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 characterized by a single proline-to-serine amino acid substitution. Cell lines and tissues from organisms genotyped as homozygous for the NQO1*2 polymorphism are deficient in NQO1 activity. In studies with cells homozygous for the wild-type allele and cells homozygous for the mutant NQO1*2 allele, no difference in the half-life of NQO1 mRNA transcripts was observed. Similarly, in vitro transcription/translation studies showed that both wild-type and mutant NQO1 coding regions were transcribed and translated into full-length protein with equal efficiency. Protein turnover studies in NQO1 wild-type and mutant cell lines demonstrated that the half-life of wild-type NQO1 was greater 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 immunoreactive NQO1 protein, suggesting that mutant protein may be degraded via the proteasome pathway. Additional studies were performed using purified recombinant NQO1 wild-type and mutant proteins incubated in a rabbit reticulocyte 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 and was ATP-dependent. Mutant NQO1 incubated in rabbit reticulocyte lysate with MG132 resulted in the accumulation of proteins with increased molecular masses that were immunoreactive for both NQO1 and ubiquitin. These data suggest that wild-type NQO1 persists in cells whereas mutant NQO1 is rapidly degraded via ubiquitination and proteasome degradation.
Siegel
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D
Department of Pharmaceutical Sciences, School of Pharmacy and Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA. siegel@uchsc.edu
Anwar
A
A
Winski
S L
SL
Kepa
J K
JK
Zolman
K L
KL
Ross
D
D
D000818:Animals; D001704:Biopolymers / Q000378:metabolism; D018938:Caco-2 Cells; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D019073:HT29 Cells; D006801:Humans; D009097:Multienzyme Complexes / Q000378:metabolism*; D009154:Mutation; D011110:Polymorphism, Genetic; D046988:Proteasome Endopeptidase Complex; D011808:Quinone Reductases / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D014452:Ubiquitins / Q000378:metabolism*
D001704:Biopolymers; D009097:Multienzyme Complexes; D014452:Ubiquitins; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex