Analysis of Ahr Expression and Stability in a Recombinant Yeast Model System.

摘要

The aryl hydrocarbon receptor (Ahr) and the aryl hydrocarbon receptor nuclear translocator (Arnt) are well characterized bHLH-PAS transcription factors shown to regulate expression of xenobiotic metabolism genes. Extensive study has shown that upon treatment with certain aromatic hydrocarbons, mammalian cells rapidly activate the Ahr signaling pathway in order to stimulate gene expression and attempt to metabolize the xenobiotic compounds. It has been shown that after DNA-binding, the Ahr but not the Arnt protein, is quickly eliminated from the nuclear compartment thereby attenuating the dose of gene regulation administered by the Ahr·Arnt transcription factor complex. Previous studies have implicated involvement of the 26S proteasome complex in the degradation process, but the exact identity of the intermediary proteins and/or ligases remains to be defined. Identification and characterization of the protein(s) involved in degrading the receptor is essential for understanding the signaling pathway in its entirety including the mechanism for regulating the genetic response to Ahr ligands.;The model organism, Saccharomyces cerevisiae, was used in order to characterize the Ahr signaling pathway and degradation mechanism in a more simplified cellular setting in which the major processes required for growth and development are conserved. First, the AHR and ARNT cDNAs were stably inserted into the yeast genome such that protein expression was inducible. A time course of induction demonstrated detectable levels of Ahr and Arnt proteins via western blotting while protein function was confirmed by detection of ligand-dependent reporter activity in an expressor strain carrying the pLXRE5-Z beta-galactosidase reporter plasmid. Additionally, a rapid reduction in protein levels was observed upon turning off the inducible GAL1 promoter located upstream of both AHR and ARNT cDNAs.;Studies in mammalian cell culture have demonstrated that disrupting receptor chaperoning results in rapid Ahr protein turnover, as demonstrated by treatment with Hsp90 inhibitors. In order to determine if reduced Ahr protein expression in the yeast system was attributed to improper chaperoning of the exogenous protein; human heat shock proteins were constitutively expressed from yeast expression vectors in the Ahr and Arnt expressing strains, but did not confer any effect on Ahr stability when protein levels were evaluated by western blotting. Additionally, a strain of yeast was constructed such that the gene encoding the cell-wall protein, ERG6, was deleted from the yeast genome to allow for permeation of proteasome inhibitors. Treatment of this strain with proteasome inhibitors blocked the receptor degradation, therefore implicating the 26S proteasome in Ahr degradation when expressed exogenously in yeast.