Interaction of the molecular chaperone Hsp70 with human NAD(P)H:quinone oxidoreductase 1.
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.
Mesh Terms:
Alleles, Amino Acid Sequence, Catalysis, Cell Line, Cysteine Endopeptidases, Electrophoresis, Polyacrylamide Gel, FMN Reductase, HSP40 Heat-Shock Proteins, HSP70 Heat-Shock Proteins, Heat-Shock Proteins, Humans, Immunoblotting, Molecular Sequence Data, Multienzyme Complexes, Mutagenesis, Site-Directed, Mutation, NADH, NADPH Oxidoreductases, Plasmids, Precipitin Tests, Proteasome Endopeptidase Complex, Protein Binding, Protein Biosynthesis, Protein Folding, Protein Structure, Tertiary, Time Factors, Transcription, Genetic, Tumor Cells, Cultured
Alleles, Amino Acid Sequence, Catalysis, Cell Line, Cysteine Endopeptidases, Electrophoresis, Polyacrylamide Gel, FMN Reductase, HSP40 Heat-Shock Proteins, HSP70 Heat-Shock Proteins, Heat-Shock Proteins, Humans, Immunoblotting, Molecular Sequence Data, Multienzyme Complexes, Mutagenesis, Site-Directed, Mutation, NADH, NADPH Oxidoreductases, Plasmids, Precipitin Tests, Proteasome Endopeptidase Complex, Protein Binding, Protein Biosynthesis, Protein Folding, Protein Structure, Tertiary, Time Factors, Transcription, Genetic, Tumor Cells, Cultured
J. Biol. Chem.
Date: Apr. 19, 2002
PubMed ID: 11821413
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