Binding studies using Pichia pastoris expressed human aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator proteins.

The aryl hydrocarbon receptor (AHR) is a transcription factor which activates gene transcription by binding to its corresponding enhancer as the heterodimer, which is consisted of AHR and the aryl hydrocarbon receptor nuclear translocator (ARNT). Human AHR can be rather difficult to study, when compared among the AHR of other species, since it is relatively unstable and less sensitive to some ligands in vitro. Overexpression of human AHR has been limited to the baculovirus expression, which is costly and tedious due to the need of repetitive baculovirus production. Here we explored whether we could generate abundant amounts of human AHR and ARNT in a better overexpression system for functional study. We observed that human AHR and ARNT can be expressed in Pichia pastoris with yields that are comparable to the baculovirus system only if their cDNAs are optimized for Pichia expression. Fusion with a c-myc tag at their C-termini seems to increase the expression yield. These Pichia expressed proteins can effectively heterodimerize and form the ternary AHR/ARNT/enhancer complex in the presence of ß-naphthoflavone or kynurenine. Limited proteolysis using thermolysin can be used to study the heterodimerization of these human AHR and ARNT proteins.

Aryl hydrocarbon receptor (AHR): From selected human target genes and crosstalk with transcription factors to multiple AHR functions.

Accumulating evidence including studies of AHR-deficient mice and TCDD toxicity suggests multiple physiologic AHR functions. Challenges to identify responsible mechanisms are due to marked species differences and dependence upon cell type and cellular context. Transient AHR modulation is often necessary for physiologic functions whereas TCDD-mediated sustained receptor activation has been demonstrated to be responsible for toxic outcomes. To stimulate studies on responsible action mechanisms the commentary is focused on human AHR target genes and crosstalk with transcription factors. Discussed AHR functions include chemical and microbial defense, organ development, modulation of immunity and inflammation, reproduction, and NAD+-dependent energy metabolism. Obviously, much more work is needed to elucidate action mechanisms. In particular, studies of pathways leading to NAD+-dependent energy metabolism may shed light on the puzzling species differences of TCDD-mediated lethality and provide options for treatment of obesity and age-related degenerative diseases.