Proteasomal degradation of human CYP1B1: effect of the Asn453Ser polymorphism on the post-translational regulation of CYP1B1 expression.
Allelic variations in CYP1B1 are reported to modulate the incidence of several types of cancer. To provide a mechanistic basis for this association, we investigated the impact of nonsilent allelic changes on the intracellular levels and post-translational regulation of CYP1B1 protein. When transiently expressed in COS-1 cells, either in the presence or absence of recombinant cytochrome P450 reductase, the cellular level of the CYP1B1.4 allelic variant (containing a Ser at the amino acid position 453; Ser453) was 2-fold lower compared with the other four allelic CYP1B1 proteins (containing Asn453), as analyzed by both immunoblotting and ethoxyresorufin O-deethylase activity. This difference was caused by post-translational regulation; as in the presence of cycloheximide, the rate of degradation of immunodetectable and enzymatically active CYP1B1.4 was distinctly faster than that of CYP1B1.1. Pulse-chase analysis revealed that the half-life of CYP1B1.4 was a mere 1.6 h compared with 4.8 h for CYP1B1.1. The presence of the proteasome inhibitor MG132 [N-benzoyloxycarbonyl (Z)-Leu-Leuleucinal] increased the stability not only of immunodetectable CYP1B1, but also--unexpectedly given the size of the proteasome access channel--increased the stability of enzymatically active CYP1B1. The data presented herein also demonstrate that CYP1B1 is targeted for its polymorphism-dependent degradation by polyubiquitination but not phosphorylation. Our results importantly provide a mechanism to explain the recently reported lower incidence of endometrial cancer in individuals carrying the CYP1B1*4 compared with the CYP1B1*1 haplo-type. In addition, the mechanistic paradigms revealed herein may explain the strong overexpression of CYP1B1 in tumors compared with nondiseased tissues.
Bandiera
Silvio
S
Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee, UK.
Weidlich
Simone
S
Harth
Volker
V
Broede
Peter
P
Ko
Yun
Y
Friedberg
Thomas
T
D000483:Alleles; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001216:Asparagine / Q000235:genetics*; D019556:COS Cells; D002522:Chlorocebus aethiops; D065633:Cytochrome P-450 CYP1B1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D011110:Polymorphism, Genetic / Q000235:genetics*; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D012694:Serine / Q000235:genetics*
D012694:Serine; D001216:Asparagine; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; D065633:Cytochrome P-450 CYP1B1; D046988:Proteasome Endopeptidase Complex