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Date of Award

2010

Document Type

Thesis - Pacific Access Restricted

Degree Name

Master of Science (M.S.)

Department

Pharmaceutical and Chemical Sciences

First Advisor

William K. Chan

First Committee Member

John C. Livesey

Second Committee Member

Jesika S. Faridi

Abstract

Aryl hydrocarbon receptor nuclear translocator (Arnt) belongs to the basic helix-loop-helix/Per-Arnt-Sim protein family. Our lab is interested in identifying the protein factors that modulate the Arnt-dependent pathway. We identified a novel Arnt-interacting peptide Ainp2 with an estimated size of 9 kDa. The endogenous Ainp2 protein is not found in MCF-7 cells. Another protein that is of interest to us is β-tubulin. Beta-tubulin is the monomeric subunit of microtubules. I have presented my thesis in two parts: the first part is focused on Ainp2 and the second part is on β-tubulin. The purpose of the first part of my thesis was to investigate whether Ainp2 affects the Arnt-dependent estrogen receptor (ER) pathway via an Arnt-related mechanism. Transient transfection studies in MCF-7 cells revealed that transfected Arnt increases E2-induced, ERE-driven luciferase activity in a dose-dependent manner; however, the enhancement effect of Arnt is significantly suppressed in the presence of Ainp2. The Ainp2 protein was successfully expressed in Sf9 cells and was affinity purified by the TALON metal resin method. Data from co-immunoprecipitation experiments showed that Ainp2 suppresses the interaction between Arnt and ERα in the presence or absence of E2. The aim of the second part of my thesis was to explore the effect of β-tubulin on the Arnt-dependent aryl hydrocarbon receptor (AhR) pathway. My data indicates that human β4-tubulin inhibits 3MC-driven, AhR-dependent luciferase expression. This suppressive effect of β4-tubulin is likely caused by a reduction in the nuclear Arnt content resulting from Arnt retention in the cytoplasm. The findings are certainly intriguing that the Arnt-mediated pathway can be modulated by either Ainp2 or β-tubulin. Limiting the nuclear Arnt function can be an approach to block Arnt-dependent signaling events which are crucial for cancer growth. These findings provide a means for rational drug development.

Pages

99

ISBN

9781124523569

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