The Role of Galactose-1-Phosphate Uridylyltransferase in Drosophila melanogaster Development and Homeostasis Öffentlichkeit

Abstract

Abstract
The Role of Galactose-1-Phosphate Uridylyltransferase in Drosophila melanogaster
Development and Homeostasis

By Rebekah Felice Kushner
Galactose metabolism occurs primarily through the Leloir pathway, which is highly
conserved from E. coli to humans and is catalyzed by three enzymes: galactokinase
( GALK, EC 2.7.1.6), galactose-1-phosphate uridylyltransferase ( GALT, EC 2.7.7.12),
and UDP-galactose 4' epimerase ( GALE, EC 5.1.3.2). In humans, impairment in any
one of the Leloir enzymes results in the metabolic disorder, galactosemia. The most
common clinically severe form of galactosemia is classic galactosemia, a potentially
lethal disorder resulting from profound GALT impairment. The current standard of care
remains the lifelong dietary restriction of galactose, which prevents or reverses the acute
and potentially lethal symptoms of the disorder, but is insufficient to prevent the long-
term complications, which include cognitive, motor, speech, and female reproductive
dysfunction. Decades of research have been hindered by the lack of a genetic animal
model that recapitulates the human phenotype, and the underlying pathophysiology of
this disorder remains poorly understood. Here we report the creation and initial
characterization of a Drosophila melanogaster model of classic galactosemia that mimics
aspects of the human disorder. Like humans, GALT-deficient flies survive under
conditions of galactose restriction but die in a dose dependent manner when exposed to
galactose during development. These animals also exhibit neurological complications
despite dietary restriction of galactose. Both the acute and long-term phenotypes
observed can be rescued by the ubiquitous transgenic expression of wild-type human
GALT. Using this D. melanogaster model, we have begun to dissect the timing and
extent of the galactose sensitivity in GALT-null animals, as well as the role(s) of GALT
function in organismal development and homeostasis. The existence of an animal model
may identify new targets for novel and effective treatments, enabling a better quality of
life for patients.

Table of Contents

I. Chapter I: Introduction 1-35 A. Galactose Metabolism 3-6

B. Galactosemia 7-11

1. GALK deficiency galactosemia 7-8

2. GALT deficiency galactosemia 8-11 3. GALE deficiency galactosemia 11 C. GALK Protein 12-14 D. GALT Protein 14-16

E. Pathophysiology of classic galactosemia 16-27

1. Metabolites 17-22 2. Inhibition of enzymes 22-24 3. Glycosylation 24-27

F. Model Systems 27-34

1. Saccharomyces cerevisiae 28-30

2. Mammalian cell culture 30-31

3. Mus musculus 31-32

4. Drosophila melanogaster 32-34

G.Significance 34-35

H. Figures and Figure Legends 1. Figure 1.1. The Leloir pathway of 4 galactose metabolism

2. Figure 1.2. Alternative pathways of 6

galactose metabolism

II. Chapter II: A Drosophila melanogaster model 36-70

of classic galactosemia A. Introduction 37-40

B. Materials and Methods 41-49

B. Results 50-64

C. Discussion 64-70

D. Figures and Figure Legends

1. Figure 2.1. The Leloir pathway of 38 galactose metabolism 2. Figure 2.2. Creation of an imprecise 45 excision allele of dGALT 3. Figure 2.3. Loss of dGALT results in 54 galactose sensitivity in D. melanogaster 4. Figure 2.4. Timing of death in dGALT-deficient 58

D. melanogaster exposed to galactose

5. Figure 2.5. Window of galactose sensitivity 60 of dGALT^Δ1AP2 /dGALT^Δ1AP2 imprecise excision homozygotes 6. Figure 2.6. dGALT-null flies demonstrate 65-66

an impaired negative geotaxic

response despite dietary restriction

of galactose

7. Table 2.1. D. melanogaster stocks and alleles 42

used in this study

8. Table 2.2. Leloir enzyme activities in 51 D. melanogaster

9. Table 2.3. Accumulation of 63

Gal-1P in larvae and flies

exposed to galactose

III. Chapter III: The Creation of a Simple, 71-94

Quantitative Assay for the Identification

of Human Galactokinase Inhibitors A. Introduction 72-76

B. Materials and Methods 76-78

B. Results 78-93 C. Discussion 93-94 D. Figures and Figure Legends 1. Figure 3.1. The secondary deletion of GALK 74-75 prevents galactose sensitivity in GALT-null yeast 2. Figure 3.2. A simple, coupled enzyme 80

assay for hGALK activity

3. Figure 3.3. hGALK and galactose are 81-82 limiting in the hGALK assay 4. Figure 3.4. hGALK assay is linear 84 for at least 90 minutes 5. Figure 3.5. Stability of the reaction 86 components at room temperature 6. Figure 3.6. The impact of DMSO on 87 the hGALK assay 7. Figure 3.7. and Table 1. S:B, S:N, and Z' 89 parameters for the hGALK assay meet criteria for high-throughput screening 9. Figure 3.8. Inhibition of hGALK by 91 six predicted small molecule inhibitors

IV. Chapter IV: Concluding Remarks and Future Direction 95-108

References 109-129

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Genetics
Stichwort	Drosophila melanogaster Galactose metabolism Classic galactosemia
Committee Chair / Thesis Advisor	Fridovich-Keil, Judith, Emory University
Committee Members	Moberg, Kenneth H, Emory University Matsumura, Ichiro, Emory University Caspary, Tamara, Emory University Fu, Haian, Emory University

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