Document Type

Dissertation

Date of Degree

2013

Degree Name

PhD (Doctor of Philosophy)

Degree In

Pharmacy

First Advisor

David L. Roman

Abstract

Regulators of G-protein signaling (RGS) proteins are a family of proteins that act as GTPase accelerating proteins (GAPs) through their interaction with GΑ subunits, including GΑo, GΑi, and GΑq but not GΑs. This increased rate of hydrolysis of GTP to GDP temporally regulates G-protein coupled receptor (GPCR) signaling. A member of this family, RGS4, has been implicated in several neurological disorders including Parkinson's Disease (PD). A hallmark of PD is the induction of oxidative stress within dopaminergic neurons. In this thesis, we evaluate the role of oxidative stress, including lipid peroxidation products with 4-hydroxy-2-nonenal (4HNE) as a model, in regulating RGS4 activity within neurons. Utilizing transfected RGS4, we evaluated whether RGS4 is readily modified by physiologically relevant concentrations of 4HNE by immuonoprecipitation of RGS4 from 4HNE treated cells. Further examination of recombinant RGS4 by mass spectrometry, revealed that RGS4 is readily modified at several cysteine residues by 4HNE, including C148. Modification at this residue has been shown to be a critical site for allosteric regulation of RGS4. This is confirmed through a malachite green based phosphate generation assay we developed to observe the GAP activity of RGS4 on its native binding partner GΑi. This malachite green based assay was then adapted for high throughput screening. The assay was successfully miniaturized to a 1536-well format. In a screen of 2300 compounds, 4 were identified as hits. The development of this simple and cheap assay can be adapted for usage with a variety of RGS proteins with little work to interrogate other pathways and identify novel RGS modulators.

Finally, expansive study of PD has linked oxidative stress to the pathology of both diseases. What has not been discerned is the potential relationship between oxidative stress and the induction of RGS4. In support of the hypothesis, we evaluated the potential relationship between oxidative stress and RGS4 expression. This was accomplished by evaluating two striatal neuron like cell lines, SH-SY5Y and HCN-1A. After treatment with hydrogen peroxide, both cell lines showed increased RGS4 in response to oxidative stress. This response is not however related to mRNA expression, indicating this change is most likely an adjustment of proteasomal regulation of RGS4. This phenomenon may explain the rapid onset of Parkinsonian motor symptoms in reserpine treated animal models of PD, as excess dopamine in the cytoplasm may be rapidly metabolized in reactive products.

Keywords

4HNE, GPCR, HTS, Oxidative Stress, RGS4

Pages

x, 125 pages

Bibliography

Includes bibliographical references (pages 114-125).

Comments

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Copyright

Copyright 2013 Carlos Aaron Monroy

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