Regulation of Endothelial Tetrahydrobiopterin in Normal and Pathophysiological States 公开

Abstract

ABSTRACT

Regulation of Endothelial Tetrahydrobiopterin in Normal and Pathophysiological States

By Li Li

Tetrahydrobiopterin (BH4) is an essential cofactor for NO synthases (NOS). In the absence of BH4, NOS produces superoxide rather than NO, a situation referred to as NOS uncoupling. The rate-limiting enzyme for de novo biosynthesis of BH4 is GTP Cyclohydrolase I (GTPCH-1). GTPCH-1 activity is regulated via interaction with the GTP cyclohydrolase feedback regulatory protein (GFRP). We have shown that laminar shear stress stimulates GTPCH-1 phosphorylation by Casein Kinase 2. The role of GFRP in modulating GTPCH-1 phosphorylation and activity in the endothelium and how phosphorylation affects GTPCH-1 activity remained undefined. My dissertation demonstrates that GFRP inhibits endothelial GTPCH-1 phosphorylation, BH4 levels, and NO production. Phosphorylation of GTPCH-1 increases its activity, reduces its binding to GFRP and confers resistance to inhibition by GFRP. Moreover, laminar, but not oscillatory shear stress, causes dissociation of GTPCH-1 and GFRP, which promotes GTPCH-1 phosphorylation and activation of the enzyme.

I found that disturbed flow produced by partial carotid ligation in mice reduces GTPCH-1 phosphorylation and BH4 levels in vivo. BH4 deficiency caused by disturbed flow leads to NOS uncoupling and induces accelerated atherosclerosis at these sites. BH4 deficiency and NOS uncoupling contribute to vascular superoxide production, endothelial dysfunction, vascular inflammation and abnormal cytokine milieu at sites of disturbed flow.

Since interaction of GFRP with GTPCH-1 inhibits its phosphorylation and enzyme activity, we propose that agents that disrupt GTPCH-1/GFRP binding would increase cellular BH4 levels. I thus developed a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor GTPCH-1/GFRP interaction. This assay has achieved a signal-to-background ratio of 12 and a Z' factor of 0.8. By adapting this assay for high throughput screening (HTS) and subsequent testing in confirmatory assays, I discovered compounds that inhibit GTPCH-1/GFRP interaction and increase cellular BH4 levels.

In summary, these data revealed a novel mechanism of GTPCH-1 regulation involving GTPCH-1 phosphorylation and its interaction with GFRP. This mechanism modulates in vivo BH4 levels and ultimately atherosclerosis. Our newly developed TR-FRET assay could be applied in future HTS to search for molecules that increase cellular BH4 levels.

Table of Contents

TABLE OF CONTENTS

Page
Chapter 1: Introduction 1
1.1 Overview 2
1.2 Shear stress 3
1.2.1 Laminar shear stress 3
1.2.2 Oscillatory shear stress 4
1.2.3 Experimental models of shear stress 5
1.3 Tetrahydrobiopterin (BH4) 9
1.3.1 Biosynthesis of BH4 9
1.3.1.1 De novo synthesis pathway 9
1.3.1.2 Salvage pathway 11
1.3.2 Metabolism of BH4 14
1.3.3 Biological functions of BH4 15
1.3.3.1 Cofactor functions 15
1.3.3.2 Cellular functions 18
1.4 Nitric Oxide Synthase (NOS) 18
1.4.1 Role of BH4 in NOS function 19
1.4.2 NOS uncoupling and production of superoxide 21
1.4.3 Role of NOS uncoupling in vascular diseases 22
1.5 GTP Cyclohydrolase I (GTPCH-1) and regulation of BH4 synthesis 25
1.5.1 GTPCH-1 structure 26
1.5.2 Regulation of GTPCH-1 gene expression 26

1.5.3 Post-translational modification of GTPCH-1 26
1.5.4 GTPCH-1 protein interactions 28
1.6 Summary 28
1.7 Objectives of this dissertation 29

Chapter 2: The interplay between GTPCH-1 phosphorylation and GFRP and
their roles in regulating endothelial cell BH4 levels and NO production 31
2.1 Introduction 32
2.2 Materials and methods 33
2.3 Results 41
2.4 Discussion 69

Chapter 3: Regulation of GTPCH-1 phosphorylation and BH4 levels by
disturbed flow in vivo and their roles in augmented atherosclerosis at sites of
disturbed flow 78
3.1 Introduction 79
3.2 Materials and methods 81
3.3 Results 86
3.4 Discussion 102

Chapter 4: Identification of inhibitors of GTPCH-1 and GFRP interaction and
development of a homogenous time-resolved fluorescence resonance energy
transfer assay to monitor GTPCH-1 and GFRP interaction 109
4.1 Introduction 110
4.2 Materials and methods 115

4.3 Results 121
4.4 Discussion 135

Chapter 5: Discussion and future directions 145
5.1 What are other mechanisms of GTPCH-1 regulation? 146
5.2 What is the role of BH4 deficiency and NOS uncoupling in atherosclerosis? 149
5.3 Is BH4 supplementation effective in long-term treatment of human diseases? 151
5.4 Development of new therapeutic agents that disrupt GTPCH-1/GFRP interaction and increase intracellular BH4 157
5.5 Future directions 159

Chapter 6: References 162

About this Dissertation

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• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Molecular & Systems Pharmacology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Health Sciences, Pharmacology
关键词	Assay Development High Throughput Screening Reactive Oxygen Species Shear Stress Cardiovascular diseases Nitric Oxide Synthase Tetrahydrobiopterin Endothelium
Committee Chair / Thesis Advisor	Harrison, David G, Emory University
Committee Members	Sutliff, Roy, Emory University Taylor, Kathy, Emory University Fu, Haian, Emory University

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