Evolutionary Conservation and Characterization of the Metazoan Amino Acid Response By Maja ...

102 Pages·2017·5.85 MB·English

Evolutionary Conservation and Characterization of
the Metazoan Amino Acid Response

By

Maja Lena Edenius

B.A., Boston University, Biology (2008)

Submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
and the
WOODS HOLE OCEANOGRAPHIC INSTITUTION
February 2018

© 2018 Maja Lena Edenius. All rights reserved.

The author hereby grants to MIT and WHOI permission to reproduce and to distribute publicly paper and
electronic copies of this thesis document in whole or in part in any medium now known or hereafter
created.

Signature of Author ………………………………………………………………………………………….
Joint Program in Oceanography/Applied Ocean Science and Engineering
Massachusetts Institute of Technology and Woods Hole Oceanographic Institution
February 5, 2018

Certified by ………………………………………………………………………………………………….
Dr. Malcolm Whitman
Professor of Developmental Biology
Harvard School of Dental Medicine
Thesis Supervisor

Certified by ………………………………………………………………………………………………….
Dr. Mark Hahn
Senior Scientist in Biology
Woods Hole Oceanographic Institution
Thesis Supervisor

Accepted by ………………………………………………………………………………………………….
Dr. Ann M. Tarrant
Chair, Joint Committee for Biological Oceanography
Woods Hole Oceanographic Institution
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Evolutionary Conservation and Characterization of
the Metazoan Amino Acid Response

by
Maja Lena Edenius

Submitted to the MIT/WHOI Joint Program in Oceanography/Applied Ocean Science and Engineering on
February 5, 2018 in partial fulfillment of the requirements for the degree of Doctor of Philosophy in
Marine Biology

ABSTRACT

Signaling pathways that respond to stress and sense nutrient availability are highly conserved throughout
eukaryotes. In mammalian cells, these pathways have evolved to regulate immune responses,
representing important therapeutic targets. Interestingly, components of these pathways can be found in
plants, yeast and nematodes, where they also participate in response to abiotic and biotic stress. The
Amino Acid Response (AAR) pathway, an ancient response to the cellular accumulation of uncharged
tRNA, is part of the larger Integrated Stress Response (ISR) in mammals. The ISR consists of multiple
branches, each one triggered by distinct stresses that produce phospho-eIF2α signal generation. Each ISR
initiating stress results in a unique cellular response due to activation of both the ISR and additional
parallel pathway(s) by the initiating stress, but, to date, no such alternate pathway has been identified for
the AAR pathway. Despite its integral role in stress adaptation, the ISR has not been studied in early
diverging animals. I have identified a highly conserved phosphorylation site in the protein eIF2α, the
signature ISR effector, which allowed me to use a mammalian antibody to identify and characterize the
ISR in the basal metazoan, Nematostella vectensis, revealing that the core components of the mammalian
ISR were present over 550 million years ago in the common ancestor of cnidarians and bilaterians.
Additionally, our lab has discovered a novel branch of the AAR pathway that regulates key tissue
protective signals. Using evolutionary conservation of this pathway in model organisms, I have identified
GCN1 as the branch point that links the signal generation components of the AAR pathway to
downstream therapeutic effects. I then used transcriptomic and protein interaction analyses to begin to
understand the scope of this pathway and identify key pathway regulators.

Thesis Supervisors:

Malcolm Whitman
Professor of Developmental Biology
Harvard School of Dental Medicine

Mark Hahn
Senior Scientist in Biology
Woods Hole Oceanographic Institution

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Thesis Advisors:

Professor Malcolm Whitman
Department of Developmental Biology
Harvard School of Dental Medicine

Dr. Mark Hahn
Department of Biology
Woods Hole Oceanographic Institution

Thesis Committee:

Professor Dennis Kim
Department of Biology
Massachusetts Institute of Technology

Professor Gerald Thomsen
Department of Biochemistry and Cell Biology
Stony Brook University

Thesis Defense Chair:

Professor Martin Polz
Department of Civil and Environmental Engineering
Massachusetts Institute of Technology

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ACKNOWLEDGEMENTS

This thesis wouldn’t have been possible without my incredible and supportive mentors, colleagues,
friends, and family. It would be impossible to thank all the people who have helped and guided me
through the last five and a half years and below is just an attempt to begin to thank some of these people,
who have together made this adventure worthwhile and possible.

Thank you to my advisor, Malcolm Whitman for showing me how to be a scientist. For teaching me to
see the important details in each experiment and to not just follow the accepted methods and ideas, but to
consider them for myself and explore all the possibilities. Thank you for always having your door open,
no matter how busy you were, and no matter how many questions I had. For always being interested and
available to discuss my experiments, whether it was how to best extract protein from sea anemones or
how to isolate ribosomes from human synoviocytes. Thank you for teaching me how to be a good
scientist, both as a biologist and a person, and for giving my sea anemones a home in a dental school.

To my mentor, Tracy Keller, for teaching me how to think, write, and talk about science. You make
science fun and exciting! Thank you for all the interesting discussions over the years, and above all for
always believing in me. For supporting my interests in marine biology and discussing and developing
ideas on anything ranging from sea stars to pharmaceuticals. You are a true scientist, able to ask and
develop ideas even in areas not your area of expertise. I hope a little bit has rubbed off on me.

To my co-advisor, Mark Hahn. Thank you for all the data discussions and for keeping me on track. No
matter how confusing my data was you always cut straight to the important facts and details, providing
important insight and advice. Thank you for the patient support and encouragement. To my thesis
committee, Dennis Kim and Gerald Thomsen, for your input and support, and for all the traveling and
phone calls!

And I couldn’t have done this without my “magical” lab. To Chang-Yeol Yeo for teaching me cell
biology. Thank you for all your help over the years, you were introduced to me as “the cell biology god”
and you have lived up to your name. To Yeon-Jin Kim whose work has been integral to the work of my
thesis. Thank you for your help, always with a smile, and a helpful hand. To Kristen Powers and Leila
Revollo for the many motivational sticky notes and unicorns. Couldn’t have done it without the sticky
notes! An additional thanks to Kristen, for helping me with ordering and caring for my cells when I was
at WHOI. To Minjin Bahn for the much needed coffee trips and Changqian Zhao for finally getting our
CRISPRs to work. And finally, to Mattia Bordoli for keeping the lab on schedule and answering my
many, many questions over the years.

To my classmates and WHOI colleagues, in particular, Amy Maas, Amalia Aruda Almada, and Harriet
Alexander, for your scientific and moral support.

To my friends, who will remain nameless here, thank you for the encouragement and distractions. I am
very lucky to have you all. Thank you for keeping me happy!

And finally, tack ja kiitos to my family, for teaching me that nature and the natural world are not separate
from us, but an integral part of our lives. And thank you aiti for the Finnish sisu.

--------------------------------------

Funding for this research was provided by the National Science Foundation Graduate Research
Fellowship Program, Allied Bristol Life Sciences (to Malcolm Whitman), the WHOI Academic Programs
Office, and the WHOI Ocean Venture Fund.
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TABLE OF CONTENTS

Abstract………………………………………………………………………………………………….... 3

Acknowledgements ………………………………………………………………………………………. 5

Chapter One: Introduction…………………...…………………………………………………………... 9
Figures……………………………………………………………………………………………15
References………………………………………………………………………………………..17

Chapter Two: Characterization of Integrated Stress Response (ISR) Pathway and its Activation in the
Basal Metazoan Lineage, Nematostella vectensis…………………………………………………………21
Tables and Figures……………..………………………………………………………………..35
References………………………………………………………………………………………..46

Chapter Three: The Evolutionarily Conserved Protein GCN1 is a Novel Branch Point in the Mammalian
Amino Acid Response (AAR) Pathway…………………………………………………………….……..53
Tables and Figures……………..………………………………………………………………..67
References………………………………………………………………………………………..77

Chapter Four: Characterization of a Novel Mammalian Nutritional Stress Response Pathway….……..81
Tables and Figures……………..………………………………………………………………..89
References……………………………………………………………………………………..…96

Chapter Five: Conclusions .……………..……………………………………………………………….97
References………………………………………………………………………………………101

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CHAPTER ONE

Introduction

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