Resurrection of Ancestral Effector Proteases.

216 Pages·2017·8.12 MB·English

ABSTRACT

GRINSHPON, ROBERT DANIEL. Evolutionary Biochemistry of the Caspase: Resurrection
of Ancestral Effector Proteases. (Under the direction of Dr. Robert Rose.)

Caspase genes are ancient cysteinyl aspartate-specific proteases that are known to facilitate a
programmed cell death (PCD) phenotype called ‘apoptosis’. Caspases organize organ tissue
and maintain cellular homeostasis by balancing proliferation with apoptosis. Dysregulation
of caspase signaling leads to many human health disorders. Cancer characteristically evades
apoptotic activity; conversely, excessive apoptotic activity is associated with neuro-
degenerative disorders. ‘Caspase activity’ involves the proteolytic cleavage of target
tetrapeptide substrates C-terminal to P1 (N’-P4-P3-P2-P1-C’) in the cognate substrate
binding grooves (S4-S3-S2-S1). Each caspase has evolved discrete cellular roles while
conserving high specificity for cleavage after aspartate residues at P1, so caspase substrate
specificity is categorized based on amino acid preference at P4: group I prefers a bulky
residue (W/H); group II prefers hydrophilic residues (D/E); and group III prefers aliphatic
residues (I/L/V). The effector caspase genes (-3, -6, and -7) were fixed into the Chordata
phylum sometime before the emergence of ray-finned fish and have persisted throughout
mammalian evolution. Caspase -3 and -7 evolved to prefer a hydrophilic P4 residue and
caspase-6 prefers a hydrophobic P4 residue. The goal of this project was to understand how
substrate specificity diverged between relatively closely related caspases, while maintaining
three structurally important features that determine caspase function; the overall
hemoglobinase fold, residues in the S1 pocket that determine aspartate specificity, and proper
orientation of the catalytic residues.
The work presented here is pioneering the evolutionary biochemistry of caspase genes. To
begin, a curated database of caspase sequences from public databanks was developed to
organize the available data, and a web tool that rapidly compiles large datasets is made
available at CaspBase.org. Then, functional divergence analysis, and ancestral state
reconstruction (ASR) were executed to determine which amino acids are involved in the
evolution of substrate specificity. The common effector caspase (Node166) and the common
ancestor of chordate caspase-6 (Node167) were resurrected, and their substrate specificity
was determined with substrate phage display and Michaelis-Menten kinetics. Results show
that Node166 promiscuously cleaves hydrophobic and aliphatic residues at P4, and caspase-6
shifted to Group III specificity early in its evolution. Functional divergence analysis flagged
13 of 250 positions between human caspase-3 and -6, and only seven were located near the
S4 pocket when mapped onto a PDB model. A highly conserved interaction network was
revealed that increases the hydrophobicity of the caspase-6 S4 pocket. Only three of those
seven residues changed between Node166 and Node167. Three mutations in Node166 have
been introduced to reflect the H-bond network that evolved early in Node167, and two
additional residues that form a salt bridge observed in WT caspase-6 loop-4. Two mutations
for the interaction network mutant were sufficient to increase catalytic efficiency of Node166
but did not significantly change specificity for VEID over DEVD; however, the K for VEID
M
substrate was reduced 24-fold and the catalytic efficiency increased 36-fold when all five
mutations were combined, while the efficiency for DEVD did not change significantly. The
research conducted here sheds light on how caspase substrate specificity evolved and will
support rational drug design efforts to regulate caspase activity
Evolutionary Biochemistry of the Caspase: Resurrection of Ancestral Effector Proteases

by
Robert Daniel Grinshpon

A dissertation submitted to the Graduate Faculty of
North Carolina State University
in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy

Biochemistry

Raleigh, North Carolina

2018

APPROVED BY:

Dr. Robert Rose Dr. Michael B. Goshe
Committee Chair

Dr. Flora Meilleur Dr. Paul Hamilton
DEDICATION

Dedicated to my grandparents; Leopold and Ela Weissberger, and Michail and Manya
Grinshpon. Ela, my only living grandparent for the last 20 years, passed away one week after
my defense on March 30th, 2018, the first night of Passover. A Hollywood movie of her life
story is currently being produced.

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BIOGRAPHY

Robert ‘Bob’ Grinshpon, son of Tamar and Grigory Grinshpon, is a first-generation Jewish
American scholar. Grandson of Holocaust survivors and religious pilgrims of the former
USSR, he cannot fully express his gratitude for the opportunity that was paved for him
through their hardships. Bob’s interest in biochemistry began with the insufficient depth of
general science education textbooks, however the rabbit hole went deeper than he could have
imagined. After graduating from East Carolina University with his degree in chemistry and
minor in biology, he rejected his offer to attend a pharmacy program due to the mundane
nature of the occupation. “Sometimes science is more art than science”. A serendipitous re-
acquaintance with his childhood friend’s older sister, Dr. Sarah MacKenzie, lead Bob to join
Dr. Clay Clark’s lab in the fall of 2011. Bob plans to continue his career as a scientist in the
industrial sector after he graduates, where he can offer his unique skillset to solving real-
world problems by engineering proteins with principles of evolutionary theory.

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ACKNOWEDGEMENTS

I would like to thank Dr. Clay Clark for his mentorship throughout the years, and Dr. Sarah
MacKenzie for recruiting me into his lab. I would also like to thank my committee members;
Dr. Bob Rose, Dr. Mike Goshe, Dr. Paul Hamilton, and Dr. Flora Meilleur for monitoring my
progress as a scientist and giving advice along the way. I could not have completed my
dissertation without Dr. Anya Williford for her help on the CaspBase, and Dr. Paul Swartz -
the crystallography guru. Thanks to my former peers and those present for the good company
and contributions to my dissertation work; Dr. Joe Maciag, Dr. Melvin Thomas, and future
doctors James McQuillan, Lauren Fuess, Suman Shrestha, and Liqi Yao. I’d also like to
thank my undergraduates; Colin Woolard and Jessica Tung for their assistance.

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TABLE OF CONTENTS

LIST OF TABLES .………………………………………………………………..…..……. ix
LIST OF FIGURES ………………………………………………………….………...……. x
CHAPTER 1: EVOLUTIONARY BIOCHEMISTRY OF THE CASPASE ..….....……….. 1
A. INTRODUCTION ………….……………………………………..…………….. 1
i. Big Data ………………… ….....…………………………..……………. 2
ii. Evolution of Evolutionary Theory ...……………………..……………… 3
iii. Molecular Phylogenetics ………………………………..……………….. 6
iv. Functional Divergence Analysis ……………………….………….…….. 8
B. CASPASES DISSECTED ……………………………………………………... 10
i. Review of Caspase Phylogeny and Sequence Analysis ..………………. 12
ii. The Effector Caspase-6 …………....………………………...…………. 14
C. REFERENCES ……………………………………………………………….... 17
D. TABLES AND FIGURES .…………………………………………………….. 22
CHAPTER 2: INTEGRATION OF EVOLUTIONARY THEORY INTO CANCER
BIOLOGY AND CASPASE SIGNALING ...……………………………………………... 29
A. ABSTRACT ..……………………………………………………………….….. 29
B. INTRODUCTION ………….....………………………………………………… 30
i. Caspases and Apoptosis .……………………………………………...... 31
C. EVOLUTIONARY BIOLOGY: WHY DO WE CARE ABOUT
CASPASES IN OTHER ORGANISMS? ..…………………………………….. 35
i. Phylogenetic Inference of Evolutionary Relationships ………………… 35
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ii. Molecular Evolution and Functional Genomics .………………………. 36
iii. Evolutionary Medicine …………………………………………………. 40
iv. Peto’s Paradox and Comparative Genomics …………………………… 44
D. CASPASE SIGNALING AND CANCER …..………………………………… 46
i. Origins of the Apoptotic Pathway ……………………………………… 46
ii. Diversity in the Caspase Family ……………………………………….. 48
iii. Caspases in Cancer …………………………………………………….. 49
iv. Caspase Polymorphisms in Cancer Prognosis …………………………. 52
E. THE CASPASE STORY IS INCOMPLETE .…………………………………. 54
F. REFERENCES ……………………………………………………………….... 60
CHAPTER 3: THE CASPBASE: A CURATED DATABASE FOR THE
ACQUISITION OF CASPASE SEQUENCES ..………………………….………………. 71
A. ABSTRACT ……………………………………………………………...……. 72
B. INTRODUCTION …………………………………………………………...... 73
i. Why are Manually Curated Databases Necessary? ................................. 74
ii. Caspase Defined and Criteria for Curation ….….………………….….. 75
iii. The Common Position (CP) System …………………………………… 77
iv. Demonstration of Utility: Ancestral State Reconstruction …………….. 78
C. METHODS …………………..……………………………………………..,.… 79
i. Curation of the Database ……………………………………………….. 80
ii. The Common Position (CP) system …………………………………..... 81
iii. Ancestral State Reconstruction ………………………………………… 82
D. RESULTS ..…………………………………………………………………...... 83
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i. The CaspBase .………………………………………………………….. 83
ii. The Common Position Multiple Sequence Alignment (CP_MSA) ……. 85
iii. The Common Position (CP) Numbering System ………………………. 87
iv. Ancestral State Reconstruction of D. rerio Caspase-3a and -3b ………. 88
E. DISCUSSION .…………..……………………………………………….…..... 89
i. The CaspBase ………………………………………………….………. 89
ii. The CP System ………………………………………………………… 91
F. REFERENCES .………………...………………………………………..…….. 93
G. SUPPLEMENTARY MATERIALS …………………………………………. 105
CHAPTER 4: RECONSTRUCTION AND RESURRECTION OF ANCESTRAL
EFFECTOR CASPASES ..……………….……………………………….……...……….. 118
A. INTRODUCTION .………………………………………………….……….. 118
B. MATERIALS AND METHODS ………………………….…………………. 120
i. Ancestral State Reconstruction ………………………….…………… 120
ii. Design, Cloning, Expression and Purification ………….……………. 121
iii. Crystallization …………………………………………….…….……. 122
iv. Substrate Phage Display …………………………………….……..…. 123
v. Michaelis-Menten Kinetics ……………………………….…………... 124
C. RESULTS ..……………………………………………………………….…... 124
i. Resurrection and Characterization .…………………………………… 124
ii. Evolutionary Sequence Analysis of S1-S4 …………………………… 126
iii. Functional Divergence Analysis ……………………………………… 126
iv. The C-terminus of Helix-5 and Conserved Network in Caspase-6 …... 127
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D. DISCUSSION ..…………..……………………………………………….…... 129
E. REFERENCES ………………...……………………………………………... 133
F. TABLES AND FIGURES ……………………………………………………. 136
CHAPTER 5: ENGINEERING OF CASPASE-6 HELIX-3 RESEARCH …………….. 162
A. SUMMARY OF HELIX-3 OF CASPASE-6 RESEARCH .…………………. 162
B. METHODS …………………………………………………………………… 164
i. IC50 Assays ………………………………………………………… 164
ii. Cloning the Constitutive Two-Chain Construct ………………………165
C. RESULTS .………………………………………………………….………… 166
i. Evolutionary Analysis …………………………….………………….. 166
ii. Engineering Helix-3 Mutants …………………….…………………… 169
iii. Zinc IC 50 Assays ………………………………….…………………. 170
D. REFERENCES ...……………..………..…………….………….……………. 172
APPENDICES: ………………………………………………...….…………………..…. 179
A. ABBREVIATIONS ………………………………………………………. 180
B. CASPASE ANCESTOR PURIFICATION …….………………………… 180
C. ACTIVITY ASSAYS ...…………………………………………………... 184
D. DATA ANALYSIS WITH KALEIDAGRAPH ..………………………… 188
E. PHAGE DISPLAY …………………………………………………….…. 189
F. SEQUENCING …………………………………………………………….195
G. ASR ANALYSIS ……………………………………………………….… 197

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