Document Type

Dissertation

Date of Degree

Spring 2015

Degree Name

PhD (Doctor of Philosophy)

Degree In

Anatomy and Cell Biology

First Advisor

Francis J. Miller

Abstract

Nox1 is of considerable importance because of its involvement in a wide variety of pathologies. Activation of Nox1 induces generation of reactive oxygen species (ROS) and cell migration, events critical for the pathogenesis of cardiovascular disease, amyotropic lateral sclerosis, gastrointestinal disease, immunological disorders, and multiple forms of cancer [1-8]. In order to best determine how to treat Nox1-mediated disease, we must gain a better understanding of the mechanisms that control Nox1 activation. Within the last decade, many studies have found that protein phosphorylation and protein trafficking are critical regulatory mechanisms that control the activation of multiple Nox proteins. Yet, to date, no studies have characterized Nox1 phosphorylation or trafficking. We hypothesized that the activity of Nox1 is controlled by its phosphorylation at specific residues and by its sub-cellular localization; and that modifying Nox1 phosphorylation or localization will alter Nox1-dependent signaling. To test this hypothesis, we utilized both in vivo and in vitro approaches. We found that phosphorylation of Nox1 is significantly increased under pathological conditions in three in vivo models: (1) in atherosclerotic vs. normal aorta from monkey, (2) in neointimal vascular smooth muscle cells (VSMCs) vs. medial VSMCs from rat following aortic balloon injury, and (3) in ligated vs. normal carotid from mouse. Studies using mass spectroscopy, pharmacological inhibition, siRNA, and in vitro phosphorylation identify PKC-βI as a kinase that mediates Nox1 phosphorylation and subsequent ROS production and VSMC migration. Site-directed mutagenesis of predicted Nox1 phospho-residues revealed that cells expressing mutant Nox1 T429A have a significant decrease in TNF-α-stimulated ROS production, VSMC migration and Nox1 NADPH oxidase complex assembly compared to cells expressing wild-type Nox1. Isothermal calorimetry (ITC) revealed that a peptide containing the Activation Domain of NoxA1 (LEPMDFLGKAKVV) binds to phosphorylated Nox1 peptide (KLK-phos-T(429)- QKIYF) but not non-phosphorylated Nox1 peptide. These findings indicate that phosphorylation of Nox1 residue T429 by PKC-βI promotes TNF-α-induced Nox1 NADPH oxidase complex assembly, ROS production, and VSMC migration. Nox1 localization and trafficking studies reveal that Nox1 endocytosis is necessary for TNF-α-induced Nox1 ROS production; and that mutation of a Nox1 VLV motif inhibits Nox1 endocytosis and ROS production. These studies have provided new evidence that phosphorylation and sub-cellular localization are involved in the regulation of Nox1 ROS production and cell migration and offer new insights as to how Nox1 activity can be targeted for the purpose of treating Nox1-mediated diseases.

Public Abstract

Nox1 is an enzyme present in blood vessels that is of considerable importance. Activation of Nox1 causes cellular signaling events critical for the development of cardiovascular disease. In order to best determine how to cure disease caused by Nox1 signaling, we must gain a better understanding of the mechanisms that control Nox1 activation. We hypothesized that Nox1 activation is controlled both by molecular modifications to the enzyme and by its location within the cell; and that preventing its molecular modification or altering its location in the cell will halt Nox1-dependent signaling involved in disease progression. We found that Nox1 molecular modification known as phosphorylation is higher in animal models of cardiovascular disease, and that preventing Nox1 phosphorylation decreases Nox1 cellular signaling and subsequent vascular cell migration, which are critical for cardiovascular disease progression. We also found that Nox1 must be localized to a part of the cell known as the endosome in order to become activated. We were able to identify a domain within the enzyme that is necessary for this localization and modify this domain to prevent Nox1 activation. These studies have provided new evidence that phosphorylation and sub-cellular localization are involved in the regulation of Nox1 signaling and cell migration and offer new insights as to how Nox1 activity can be targeted for the purpose of treating Nox1-mediated diseases.

Keywords

publicabstract, NADPH Oxidase, Phosphorylation, Protein Kinase, Vascular Smooth Muscle Cells

Pages

xi, 99 pages

Bibliography

Includes bibliographical references (pages 86-99).

Copyright

Copyright 2015 Jennifer Streeter

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