title: Interaction of the molecular chaperone Hsp70 with human NAD(P)H:quinone oxidoreductase 1.
abstract: NAD(P)H:quinone oxidoreductase 1 (EC; DT-Diaphorase, NQO1) is predominantly a cytosolic flavoenzyme that catalyzes a two-electron reduction. Using human tumor cell lines devoid of NQO1 enzymatic activity, we have previously identified a single nucleotide polymorphism (NQO1*2 allele) in the human NQO1 gene. This mutation has been characterized as a genetic polymorphism (NQO1*2), which leads to greatly diminished levels of protein due to rapid degradation of the NQO1*2 protein by the ubiquitin proteasomal pathway (UPP). In an attempt to decipher the mechanism responsible for the differential stability of wild-type NQO1*1 and mutant NQO1*2 proteins, we have investigated the interactions of these proteins with molecular chaperones of the Hsp family. Using co-immunoprecipitation studies (co-IPs), no association was observed between Hsp90 and either wild-type NQO1*1 or mutant NQO1*2 proteins. Hsp70, however, was found to associate with NQO1*1 protein in cells when co-IPs were performed with an anti-NQO1 antibody followed by immunoblotting with an anti-Hsp70 antibody or vice versa. Hsp40 could also be detected in the immunoprecipitated protein complex. Experiments were also performed using either the NQO1*1 or NQO1*2 coding regions in an in vitro transcription/translation system employing rabbit reticulocyte lysates (RRLs). Consistent with the cellular data, co-IP experiments in RRLs demonstrated an association of Hsp70 with wild-type NQO1*1 protein but not with NQO1*2 protein. To further elucidate the role of the association of Hsp70 with the NQO1*1 protein, site-directed mutagenesis was used to modify a proposed Hsp70 binding site near the N terminus of the NQO1 protein. We generated a plasmid containing an NQO1*1 coding region with a mutated Hsp70 binding site (isoleucine to aspartic acid at position 8, NQO1*1/I8D). In contrast to the NQO1*1 protein translated in RRLs, the NQO1*1/I8D protein did not associate with Hsp70, as demonstrated by co-IP, was catalytically inactive, and was degraded by the UPP. These data suggest that the association of Hsp70 with NQO1*1 may play an important role in the stability and functionality of the NQO1 protein.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy and Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
authors.forename: Adil
authors.initials: A
authors.lastname: Anwar
authors.forename: David
authors.initials: D
authors.lastname: Siegel
authors.forename: Jadwiga K
authors.initials: JK
authors.lastname: Kepa
authors.forename: David
authors.initials: D
authors.lastname: Ross
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D002384:Catalysis; D002460:Cell Line; D003546:Cysteine Endopeptidases; D004591:Electrophoresis, Polyacrylamide Gel; D038181:FMN Reductase; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D014176:Protein Biosynthesis; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
chemical_list: C494647:DNAJB1 protein, human; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D009097:Multienzyme Complexes; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex