REGULATION OF THE ARYL HYDROCARBON RECEPTOR PROTEIN LEVELS AND SIGNAL TRANSDUCTION PATHWAYS
Open Access
- Author:
- Morales, Jose Luis
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 30, 2007
- Committee Members:
- Craig Richard Baumrucker, Committee Member
Gary H Perdew, Committee Chair/Co-Chair
Avery August, Committee Member
Richard John Frisque, Committee Member
Jeffrey Maurice Peters, Committee Member - Keywords:
- AhR
hsp90
CHIP
Ubiquitin
Immunity
NF-kB - Abstract:
- The aryl hydrocarbon receptor is a ligand-activated transcription factor that mediates most of the toxic effects of numerous halogenated and non-halogenated polycyclic aromatic hydrocarbons (e.g., chlorinated dibenzo-p-dioxins). In its ligandbound state, the AhR rapidly accumulates in the nucleus where it dissociates from the hsp90/XAP2 complex and heterodimerizes with ARNT primarily to upregulate genes encoding metabolic enzymes implicated in the carcinogenic activation or detoxification of endogenous and exogenous substances. Interestingly, the AhR protein levels are rapidly depleted after its activation by full agonists in an ubiquitin and proteasomedependent manner. Given the potential role of the AhR in normal vascular, liver, and immune system development and/or regulation, as well as in mediating the toxicity of numerous HPAH and PAHs, the study of AhR protein level regulation and activation in the cell may be essential to our understanding of the mechanisms of toxicity elicited by persistent AhR activators (e.g., TCDD). The first project in this thesis explored the question of whether the carboxyl-terminus of hsc70 interacting-protein (CHIP) was involved in the regulation of the ligand-mediated degradation of the AhR in an ubiquitin and proteasome-dependent process. CHIP associates with chaperones such as hsp90 and hsc70 and negatively regulates their ability to function as protein folding complexes, causing client proteins (e.g., estrogen receptor) to be degraded through the proteasome. Immunoprecipitates of the AhR revealed that CHIP could also associate with the AhR protein complex at cellular levels and the transient expression of CHIP in cell cultures resulted in AhR protein turnover. Through the use of in vitro reconstitution assays, iv sucrose gradient fractionation, and RNA silencing methods we established that the E3 ubiquitin ligase CHIP can mediate ubiquitination of the AhR and hsp90. However, CHIP did not seem to regulate the steady-state protein levels of the AhR nor the ligandmediated degradation of the AhR. Rather, it appears that CHIP is capable of directly mediating ubiquitination of the hsp90 and, perhaps through this mechanism, it may affect the ability of the hsp90 chaperone to protect the AhR from ubiquitination and degradation through the proteasome. A second question explored whether the benzoimidazole-derived anti-asthmatic drugs termed M50354 and M50367 mediated their AhR-dependent therapeutic roles as partial agonists for the AhR and in a non-dioxin responsive element (DRE)-driven process. Previous published work suggested that these substances could not mediate classical DRE-driven gene activation of CYP1A1 to the same degree as established high-affinity AhR ligands. Presumably, these two drugs were also capable of mediating AhR-dependent immunomodulatory functions not entirely elicited by other established AhR ligands, possibly through a non-genomic role of the AhR. However, in contrast to previous observations, we demonstrated that both substances are full but transient AhR agonists. It is currently unknown whether the suggested ability of these drugs to modulate the differentiation of naïve T helper cells in an AhR-dependent manner is through the direct modulation of an immune system associated factor(s) through physical interactions with the ligand-activated AhR and/or through an AhR-regulated DRE-driven gene product. Therefore, this work concludes that further structure-activity relationship analysis is necessary to determine possible role of the liganded AhR in immune system regulation.