Table Of ContentSTUDIES OF PROTEIN PRENYLATION IN LIVING SYSTEMS: TOWARD THE
BETTER UNDERSTANDING OF DISEASES
A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE
SCHOOL OF THE UNIVERSITY OF MINNESOTA BY
JOSHUA DANIEL OCHOCKI
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
ADVISOR: DR. MARK D. DISTEFANO
AUGUST, 2012
© Joshua Daniel Ochocki 2012
Acknowledgements
There are several people in my life who have helped shape the decisions I
have made and supported me on my journey thus far. I would like to take a
moment to acknowledge those that have been instrumental in my life and career.
Several of my grade school teachers deserve many thanks for making
science fun and interesting, and feeding my curiosity with fascinating science
demonstrations on simple subjects from electricity and batteries (Mr. Garbe) to
baking soda volcanoes (Mr. Wiger). Those teachers are Mr. Garbe, Mr. Wiger,
Mrs. Volker, and Mr. Larson. Additionally, my high school teachers were also
responsible for molding my curiosity and encouraging my interest in science,
especially Mr. Walker, Mrs. Fier-Hansen, and Mrs. Knutson. Two of my
undergraduate professors deserve many thanks as well, Professors Lee Sanow
and Jay Brown. To Professor Sanow, for guiding me in choosing science as a
career and showing me the truly remarkable possibilities it held from an
intellectual and career standpoint. To Professor Brown, for being an excellent
research mentor, for teaching me the value of accuracy in science, and the
importance of knowing when exact precision is or isn’t needed.
To my advisor, Mark Distefano, whom I owe a great debt to. You have
always been supportive and encouraging, from allowing me to study at a
u n i v e r s i t y i n J a p a n f o r t h r e e w e e k s t o h e l p i n g m e g e t a n i n t e r n s h i p a t G e n e n t e c h
f o r t h r e e m o n t h s , I h a v e a l w a y s a p p r e c i a t e d y o u r s u p p o r t f o r s u c h e n d e a v o r s a n d
t h e f l e x i b i l i t y t o a l l o w m e t o d o t h e m . Y o u ’ v e a l s o t a u g h t m e a b o u t t h e p o l i t i c s o f
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science, and the realities a scientist faces in every aspect of their daily life. It
seems like every group meeting you always gave some piece of advice, and, in
time, I have found nearly all of them to be worthwhile and helpful. You have been
the best of mentors and I’m proud to have been able to start my career under
your guidance.
To my family: you may not have always understood what I was doing or
why I was doing it, but you have always been behind me and I have appreciated
it. To my friends: you constantly provided me with much needed distractions and
were always a sounding board for my struggles and I will never forget it. I’ve
been constantly motivated by the famous words of Jon Dozier (via Yoda), “do or
do not, there is no try.” You have all continuously reminded me that it’s important
to laugh often and not take life too seriously, or you’ll never get out alive. I have
truly been fortunate throughout my education, and life, to be surrounded by
incredibly bright, encouraging, and enthusiastic people. All that you have done for
me will never be forgotten.
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Abstract
Protein prenylation is a post-translational modification that is present in a
large number of proteins; it has been proposed to be responsible for membrane
association and protein-protein interactions which contributes to its role in signal
transduction pathways. Research has been aimed at inhibiting prenylation with
farnesyltransferase inhibitors (FTIs) based on the finding that the farnesylated
protein Ras is implicated in 30% of human cancers. Despite numerous studies on
the enzymology of prenylation in vitro, many questions remain about the process
of prenylation as it occurs in vivo. This dissertation describes our efforts to better
understand the enzymology of protein prenylation in living systems. Initially, we
prepared fluorescently labeled farnesylated peptides, based on the C-terminus of
the naturally prenylated protein CDC42, to serve as substrates of the prenylation
enzymes in living cells. These peptides are cell-permeable, can be imaged with
confocal microscopy, quantified in cells with flow cytometry, and be detected by
capillary electrophoresis after they have been processed by the cells own
machinery. In addition to these peptides, we have also developed unnatural
azide and alkyne containing isoprenoid moieties to serve as substrates for the
prenyltransferase enzymes. Using the ‘click’ reaction to a fluorophore we can
quantify the levels of prenylated proteins in living systems and we show that this
method is applicable to study the connection between protein prenylation and
neurodegenerative disorders such as Alzheimer’s disease. Taken together, our
results highlight the applicability of these peptides and unnatural isoprenoid
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analogs as a platform for further study to better understand the enzymology of
protein prenylation in living systems and to elucidate its role in certain diseases.
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Table of Contents
Acknowledgments………………………………………………………………………..i
Abstract…………………………………………………………………………………..iii
Table of Contents………………………………………………………………………..v
List of Tables………………………………………………………………………..……x
List of Figures……………………………………………………………………………xi
List of Abbreviations.............................................................................................xv
Chapter 1: Background..……………………………………………………….……....1
1.1 Post-translational Protein Modification………………...…………………1
1.2 Protein Prenylation…………………………………..............................…1
1.3 Biochemistry of Protein Prenylation……………………………………....6
1.4 Substrate Specificity of the Prenyltransferase Enzymes.....................10
1.5 Biological Significance of Protein Prenylation………………………….13
Chapter 2: Prenyltransferase Inhibitors for Disease Therapies…………………..15
2.1 Types of Prenyltransferase Inhibitors…………………………………...15
2.2 Evolution of FTIs…….………………………….....................................18
2.3 Inhibitors for GGTase I……………………………………......................24
2.4 RabGGTase Inhibitor Development……………………………………..26
2.5 Prenyltransferase Inhibitors as Cancer Therapeutics.........................32
2.6 FTIs for the Treatment of Progeria……………………………………....35
2.6.1 Hutchinson-Gilford Progeria Syndrome...............................36
2.6.2 Treating Progeria with FTIs…………………………………...38
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2.6.3 Clinical Trials with FTIs to Treat Progeria............................39
2.7 Other Therapies Utilizing Prenyltransferase Inhibitors........................40
2.7.1 FTIs for Parasitic Infections…………………………………..40
2.7.2 Prenyltransferase Inhibitors in Hepatitis Treatment.............41
2.7.3 Other Therapeutic Uses of FTIs……………………………...42
Chapter 3: Unnatural Isoprenoid Analogs to Study Protein Prenylation in
Living Cells……………………………………………………………………..44
3.1 Introduction………………………………………………………………...44
3.2 Research Objectives…………………………………….........................45
3.3 Results and Discussion………………… …….......................................45
3.3.1 Unnatural Isoprenoid Analogs………………………...............45
3.3.2 Metabolic Lableing in HeLa Cells and Response to
Inhibitors………………………..............................................47
3.3.3 Effects of the Addition of Lovastatin During Metabolic
Labeling………………………...............................................50
3.3.4 Identifying Proteins Labeled with Unnatural Isoprenoids.......52
3.4 Conclusions………………………................................................ ........56
3.5 Experimental……………………….......................................................56
3.5.1 General Materials……….......................................................56
3.5.2 Cell Growth and Lysis……….................................................58
3.5.3 Prenylation Changes Upon Inhibitor Exposure......................58
3.5.4 Two-dimensional Gel Electrophoresis...................................59
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3.5.5 Cell Growth and MS Analysis to Identify Prenylated
Proteins...............................................................................60
Chapter 4: Imaging and Quantification of the Prenylome…………………………63
4.1 Introduction…………………………………………………..................…63
4.2 Research Objectives…………………………..................…..................67
4.3 Results and Discussion……………………..................….....................67
4.3.1 Characterization of Alkyne Substrates……………….............68
4.3.2 Cellular Labeling and Visualization of Prenylated Proteins...72
4.3.3 Quantitative Analysis of Prenylated Protein Levels Using
Flow Cytometry in Different Cell Types...............................77
4.3.4 Effects of Prenylation Inhibitors on the Extent of Labeling....86
4.3.5 Measurement of Prenylation Levels in a Cellular Model
for Aging..............................................................................89
4.4 Conclusions.........................................................................................92
4.5 Experimental ......................................................................................92
4.5.1 General Materials..................................................................93
4.5.2 Western Blot Analysis...........................................................94
4.5.3 Imaging Prenylated Proteins.................................................95
4.5.4 Quantifying the Prenylome....................................................95
4.5.5 L6 Aging Model......................................................................97
4.5.6 Primary Neuron Preparation..................................................97
4.5.7 TAMRA-PEG-N3 Synthesis...................................................98
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Chapter 5: Fluorescently Labeled Prenylated Peptides for in vivo analysis…...100
5.1 Introduction........................................................................................100
5.2 Research Objectives.........................................................................101
5.3 Results and Discussion.....................................................................102
5.3.1 Peptide Synthesis................................................................102
5.3.2 Peptide Design and Uptake in HeLa Cells..........................105
5.3.3 Utility of the Peptides in Diverse Cell Types........................110
5.3.4 Evaluation of a Probe to Study in vivo Prenylation..............114
5.4 Conclusions.......................................................................................117
5.5 Experimental ....................................................................................117
5.5.1 General Materials................................................................118
5.5.2 Synthesis.............................................................................118
5.5.3 Cell Experiments.................................................................122
5.5.4 Octanol/water (LogP) Measurement....................................125
Chapter 6: Prenylated Peptides Lacking a Fluorophore for in vivo analysis…..126
6.1 Introduction........................................................................................126
6.2 Research Objectives.........................................................................127
6.3 Results and Discussion.....................................................................127
6.3.1 Peptide Synthesis................................................................127
6.3.2 Uptake studies in HeLa cells...............................................130
6.3.3 Localization of Peptides Inside Cells...................................132
6.3.4 Quantification of Peptide Uptake.........................................134
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