Identifying Dysregulated Protein Activities Using Activity-Based Proteomics

261 Pages·2016·20.72 MB·English

Identifying Dysregulated Protein
Activities Using Activity-Based
Proteomics
Author: Julianne Martell
Persistent link: http://hdl.handle.net/2345/bc-ir:106812
This work is posted on eScholarship@BC,
Boston College University Libraries.
Boston College Electronic Thesis or Dissertation, 2016
Copyright is held by the author, with all rights reserved, unless otherwise noted.
Identifying Dysregulated Protein Activities
Using Activity-Based Proteomics

Julianne Martell

A dissertation

submitted to the Faculty of

the department of Chemistry

in partial fulfillment

of the requirements for the degree of

Doctor of Philosophy

Boston College
Morrissey College of Arts and Sciences
Graduate School

April 2016

© copyright by JULIANNE MARTELL
2016
Identifying Dysregulated Protein Activities Using Activity-Based Proteomics

by

Julianne Martell

Thesis Advisor: Eranthie Weerapana

Abstract
Activity-based protein profiling (ABPP) is a chemical proteomic technique that
allows for selective labeling, visualization, and enrichment of the subset of active
enzymes in a complex proteome. Given the dominant role of posttranslational
modifications in regulating protein function in vivo, ABPP provides a direct readout of
activity that is not attained through traditional proteomic methods. The first application of
chemical proteomics in C. elegans was used to identify dysregulated serine hydrolase and
cysteine-mediated protein activities in the long-lived daf-2 mutant, revealing LBP-3,
K02D7.1, and C23H4.2 as novel regulators of lifespan and dauer formation. The tools of
ABPP were also utilized in studying protein interactions at the host-pathogen interface of
V. cholerae infection, discovering four pathogen-secreted proteases that alter the
biochemical composition of the host, decrease the activity of host serine hydrolases, and
inhibit bacterial binding by a host-secreted lectin. Lastly, ABPP was used to study the
targets of protein arginine deiminases (PADs) using a citrulline-specific activity-based
probe (ABP), highlighting its utility in detecting biologically relevant PAD substrates as
well as identifying mRNA processing factors as previously unknown targets of PAD.
Taken together, these studies demonstrate the ability of ABPP to discover novel protein
regulators of physiological and pathological processes.
Acknowledgements
The first and most important thank you goes to my advisor, Professor Eranthie
Weerapana. My transition into a lab took some detours but I am so thankful that I had the
opportunity to join yours. I appreciate your patience and willingness to answer the
countless questions I had in the beginning of my research. I’m grateful for the
experiences I had being a member of your lab, from traveling to New York to learn about
C. elegans to meeting and becoming good friends with some really great people through
collaborations. I genuinely enjoyed working for you! Thank you so much to my thesis
committee Jianmin Gao and Abhishek Chatterjee for reading my thesis. While
unfortunately unable to be on my thesis committee, I’d also like to say thanks to Mary
Roberts for letting me do research in your lab before joining Eranthie, as well as for all
your advice and enthusiastic interest in my worms.
Thank you to all the collaborators I worked with. A special thanks goes to Dr.
Stavroula Hatzios, over the several years of collaborating I always enjoyed our
conversations, you were an absolute pleasure to work with. I also want to thank Dr. Heidi
Tissenbaum and the members of her lab, especially Dr. YongHak Seo, for teaching me so
much and allowing me to work and be a part of your lab over the summer.
I need to thank all the Weerapana lab members for everything you’ve done to help
me, to teach me, and for making coming to lab each day so fun and enjoyable: Katie,
Tyler, Kyle, Lisa (Stanks), Shalise, Alex, Nick, Yani, Ranjan, Haley, Dan B, and
Masahiro. You are strange people and absolutely wonderful friends. The thing I will miss
most about grad school is getting to hang out with you all everyday. To the OG
Weerapana lab (Yani, Nick, and Alex): Your positivity, ambition, and camaraderie was
i
remarkable and so much appreciated while we all were pretty much making it up as we
went along. I am so thankful that you were the people I had the opportunity to experience
grad school with. Thank you to all the other friends I’ve made at BC who are on to bigger
and better things: Hilan, Dan S, Kevin, Carolyn, and Hekla. You are fabulously
incredible people and helped me create so many memorable experiences that I’ll
remember forever.
Thank you to all the Boston College Chemistry Department staff that I got the
chance to interact with throughout my years at BC: Steve, Lori, Ginny, Dale, Lynne P,
Lynne O, Howard, Ian, Jen, and Bill. Your constant enthusiasm and encouragement were
really appreciated.
Finally, I’d like to give special thanks to my family. Mom and Dad, you both are
exceptionally supportive and have been my entire life. While explaining my research may
have left you even more confused, I truly appreciate your interest and excitement in
everything that I do. I want you to know that a significant driving force behind
completing my degree was to make you proud. Also, thank you so much to my twin sister
Katie. I’m so grateful for the friendship we have and everything you have done for me. I
am constantly impressed by the amazing things you’re able to accomplish, hopefully
getting a PhD will help raise me toward your totally unreachable level of awesomeness.
Lastly, I was to acknowledge my cousin Marc (sup cuz). Our entire lives we have
been fascinated in how the universe works, and I loved all the conversations we had
trying to figure it out. Your eagerness in seeking knowledge was infectious and you
helped me understand how cool being a complete and utter geek could be. I wish you
were here to celebrate science and life with me. “Shine on you crazy diamond”.
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Table of Contents
Acknowledgements .............................................................................................................. i
Table of Contents ............................................................................................................... iii
List of Figures ................................................................................................................... vii
List of Tables ..................................................................................................................... xi
List of Abbreviations ....................................................................................................... xiii
Chapter 1. Activity-Based Protein Profiling ......................................................................1
Introduction .....................................................................................................................2
Electrophilic enzyme family-directed ABPs ...................................................................4
Directed ABPs to target functional amino acids .............................................................9
Non-electrophilic ABPs for enzyme family active sites ...............................................12
Non-directed ABP libraries ...........................................................................................13
Directed ABPs for modified amino acids ......................................................................14
CuAAC-mediated ABPP ...............................................................................................16
Applications of CuAAC in Mass Spectrometry-Based ABPP ......................................19
Conclusion .....................................................................................................................20
References .....................................................................................................................21
Chapter 2. An Isotopically Tagged Azobenzene-Based Cleavable Linker for Quantitative
ABPP .................................................................................................................................31
Introduction ...................................................................................................................32
Isotopically Tagged Azobenzene-Based Cleavable Linker ...........................................37
Evaluation of the efficiency of cleavage of the Azo-L and Azo-H tags .......................39
Evaluation of the quantitative accuracy of the isotopic Azo-tag platform ....................40
iii
Application of the isotopic Azo-tag platform to compare two biologically independent
samples ..........................................................................................................................42
Release of intact proteins from streptavidin beads ........................................................43
Conclusion .....................................................................................................................46
Experimental Procedures ...............................................................................................47
References .....................................................................................................................56
Chapter 3. Identifying Dysregulated Cysteine-Mediated Protein Activities During
Impaired Insulin/IGF-1 Signaling in C. elegans ................................................................61
Introduction ...................................................................................................................62
Insulin signaling and its role in lifespan regulation ......................................................64
Global profiling of DAF-16 transcriptional targets .......................................................68
Reactive-cysteine profiling reveals functional cysteines in C. elegans .........................71
Chemical-proteomic analysis identifies changes in cysteine reactivity between daf-2
and daf-16;daf-2 mutants ..............................................................................................76
RNAi-mediated knockdown of lbp-3 and K02D7.1 results in modulation of lifespan
and dauer formation .......................................................................................................81
C. elegans lipid binding protein-3 .................................................................................85
C. elegans protein K02D7.1 ..........................................................................................87
Conclusion .....................................................................................................................88
Experimental procedures ...............................................................................................90
References .....................................................................................................................98
Chapter 4. Fluorophosphonate ABPs to Investigate the Roles of Serine Hydrolases in
Impaired Insulin Signaling/Lifespan Regulation in C. elegans ......................................110
iv
Introduction .................................................................................................................111
Investigating serine hydrolase activity changes in impaired insulin/IGF-1 signaling in
C. elegans ....................................................................................................................112
Chemical-proteomic analysis reveals previously unidentified changes in SH activity
between daf-2 and daf-16;daf-2 mutants .....................................................................113
RNAi-mediated knockdown of ZK370.4 and C23H4.2 affects lifespan and dauer
formation .....................................................................................................................115
Conclusion ...................................................................................................................118
Experimental Procedures .............................................................................................118
References ...................................................................................................................121
Chapter 5. Chemoproteomic Profiling of Host and Pathogen Enzymes Active in Cholera
..........................................................................................................................................125
Introduction .................................................................................................................126
ABPP identifies serine hydrolases active in cholera ...................................................127
IvaP influences the activity of other serine hydrolases ...............................................130
V. cholerae proteases alter host protein abundance ....................................................131
Intelectin binds V. cholerae and other enteric bacteria ...............................................133
V. cholerae proteases decrease intelectin binding in vivo ...........................................134
Conclusion ...................................................................................................................135
Experimental Procedures .............................................................................................136
References ...................................................................................................................139
Chapter 6. Chemical Proteomic Platform To Identify Citrullinated Proteins ................142
Introduction .................................................................................................................143
v