 and ubiquitin. These data su ggest that wild-type NQO1 persists in cells whereas mutant NQO1 is rapidly degraded via ubiquitination and proteasome degradation.