Rapid Polyubiquitination and Proteasomal Degradation of a Mutant Form of NAD(P)H:Quinone Oxidoreductase 1

Abstract

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.

Expression of the prodrug-activating enzyme DT-diaphorase via Ad5 delivery to human colon carcinoma cells in vitro

“…BE cells infected with Ad5.DTD 609T exhibited no major increases in MMC sensitivity, consistent with previous observations of impaired catalytic activity and protein instability of the DTD 609T isoform. 18,19 As shown in Figure 5C, exogenous expression of DTD was confirmed in cells infected with Ad5.DTD 609C by assessing DTD activities in cells that were infected in parallel with the MMC sensitivity studies described above.…”

“…-gal or Ad5.DTD A single-base transition corresponding to nucleotide 609 of DTD cDNA ( C -to -T ) has been shown to result in impaired DTD activity and decreased protein stability. 18,19 HT29 cells ( homozygous for the wild -type DTD allele ) displayed mean ± SEM (n = 5) DTD activity of 1060 ± 215 nmol / min / mg protein, whereas BE cells (homozygous for the variant DTD allele ) contained very low to undetectable DTD activity ( less than 20 nmol /min /mg protein ). Consistent with the fact that the variant allele promotes decreased DTD protein stability, 19 HT29 cells display Western blot reactivity whereas BE cells express very low to undetectable levels of DTD protein.…”

“…This base change is frequent in the population 33 and has been associated with reduced catalytic activity, protein instability, and MMC resistance. 15,18,21 One way to address this problem is to introduce a bioreductive enzyme like DTD into tumor cells using gene transfer techniques. 34 Gene therapy strategies involving the delivery of activating enzymes to sensitize human tumors to prodrugs have been explored using nonmammalian enzymes, such as herpes simplex virus thymidine kinase 35 and E. coli cytosine deaminase 36 in combination with the nucleoside analogues ganciclovir and 5 -fluorocytosine, respectively.…”

“…This base transition causes a proline-to -serine change in amino acid 187, resulting in impaired DTD catalytic activity and protein instability. 15,18 Individuals homozygous for this mutation exhibit little or no DTD activity and heterozygotes have approximately half the DTD activity observed in wild -type cells. 18,19 Homozygous mutants have a much reduced sensitivity to MMC under aerobic exposure conditions in vitro.…”

“…15,18 Individuals homozygous for this mutation exhibit little or no DTD activity and heterozygotes have approximately half the DTD activity observed in wild -type cells. 18,19 Homozygous mutants have a much reduced sensitivity to MMC under aerobic exposure conditions in vitro. 10,15 To reduce the problem of heterogeneity and to enhance DTD activity in tumor cells, recombinant adenoviral vectors were constructed, which express DTD minigenes for both wild -type and mutant ( C -to -T base change ) DTD under the control of the cytomegalovirus (CMV ) promoter.…”