Comparative Biochemical and Structural Analysis of the Alzheimer's Disease Related Proteins

164 Pages·2016·24.37 MB·English

by  Catherine Deatherage

Comparative Biochemical and Structural Analysis of the Alzheimer’s Disease Related
Proteins: Amyloid Precursor Protein and Notch1.

By

Catherine L. Deatherage

Dissertation
Submitted to the Faculty of the
Graduate School of Vanderbilt University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
in

Biochemistry
May, 2016
Nashville, Tennessee

Approved:

Charles R. Sanders, Ph.D.

Walter Chazin, Ph.D.

Bruce Carter, Ph.D.

Borden Lacy, Ph.D.

Lauren Jackson, Ph.D.
Copyright © 2016 by Catherine L. Deatherage
All Rights Reserved

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To my parents Rebecca and Jim Deatherage
To my sisters Mary and Elizabeth
Thank you for always being there.

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ACKNOWLEDGMENTS

I would like to take a moment to thank Dr. Charles Sanders for all of the time and
energy that he spent helping me in my journey to becoming an independent researcher.
He encouraged me to follow my research interests and pursue my own goals. I’d also
like to thank the members of my committee for believing that I knew what I was doing
and where I was going. Dr. Bruce Carter, Dr. Walter Chazin, Dr. Borden Lacy, and the
late Dr. Richard Armstrong; I couldn’t have done this without your support. I’d also like
to thank Dr. Lauren Jackson for stepping up and being willing to join my committee in
the final hour: you have always been helpful, no matter what questions I’ve asked. To
the members of the Sanders Lab past and present: Thank you all. This work was made
possible with the financial support of PO1 GM080513, R01GM106672 and funds
provided by Aileen M. Lange and Annie Mary Lyle Chair in Cardiovascular Research.
On a personal level I need to thank my early research mentors, Dr. Mary Berry
and Dr. Stanley May at the University of South Dakota and Dr. Andrew Byrd at the
National Cancer Institute. I wouldn’t be here without you. I would also like to thank my
entire family for being so supportive. Thank you for not asking, “When are you going to
finish?” I also need to thank my friends Reneé, Kate, and Joanna for not only listening
as I babbled about my work over the years but also remembering enough to ask about
how things are going.
To the lovely group of friends that I made here at Vanderbilt: Jim and Laura, Gus
and Rachel, Whitney and Ben, Kallie and Ryan, Shaun and Noelle, Beth, Allison, Arina,
and Drew. I enjoyed every conversation, outing, and girls’ night out.

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TABLE OF CONTENTS

Page
ACKNOWLEDGMENTS .................................................................................................. iv
LIST OF FIGURES ......................................................................................................... vii
LIST OF TABLES ............................................................................................................ ix
LIST OF ABBREVIATIONS .............................................................................................. x

Chapter

I. Introduction .................................................................................................................... 1

Statement of Purpose ............................................................................................................... 1
Introduction to Alzheimer’s Disease .......................................................................................... 1
Alzheimer’s Disease Etiology .................................................................................................... 3
APP and Cholesterol ............................................................................................................... 10
Gamma Secretase .................................................................................................................. 14
Preventing APP Processing .................................................................................................... 21
Gamma Secretase Activating Protein ................................................................................. 23
Notch ....................................................................................................................................... 25
Summary ................................................................................................................................. 32

II. Purification and Characterization of the Human γ-Secretase Activating Protein ........ 33

Introduction ............................................................................................................................. 33
Materials and Methods ............................................................................................................ 35
Results .................................................................................................................................... 40
Discussion ............................................................................................................................... 56
Acknowledgment ..................................................................................................................... 58

III. Notch Transmembrane Domain: Secondary Structure and Topology ....................... 59

Introduction ............................................................................................................................. 59
Materials and Methods ............................................................................................................ 60
Results and Discussion ........................................................................................................... 69

IV. The Notch 1 TM Segment is Structurally and Biochemically Distinct From C99. ..... 85

Introduction ............................................................................................................................. 85
Materials and Methods ............................................................................................................ 88
Results .................................................................................................................................... 94

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Discussion ............................................................................................................................. 107
Conclusion ............................................................................................................................ 114

V. Discussion and Future Directions ............................................................................ 115

γ-Secretase Activating Protein .............................................................................................. 115
GSAP Follow-up Research and Controversy .................................................................... 116
Notch 1 .................................................................................................................................. 120
Structure ........................................................................................................................... 120
Notch and Disease ............................................................................................................ 122
Notch Family Sequence Conservation .............................................................................. 124
Docking and Molecular Dynamics Analysis of γ-Secretase and Substrate Interactions ... 126
Significance and Impact ........................................................................................................ 130

REFERENCES ............................................................................................................. 132

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LIST OF FIGURES

Figure
Page

1.1- APP Processing. ....................................................................................................... 6
1.2- Amyloid Cascade Hypothesis. .................................................................................. 9
1.3- Map of how cholesterol can influence Alzheimer’s disease. ................................... 13
1.4- Gamma Secretase Components. ............................................................................ 15
1.5- Cryo-EM structure of γ-Secretase. .......................................................................... 20
1.6- Domains of Notch1. ................................................................................................. 28
1.7- Notch Processing. Notch is cleaved at site S1 to form a heterodimer. ................... 30
2.1- GSAP Purification. .................................................................................................. 46
2.2- Estimation of secondary structure from CD data. ................................................... 49
2.3- 600MHz spectrum of GSAP. ................................................................................... 51
2.4. GSAP interaction with imatinib. .............................................................................. 53
2.5. Imatinib titration by GSAP. ..................................................................................... 54
2.6. Titration of U-15N-C99 by GSAP. ............................................................................ 55
3.1. Notch 1 TM condition screening. ............................................................................ 71
3.2. Notch 1 TM construct information and NMR spectrum. .......................................... 73
3.3. Notch 1 TM chemical shift indexing. ........................................................................ 75
3.4. 15N NMR relaxation measurements. ........................................................................ 77
3.5. Notch 1 TM Membrane Topology. ........................................................................... 79
3.6. CLEANEX-PM water exchange experiment. ........................................................... 80

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3.7. Notch 1 TM/JM preliminary structural/topological model. ........................................ 84
4.1. Notch 1 TM cholesterol titration. .............................................................................. 96
4.2- Membrane Thickness. ........................................................................................... 100
4.3- Secondary structure changes. .............................................................................. 101
4.4- Top 1% of 2,000 structures. .................................................................................. 104
4.5- Average structure of Notch 1 TM during rMD simulation. ..................................... 105
4.6- X-plor vs. AMBER comparison. ............................................................................. 109
4.7- AMBER analysis. .................................................................................................. 111
4.8- Comparison of C99 and Notch 1 TM in DMPC bilayer. ......................................... 113
5.1- Clustal sequence alignment of the four human Notch family members
transmembrane segment. ..................................................................................... 123
5.2- Lipid raft induced structural perturbations. ............................................................ 129

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LIST OF TABLES

Table
Page

1.1- List of known γ-secretase substrates as of 2011* ................................................... 18
2.1- Summary of Attempts to refold Non-immobilized GSAP ......................................... 42
2.2- On-column refolding attempts ................................................................................. 44
4.1- Statistics of structural quality. The statistics for restraints, structural calculations,
and structural quality for 10 lowest energy structures of 2000 calculated using XPlor
and further refined in Amber. ................................................................................ 106
5.1- New TM segment targets ...................................................................................... 121

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LIST OF ABBREVIATIONS

HIV- Human Immunodeficiency Virus
AD- Alzheimer’s disease
Aβ- amyloid beta
APP- Amyloid precursor protein
AICD- Amyloid precursor protein intracellular domain
sAPPβ- soluble amyloid precursor protein beta fragment
sAPPα- soluble amyloid precursor protein alpha fragment
ApoE- ApolipoproteinE
LOAD- Late onset Alzheimer’s disease
ACAT- Acyl-CoA cholesterol acyltransferase
I-CLiP- intramembrane cleaving proteases
S2P- Site 2 Protease
APH-1- anterior pharynx defective-1
PEN2- presenilin enhancer-2
EM-electron microscopy
GSI- gamma-secretase inhibitor
GSM- gamma-secretase modulator
NSAID- non-steroidal anti-inflammatory drug
DAPT- N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester
NMR- nuclear magnetic resonance
SPR- surface plasmon resonance

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