Proteasomal Degradation of Human CYP1B1: Effect of the Asn453Ser Polymorphism on the Post-Translational Regulation of CYP1B1 Expression

Abstract

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

Pharmacogenetics and Regulation of Human CytochromeP450 1B1: Implications in Hormone-Mediated Tumor Metabolism and a Novel Target for Therapeutic Intervention

“…However, other functionally important CYP1B1 variants, including CYP1B1.3 and CYP1B1.4, were not evaluated, and other studies have shown that both CYP1B1.3 and CYP1B1.4 are important in determining catalytic efficiency and the protein processing of CYP1B1 in both COS-1 and other cellular expression systems. Recently, others have shown that the CYP1B1.4 variant increases the degradation efficiency of CYP1B1 by proteases and the proteasome in COS-1 cells, although the precise mechanism to explain these results remains to be elucidated (50,113). When expressed by bacteria in an ex vivo assay, the CYP1B1.3 variant alone was found have the lowest catalytically efficiency toward estrogen 4-hydroxylation reactions and the lowest ratio of 4-OHE 2 :2-OHE 2 formation.…”

“…RNA-PCR studies have shown that expression of CYP1B1 is equivalent in normal and tumor tissues, whereas immunohistochemical analysis and activity assays indicate that protein expression is increased in tumors (4,5,63,68). This could suggest that (a) a certain threshold of mRNA expression must be present before the protein can be expressed, (b) cell-specific post-transcriptional modifications must be present before protein expression is achieved, and (c) proteolytic degradation can modulate CYP1B1 protein levels (50,69). Thus, cell-specific CYP1B1 transcriptional activity must be assessed using immunologic and activity assays in conjunction with mRNA detection.…”

“…The degradation of CYP1B1 was recently shown to be mediated by the proteases, polyubiquitination, and proteasomal degradation but not by phosphorylation in COS-1 cells (50). Although it cannot be excluded that degradation of CYP1B1 in this cell line is different from the tumor degradation pathway, a polymorphism that increases the degradation efficiency of CYP1B1 (N453S) is also correlated with decreased cancer risk, suggesting that an increased rate of CYP1B1 degradation mediates a protective effect against tumorigenesis (51).…”

“…The levels of immunologically active CYP1B1.4 are a factor of 2 lower than other alleles because the 453S allele results in rapid proteolytic degradation of the CYP1B1.4 protein isoform. The increase in degradation rate can provide a rationale for the observed decreased activity of CYP1B1 in ethoxyresorufin de-ethylase assays and decreased genotoxicity from CYP1B1 metabolism (50).…”

“…The amino acid substitution does not cause an increase in ubiquitination rate, and the structural alterations responsible for this phenomenon are currently unknown (see Polymorphisms; ref. 50). …”