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Temperature triggered protein assembly enables signaling switching and peptide drug delivery
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Temperature triggered protein assembly enables signaling switching and peptide drug delivery
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i
TEMPERATURE TRIGGERED PROTEIN ASSEMBLY ENABLES
SIGNALING SWITCHING AND PEPTIDE DRUG DELIVERY
by
Zhe Li
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PHARMACEUTICAL SCIENCES)
August 2018
ii
ACKNOWLEDGEMENTS
I would like to thank my mentor Dr. J. Andrew MacKay for the constant support and guidance
throughout my PhD studies. I would also like to thank my committee members Dr. Curtis
Okamoto, Dr. Sarah Hamm-Alvarez, Dr. David Hinton, Dr. Ching-Ling Lien for their generous
advice. I would like to appreciate Dr. Parameswaran G. Sreekumar, David Tyrpak, Mincheol
Park for contributing data to my thesis and publications. I would also like to thank all the
MacKay lab members for their help and support. I would like to acknowledge all the funding
resources for my research: the National Institutes of Health R01GM114839, RO1EY01545,
P30DK048522, P30 CA014089, the Translational Research Laboratory at USC School of
Pharmacy, the USC Ming Hsieh Institute, the USC Whittier Foundation, the Gavin S. Herbert
Endowed Chair of Pharmaceutical Sciences, the Arnold and Mabel Beckman Foundation, and
USC Clinical and Translational Science Institute SC CTSI (NIH/NCRR/NCATS) Grant #
UL1TR000130. Last but not least, I would like to thank my parents, my husband and my best
friend for their unconditional support, encouragement and love, without which I would not have
come this far.
iii
TABLE OF CONTENTS
LIST OF FIGURES vii
LIST OF TABLES ix
LIST OF ABBREVIATIONS x
INTRODUCTION 1
CHAPTER 1 Molecular switches for the control of protein assembly in
mammalian cells
4
1.1 Abstract 4
1.2 Introduction 4
1.3 Chemically responsive switches 7
1.4 Optically responsive switches 8
1.5 Magnetically responsive switches 10
1.6 Thermally responsive switches 11
1.7 Conclusion 12
CHAPTER 2 A new temperature-dependent strategy to modulate the epidermal
growth factor receptor
13
2.1 Abstract 13
2.2 Introduction 14
2.3 Materials and methods 16
2.3.1 Plasmid construction 16
2.3.2 Cell culture and transfection 17
2.3.3 Immunofluorescence and confocal microscopy 17
2.3.4 Colocalization analysis 18
iv
2.3.5 Flow cytometry 18
2.3.6 Immunoblot analysis 19
2.3.7 Live cell imaging 20
2.3.8 Statistics 20
2.4 Results 20
2.4.1 Generation of thermo-responsive EGFR-ELP fusion proteins 20
2.4.2 Temperature-triggered clustering of cell surface EGFR 24
2.4.3 Temperature-triggered internalization of cell surface EGFR 29
2.4.4 Temperature-triggered activation of EGFR signaling pathways 32
2.4.5 Dynamic and quantitative control of microdomain-mediated
signaling activation
36
2.4.6 Single-cell characterization of EGFR microdomain assembly 41
2.5 Discussion 45
2.6 Conclusion 49
CHAPTER 3 Tunable assembly of protein-microdomains in living vertebrate
embryos
51
3.1 Abstract 51
3.2 Introduction 51
3.3 Materials and methods 53
3.3.1 Zebrafish husbandry and care 53
3.3.2 Plasmid construction 53
3.3.3 ELP purification and physiochemical characterization 54
3.3.4 mRNA preparation and microinjection 54
3.3.5 Immunoblotting 55
v
3.3.6 Zebrafish imaging 55
3.3.7 Image processing 56
3.3.8 Statistical analysis 56
3.4 Results 56
3.4.1 ELP expression and microdomain assembly in vivo 56
3.4.2 ELPs can co-assemble different proteins in vivo 60
3.4.3 ELP self-assembly temperature can be tuned in vivo 62
3.4.4 ELPs can be tuned for short- or long-term microdomain
assembly in vivo
65
3.5 Conclusion 68
CHAPTER 4 Multimeric peptide-polymer enables protection of RPE cells against
oxidative stress
69
4.1 Abstract 69
4.2 Introduction 70
4.3 Materials and methods 73
4.3.1 ELP gene design and construction 73
4.3.2 ELP expression and purification 73
4.3.3 Characterization of ELP phase behavior 74
4.3.4 Characterization of ELP assembly 74
4.3.5 Transmission electron microscopy (TEM) imaging 75
4.3.6 Protection of RPE cells from oxidative stress 75
4.3.7 Statistics 76
4.4 Results 76
4.4.1 Purification of ELPs fused to humanin peptide 76
vi
4.4.2 Humanin peptide shifts the phase diagram differently for V96
and S96
78
4.4.3 Humanin peptide mediates the assembly of ELP-stabilized
nanoparticles
80
4.4.4 Exogenous HN-V96 protects RPE cells from oxidative-stress
induced apoptosis
83
4.5 Discussion and future directions 87
CHAPTER 5 Pharmacokinetics of intra-vitreal αB crystallin fragment fused to an
elastin-like polypeptide
90
5.1 Abstract 90
5.2 Introduction 90
5.3 Materials and methods 95
5.3.1 Materials and reagents 95
5.3.2 ELP expression and purification 95
5.3.3 Fluorescent labelling of ELPs 96
5.3.4 Color fundus photography 96
5.3.5 Quantitative analysis of fluorescence in color fundus images 97
5.3.6 Intra-vitreal pharmacokinetics 97
5.3.7 Pharmacokinetic analysis 98
5.3.8 Data analysis 98
5.4 Results 99
5.5 Discussion 104
5.6 Conclusion 107
CONCLUSION AND FUTURE DIRECTIONS
108
REFERENCES 109
vii
LIST OF FIGURES
Figure 1 Strategies for manipulating protein assembly and disassembly. 6
Figure 2 Design and function of EGFR-ELPs. 23
Figure 3 Temperature dependent clustering of surface EGFR-ELP is fast
and tunable.
25
Figure 4 Immunofluorescence of total EGFR vs. cell surface EGFR in
293T cells transiently transfected with EGFR fusions.
26
Figure 5 Live cell imaging of temperature-dependent EGFR assembly. 27
Figure 6 EGFR-ELP microdomain assembly is retained across cell lines
and polymer species.
28
Figure 7 EGFR-ELP assembly initiates receptor internalization to early
endosomes.
30
Figure 8 Subcellular localization of internalized EGFR-V96 in 293T
cells.
31
Figure 9 EGFR-V96 assembly specifically activates EGFR and the
ERK1/2 pathway.
33
Figure 10 Inhibition of ELP-mediated ERK1/2 activation. 34
Figure 11 Subcellular localization of pERK1/2 in 293T cells. 35
Figure 12 ELP-mediated ERK signaling is transient and reversible. 37
Figure 13 EGFR-V96 mediated ERK signaling can be temporally
modulated.
40
Figure 14 GFP-ELP microdomains reveal intracellular phase separation of
EGFR-V96 using live cell imaging.
42
Figure 15 EGFR-ELPs co-expressed with cytosolic GFP-ELPs co-
assemble above the ELP phase transition temperature.
43
Figure 16 Co-expression of GFP-V60 with EGFR-V96 does not affect the
ELP-mediated ERK1/2 activation.
44
Figure 17 Optimization of signaling kinetics using different heating 48
viii
systems.
Figure 18 ELP expression and microdomain assembly in a zebrafish
embryo.
59
Figure 19 ELPs can co-assemble different proteins in vivo. 61
Figure 20 ELP self-assembly temperature can be tuned by varying
molecular weight both in vitro and in vivo.
63
Figure 21 ELP self-assembly temperature can be tuned by varying
concentrations of ELPs both in vitro and in vivo.
64
Figure 22 ELPs can be tuned for short- or long-term microdomain
assembly.
66
Figure 23 ELPs do not affect embryonic survival and development of
zebrafish.
67
Figure 24 Construction and purification of Humanin (HN) ELP fusions. 77
Figure 25 Fusion of HN peptide changes the ELP transition temperature. 79
Figure 26 Fusion to HN peptide mediates the assembly of ELP
nanoparticles.
81
Figure 27 HN-ELP fusions assemble nanoparticles below transition
temperature.
82
Figure 28 Exogenous HN-V96 protects RPE cells from oxidative stress-
induced cell death in a dose dependent manner.
84
Figure 29 Exogenous HN-V96, not V96 protects RPE cells from oxidative
stress-induced cell death.
85
Figure 30 Exogenous HN-V96, not V96 inhibits oxidative stress-induced
caspase-3 activation.
86
Figure 31 Pharmacokinetics of mini cry and crySI in mice. 101
Figure 32 Intra-vitreal crySI is retained for long durations near the retina. 102
Figure 33 Morphological characterization of crySI in phosphate buffered
solution and the mouse vitreous.
106
ix
LIST OF TABLES
Table 1 ELP protein polymers fused with EGFR. 22
Table 2 Nomenclature, amino acid sequence and phase behavior of
expressed proteins.
58
Table 3 Recombinant protein polymers evaluated in the study. 72
Table 4 Nomenclature, sequence, molecular weight and purity of mini
cry and ELP fusion proteins.
94
Table 5 Ocular pharmacokinetics of mini cry and ELPs following intra-
vitreal administration to mice.
103
x
LIST OF ABBREVIATIONS
AMD Age-related macular degeneration
AUC Area under the curve
AUMC Area under the moment curve
CHO Chinese hamster ovary
CID Chemically induced dimerization
CLC Clathrin light chain
CME Clathrin-mediated endocytosis
Cry2 Cryptochrome 2
DR4 Death receptor 4
EEA1 Early endosome antigen 1
EGF Epidermal growth factor
EGFR Epidermal growth factor receptor
ELP Elastin-like polypeptide
ERK Extracellular signal-regulated kinase
FKBP FK506 Binding Protein
GA Geographic atrophy
GFP Green fluorescent protein
GPCRs G protein-coupled receptors
HN Humanin
hpf Hour post fertilization
ITC Inverse transition cycling
LARIAT Light-activated reversible inhibition by assembled trap
LOV Light-oxygen-voltage
mini cry 20-mer αB crystallin
MAPK Mitogen-activated protein kinase
MCC Mander’s colocalization coefficient
MDP Mitochondrial-derived peptide
MEC Molecular extinction coefficient
MNPs Magnetic nanoparticles
MRT Mean residence time
OD Optical density
PBS Phosphate buffered saline
PCL Polycaprolactone
xi
PFA Paraformaldehyde
PI3K Phosphatidylinositol 3-kinase
PLCg1 Phospholipase Cg1
R
h
Hydrodynamic radius
ROI Region of interest
RPE Retinal pigment epithelium
RTK Receptor tyrosine kinase
sHSPs Small heat shock proteins
tBH tert-Butyl hydroperoxide
TCR T cell receptor
T
t
Transition temperature
UVR8 UV-B receptors
1
INTRODUCTION
This research project primarily focuses on the development of a new temperature-
dependent strategy to modulate protein/peptide assembly using a thermo-responsive elastin-like
polypeptide (ELP), with applications in manipulating signal transduction and peptide-drug
delivery. ELPs are biocompatible, biodegradable polypeptides consisting of a pentameric repeat
of (Val-Pro-Gly-X
aa
-Gly)
n
, where X
aa
specifies the ELP hydrophobicity and n determines the
number of the repeats. ELPs are emerging as a platform to manipulate protein/peptide assembly
owing to their ability to phase separate above a transition temperature, T
t
, which can be easily
tuned through the selection of X
aa
and n. When expressed in mammalian cells, ELPs rapidly
assemble and disassemble microdomains in response to temperatures, suggesting its potential for
biological tools in synthetic biology. In previous work our group has shown that ELPs fused to
effector proteins such as clathrin light chain (CLC) can reversibly inhibit clathrin mediated
endocytosis (CME).
Extending the reach of this technology, Chapter 1 summarizes the current developments
in the design of tools for manipulating protein assembly in cells and Chapter 2 presents a new
platform using ELPs as regulatory component to specifically switch on/off signaling pathways.
As a proof of concept, I engineered chimeric receptors containing epidermal growth factor
receptor (EGFR) and ELPs and demonstrated their tunable modulation of intracellular signaling
pathways through receptor clustering. Prior to thermal stimulation, EGFR-ELPs freely
distributed on the cell surface and remain ‘OFF’. Once induced to phase separate, the EGFR-
ELPs bring their catalytic domains into proximity, thereby switching downstream signals ‘ON’.
2
Unlike natural ligands or synthetic agonists, the use of ELPs has high temporal resolution and
reversibility, which enables dynamic control of ERK1/2 and provides an approach to
quantitatively investigate signal transduction dynamics.
To further pursue their application in vivo, Chapter 3, for the first time, characterizes the
tunable assembly of ELPs in a vertebrate embryo. It supports the hypothesis that ELPs retains the
ability to regulate the self-assembly and disassembly of protein microdomains when expressed in
a multicellular organism. By tuning ELP length and sequence, protein microdomains can be
induced to assemble at different temperatures, in varying sizes, or for desired periods of time
within individual cells of zebrafish embryos, which may have applications in the study and
manipulation of in vivo biological functions.
In addition to applications in protein switching, ELP mediated protein/peptide assembly
has also been explored for the delivery of therapeutic peptides. Earlier work from our group has
established that fusion of a 20-mer aB crystallin (mini cry) peptide to a nanoparticle ELP
scaffold called SI retains the chaperone activity of the peptide and protects human retinal
pigmented epithelium (RPE) cells from oxidative stress induced cell death. Similarly, Chapter 4
evaluated the potential of ELPs to deliver a mitochondria-derived peptide (MDP) for protecting
the RPE cells against oxidative stress. Furthermore, it is hypothesized that ELP mediated peptide
assembly at body temperature may provide a local depot and extend its effect over longer periods.
Therefore, Chapter 5 compares the ocular pharmacokinetic profiles of mini cry and crySI.
While mini cry is cleared form the eye with a mean residence time of 0.4 days, crySI is retained
with a mean residence time of 3.0 days, suggesting that intra-ocular crySI may provide
3
prolonged protection against aged-related macular degeneration (AMD).
4
CHAPTER 1
Molecular switches for the control of protein assembly in mammalian cells
1.1 Abstract
Regulated protein assembly and disassembly underlie nearly all cellular processes. The
ability to rapidly and reversibly control these activities would greatly enhance our understanding
of the biology. As such, many approaches have been introduced to manipulate cellular functions
by targeting these activities (Ross, Mehta, and Zhang 2016). This chapter provides a detailed
overview of the available molecular switches that are classified by their physical cues: chemical,
magnetics, light, and temperature (Figure 1).
1.2 Introduction
The precise manipulation of cellular functions can serve as an effective means to
understand biological systems and to identify therapeutic targets. Regulated assembly and
disassembly of proteins is a vital step in many signaling processes and therefore plays an
important role in modulating cellular activities. However, studying the impact of protein
assembly/disassembly on cell behavior has been challenging because of the dynamic nature of
protein-protein interactions. For example, genetic perturbations such as overexpression or RNAi
knockdown typically require 24 to 48 hours to exhibit effects, making it impossible to follow the
transient changes within the cell. To date, a variety of molecular switches have been developed
in response to this challenge. Important criteria include: 1) specificity, the switch should be
precise and target only the action of interest; 2) speed, it should be rapid and respond quickly
upon stimulation; 3) reversibility, it should enable quick return to ground state in the absence of
5
a trigger; 4) safety, it should have minimal side effects; 5) adaptability, the general strategy
should be applicable to multiple cellular targets and suitable for animal models. Although these
switches have proven to be useful in controlling cellular functions, they meet the above criteria
with various strengths. In this chapter, I summarize existing strategies used for the design of
cellular switches and offer a perspective on future improvements.
6
Figure 1. Strategies for manipulating protein assembly and disassembly. a) Chemically
induced dimerization (CID) systems are composed of two protein components (FKBP) that
only bind in the presence of an exogenous chemical agent (FK1012). b) Light-mediated Cry2
oligomerization reversibly controls target protein clustering. c) Light-induced Cry2-CIB1
hetero-dimerization can be combined with Cry2 homo-oligomerization to trigger the
formation of large protein clusters for protein inhibition. d) Magnetic nanoparticles (MNPs)
coated with molecules of interest can be clustered in the presence of magnetic field. e)
Elastin-like polypeptides (ELPs) linked to effector proteins can reversibly regulate protein
assembly in response to temperature changes.
7
1.3 Chemically responsive switches
Biochemical reagents such as receptor agonists and small molecule inhibitors have been
broadly used to activate or inactivate protein functions (Stockwell 2004; Zhou 2005; Maynard-
Smith et al. 2007). They work rapidly but are prone to off-target effects and low reversibility.
For example, many kinase inhibitors bind to the ATP binding domain, which have a high degree
of similarity between diverse kinases, thus even ‘specific’ kinase inhibitors in actuality block a
wide range of cellular signaling processes with a range of efficiency.
The chemically induced dimerization (CID) system is one alternative for compensating
those drawbacks and is based on a concept in which the proximity between proteins of interest
can be increased through addition of a small, drug-like molecule (Figure 1a). The concept of
CID was firstly introduced in a landmark paper in 1993 by Schreiber, Crabtree and their co-
workers (Spencer et al. 1993). A bivalent derivative of immunosuppressant drug FK506, called
FK1012, was shown to reversibly bring its binding partner, FK506 Binding Protein (FKBP)
together to form a homodimer. By fusing the FKBP to the z chain of the T cell receptor (TCR),
they demonstrated that T cell signal transmission can be activated by the cell-permeable
crosslinker FK1012 in the absence of antigen binding.
Since then, this toolbox has been expanded rapidly by researchers. Ligand-induced
disassembly was demonstrated a few years later through the coincidental identification of an
FKBP mutant that naturally forms a stable dimer in the absence of any ligand (Rollins et al.
2000). Interestingly, adding a monomeric ligand of FKBP, rapamycin, completely disassociates
self-dimerization and thus serves as a ligand-induced off switch. Similar concepts were cleverly
8
exploited to block b-amyloid aggregation which is associated with Alzheimer’s disease
(Gestwicki, Crabtree, and Graef 2004).
In addition to small molecule induced dimerization, there are also other species which
can induce dimerization, such as dihydrofolate reductase (Carlson et al. 2003) and a
methotrexate based ligand (Kopytek et al. 2000). The expansion of the number of dimerization
systems is important to improve the biocompatibility and reduce off-target effects such as
binding to naturally occurring proteins. For example, both FK1012 and rapamycin bind
endogenous FKBP12, which then binds the FRB domain on the mechanistic target of rapamycin
(mTOR) protein. mTOR signaling is essential to many cellular processes, thus the CID system is
accompanied by substantial off-target effects. Furthermore, due to their reliance on the
diffusible biochemical reagents to mediate assembly or disassembly, this technique still has
intrinsic difficulties, such as limited temporal and spatial resolution.
1.4 Optically responsive switches
Optogenetics is a powerful technique that allows the control of protein assembly with
light (Deisseroth 2011). Compared to the chemically induced dimerization, this method offers
more rapid responses, better reversibility and higher spatiotemporal resolution. Deisseroth et al.
first named “optogenetics” as an approach combining the use of light and genetically encoded
photoreceptors for precise control of neuronal activity (Boyden et al. 2005; Deisseroth et al.
2006). This term was later extended to other light-sensitive proteins to control the behavior of
living cells and organisms. In particular, light-mediated changes to the oligomeric states of
9
protein complexes have been applied to various cellular targets to either activate or inactivate
protein functions.
Arabidopsis thaliana Cryptochrome 2 (Cry2) is a class of photoreceptor that oligomerizes
in response to blue light (Más et al. 2000). Genetic engineering of Cry2 to target proteins has
been demonstrated to be sufficient to stimulate a variety of cellular functions (Figure 1b). For
instance, Bugaj et al. showed that when fused to Cry2, LRP6 underwent light-dependent
assembly and activated the b-catenin pathway. The modularity of this approach was further
confirmed by clustering the Rho GTPase Rac1, resulting in enhanced enzymatic activity and
effector activation (Bugaj et al. 2013; Bugaj et al. 2015). Alternatively, light-triggered protein
assembly has also been used to sequester target proteins into higher-order complexes and thereby
inactivate their function (Figure 1c). Taslimi et al. reported that fusion of a Cry2 mutant, which
has enhanced clustering capability, to clathrin light chain (CLC) allowed conditional disruption
of clathrin-mediated endocytosis (CME) with light (Taslimi et al. 2014). In the meanwhile, the
Heo group also demonstrated the use of Cry2 to inhibit protein function using a system termed
light-activated reversible inhibition by assembled trap (LARIAT). Instead of direct fusing to
Cry2, they combined the Cry2 homo-oligomerization with Cry2-CIB1 hetero-dimerization to
initiate protein assembly (Lee et al. 2014).
In addition to the Cry2 based systems, other light-sensitive proteins including
phytochromes, light-oxygen-voltage (LOV) proteins, UV-B receptors (UVR8) and fluorescent
protein-derived interaction domains have been generated to enable photo-control of protein
10
oligomerization. Therefore, researchers can now choose from a variety of systems offering
different advantages with respect to the experimental design.
1.5 Magnetically responsive switches
Another alternative way to precisely control protein assembly in space is by exploiting
magnetic manipulation of proteins bound to nanoparticles. These functionalized magnetic
nanoparticles (MNPs) are attracted by magnetic forces and could be loaded either extracellularly
or intracellularly for different applications (Figure 1d).
Cell surface receptors targeted by magnetic nanoparticles can be clustered upon magnetic
field actuation. Mannix et al. demonstrated that MNPs decorated with monovalent antigen could
induce the assembly of FceRI and activate signal transduction by a magnetic field (Mannix et al.
2008). Similar strategies were applied to trigger epidermal growth factor receptor (EGFR)
signaling (Bharde et al. 2013) and death receptor 4 (DR4) induced apoptosis (Cho et al. 2012).
Remarkably, magnetic assembly of MNPs functionalized with T cell activating proteins was
shown to enhance T cell activation and anti-tumor activity (Perica et al. 2014).
In addition to the cell surface receptors, MNPs can be used to manipulate intracellular
functions. While one study relies on the magnetic nanoparticles being endocytosed into the
endosomes to study subcellular organelle localization (Steketee et al. 2011), most the studies
have utilized magnetic nanoparticles introduced through microinjection into the cells. Examples
are cytoskeleton remodeling using TIAM-MNPs (Etoc et al. 2013) and microtubule assembly by
Ran-NPs (Hoffmann et al. 2013). Recent developments have focused on magnetogenetic tools
11
that are completely genetically encodable, which may further expand the application of this
technology.
1.6 Thermally responsive switches
Temperature-dependent protein assembly or disassembly is an emerging strategy to
reversibly manipulate cellular functions. The MacKay group has discovered that thermally
responsive elastin-like polypeptides (ELPs) assemble functional proteins when expressed in live
cells (Figure 1e) (Pastuszka et al. 2012).
ELPs are biocompatible, biodegradable polypeptides consisting of a pentameric repeat of
(Val-Pro-Gly-X
aa
-Gly)
n
, where X
aa
can be substituted with any amino acid and n determines the
number of the repeats (Shah et al. 2012a; Despanie et al. 2016). A small increase in temperature
stimulates ELPs to phase separate, and this process can be fully reversed by decreasing the
temperature. The temperature of assembly, termed the transition temperature (T
t
), is specified by
the hydrophobicity of the guest residue X and the number of repeats n. ELPs have been explored
for drug delivery (Chilkoti, Dreher, and Meyer 2002; Shi et al. 2013) and tissue engineering
(Nettles, Chilkoti, and Setton 2010); however, they have untapped potential as biological tools
with applications in synthetic biology.
When fused to an effector protein such as clathrin light chain (CLC), this temperature
sensitive behavior is retained, producing a thermo-responsive fusion rapidly cycles between
assembly and disassembly in response to temperature and enables the reversible control of
clathrin-mediated endocytosis (CME) (Pastuszka et al. 2014). In this case, ELP fusion is soluble
12
prior to thermal stimulation and clathrin-mediated endocytosis remains “ON”. Once heated
above T
t
, the assembly of ELP sequesters CLC away from its site of action and thereby switches
clathrin-mediated endocytosis “OFF”. Similarly, ELPs have also been exploited to reversibly
assemble EGFR, which enables dynamic control over downstream ERK1/2 (Chapter 2).
Furthermore, a recent in vivo characterization of this system suggests that the temperature-
mediated ELP assembly is retained in zebrafish, which may have applications in the study and
manipulation of in vivo biological functions (Chapter 3).
1.7 Conclusion
Technologies enabling the manipulation of protein assembly and disassembly within
complex cells are powerful tools for biological research. Despite their tremendous progress in the
past few decades, the toolkit needs to be further expanded, not only applying the current systems
to more cellular targets, but also optimizing the key parameters in each system such as kinetics
and sensitivity.
13
CHAPTER 2
A new temperature-dependent strategy to modulate the epidermal growth
factor receptor
2.1 Abstract
The dynamic manipulation of kinases remains a major obstacle to unraveling cell-
signaling networks responsible for the activation of biological systems. For example, epidermal
growth factor (EGF) stimulates epidermal growth factor receptor (EGFR), however, also recruits
other kinases involved with various signaling pathways. To better understand EGFR alone and
its relevance to therapies, better tools are required to dissect the activity of specific kinases.
Toward this goal I report a new strategy to selectively activate receptor tyrosine kinases fused to
elastin-like polypeptides (ELPs), which can be expressed as tools inside mammalian cells. ELPs
are high molecular weight polypeptides that phase separate abruptly in response to changes in
temperature, pressure, concentration, and other environmental factors. When an EGFR-ELP
fusion is heated, it clusters, initiates receptor internalization, phosphorylates, and initiates
downstream kinase signaling. Unlike other strategies to block EGFR (small molecule inhibitors,
RNA interference, or transcriptional regulators) EGFR-ELP clustering can be switched on/off
within minutes. This strategy was found to be both reversible and able to dynamically control the
downstream phosphorylation/activation of the ERK1/2 kinase pathway. For the first time, this
strategy enables the rational engineering of specific temperature-sensitive receptors that may
have broad applications in the study and manipulation of biological processes.
14
2.2 Introduction
Decisions on cell fate and function mainly rely on the coordinated intracellular signaling
pathways that are activated by plasma membrane receptors, such as G protein-coupled receptors
(GPCRs) and receptor tyrosine kinases (RTKs) (Gutkind 1998; Schlessinger 2000). In particular,
the dynamic behaviors of signaling networks have been regarded as the determinant for their
specificity, resulting in different gene expression patterns and diverse physiological responses
(Kholodenko 2006). A range of tools has been developed to manipulate cell signals for
understanding biological systems. Conventional approaches using chemical-based strategies or
genetic perturbation allow for activation and inhibition of selected receptors (Spencer et al. 1993;
Muthuswamy, Gilman, and Brugge 1999; Dikic, Schlessinger, and Lax 1994); however, they
also suffer from poor temporal resolution and low reversibility. This makes it challenging to
examine the dynamic properties of signaling networks in a highly specific manner. Existing
methods to address these problems use various stimuli, such as light (Chang et al. 2014; Grusch
et al. 2014; Kim et al. 2014), magnetics (Cho et al. 2012; Etoc et al. 2013; Seo et al. 2016) and
electrics (Wolf-Goldberg et al. 2013). For instance, chimeric receptors fused with light-sensing
domains can be activated in response to light in the absence of endogenous ligands. Although
these systems have been proven to control various biological events, they have drawbacks
including the introduction of exogenous materials (ligands, nanostructures, crosslinkers, etc.)
and/or the need for a specialized stimulation system (optical, electromagnetic, etc.). Therefore,
simple and convenient solutions remain highly desirable.
Recently our research group discovered that thermally responsive protein-polymers
assemble functional proteins when expressed in live cells (Pastuszka et al. 2012). A small
15
increase in temperature stimulates elastin-like polypeptides (ELPs) to phase separate, and this
process can be fully reversed by decreasing the temperature. ELPs have been explored for drug
delivery and tissue engineering (Chilkoti, Dreher, and Meyer 2002; Nettles, Chilkoti, and Setton
2010; Despanie et al. 2016); however, they have untapped potential as biological tools with
applications in synthetic biology. When expressed within cells and fused to key effector or
fluorescent proteins, ELPs assemble cytosolic microdomains in a temperature dependent manner
that can be tuned to target intracellular processes such as clathrin-mediated endocytosis
(Pastuszka et al. 2012; Pastuszka et al. 2014; Shi et al. 2014). Here, I extend this technology by
developing a protein switch for manipulating intracellular signals utilizing ELPs as a regulatory
component. In contrast to methods discussed above, the ELP-based activation is rapidly
reversible and enables withdrawal of stimuli without further disturbing the cellular environment.
To demonstrate this strategy, I utilize epidermal growth factor receptor (EGFR) and its
downstream signaling pathways as a model system. EGFR is an essential receptor tyrosine
kinase (RTK) that regulates cell proliferation, differentiation, metabolism and is implicated in
numerous human cancers (Yarden and Sliwkowski 2001; Normanno et al. 2006; Seshacharyulu
et al. 2012). Upon binding with extracellular ligands (such as EGF), EGFRs homo and hetero
dimerize with other RTKs. This leads to trans-phosphorylation of the cytosolic arms of the RTKs.
The phosphorylated tyrosine residues then recruit other downstream signaling components that
activate diverse intracellular pathways, including mitogen-activated protein kinase
(MAPK)/extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K)/Akt
and phospholipase Cg1 (PLCg1)/Ca2+ pathways. Since receptor clustering plays an important
role in both signal transduction and receptor internalization, I hypothesized that ELP-mediated
16
assembly could functionally substitute for ligands. When induced to phase separate, expressed
EGFR-ELPs might reasonably be expected to cluster and activate. To validate this strategy, this
manuscript presents a comprehensive library of fusion proteins between EGFR and ELP and
demonstrates their tunable modulation of intracellular signaling pathways through receptor
clustering (Figure 2). Prior to thermal stimulation, EGFR-ELPs remain freely distributed in the
cytoplasmic membrane at the cell surface, remaining ‘OFF’ due to their lack of self-association.
Once induced to phase separate, the EGFR-ELPs bring their catalytic domains into proximity,
thereby switching downstream signaling ‘ON’. The reversibility of ELP-mediated phase
separation enables precisely-controlled repetitive activation, providing a simple means for
manipulating signaling dynamics.
2.3 Materials and methods
2.3.1 Plasmid construct
ELP expression vectors were synthesized using recursive directional ligation in pET25b
(+) plasmid (MilliporeSigma, Burlington, MA), as previously reported.(McDaniel et al. 2010)
The pCDNA6A-EGFR WT [Mien-Chie Hung, The University of Texas] was obtained from
Addgene (#42665, Cambridge, MA) and modified by inserting DNA oligonucleotides with
EcoRV/NotI digestion sites (Top strand: 5’-ccggTGCGATATCatgatgGCGGCCGC-3’; Bottom
strand: 5’-GCGGCCGCcatcatGATATCGCA-3’) into AgeI/PmeI. Then DNA sequences
encoding ELPs were cloned into the downstream of EGFR-myc after digesting the ELP plasmid
with MslI/NotI and the modified EGFR plasmid with EcoRV/NotI. The final opening frames of
the fusion proteins were verified by DNA sequencing (Retrogen, San Diego, CA).
17
2.3.2 Cell culture and transfection
293T cells (#CRL-3216, ATCC, Manassas, VA) were maintained in DMEM (Thermo
Fisher Scientific, Waltham, MA) supplemented with 10% FBS (#35-011-CV, Corning, NY) in a
humidified incubator with 5% CO
2
at 37 °C. CHO K1 cells were grown in DMEM/F12 1:1 (GE
Healthcare Life Sciences, Logan, UT) containing 10% FBS at 37 °C with 5% CO
2
. For the
transient expression, cells were transfected with Lipofectamine 3000 (Thermo Fisher Scientific,
Waltham, MA) according to manufacturer’s instructions. After transfection, cells were cultured
at 32 °C for 36~48 hours prior to assay.
2.3.3 Immunofluorescence and confocal microscopy
To assess the assembly of cell surface EGFR, 293T cells were plated on glass coverslips
in 12-well plates and transfected with EGFR-ELPs. 24 hours after transfection, medium was
replaced with serum free DMEM. After additional 16 hours, cells were cooled at 4 °C for 30 min
with an Alexa Fluor 488-conjugated EGFR antibody (#sc-120 AF488, Santa Cruz Biotechnology,
Dallas, Texas) that specifically stains EGFR at the plasma membrane. After washing, cells were
incubated at specified temperatures (22 °C, 27 °C, 32 °C, 37° C) for 30 min with serum free
DMEM prior to fixation. Coverslips were mounted on microscope slides in fluoromount
(Diagnostic Biosystems, Pleasanton, CA) and images were captured on a LSM510 confocal
microscope (Carl Zeiss Microscopy, Thornwood, NY) with a Plan-Apochromat 63x oil objective.
For detection of phosphorylated EGFR or EEA-1, surface EGFR was firstly labeled at 4 °C and
cells were then assayed at different temperatures as described above. After 30 min incubation,
cells were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.1% Triton X-100, and
incubated with a rabbit anti-pEGFR antibody (#2234, Cell Signaling Technology, Danvers, MA)
18
or a rabbit anti-EEA1 antibody (#3288, Cell Signaling Technology, Danvers, MA) for 1 hour at
37 °C followed by another hour with Alexa Fluor 568-linked donkey anti-rabbit antibody
(#A10042, Thermo Fisher Scientific, Waltham, MA). Cells were then washed and stained with
DAPI before mounting.
2.3.4 Colocalization analysis
Colocalization analysis was performed in this study to quantify the receptor activation
and internalization using ZEN2009 software (Carl Zeiss Microscopy, Thornwood, NY). Since
the surface receptor was stained with a green fluorescent dye in both studies, only Mander’s
colocalization coefficient (MCC) for the green channel was calculated to estimate the fraction of
the surface receptor activated or internalized using the following Eq. 1.
𝑀
"#$$%
=
'
(,*+,+*-, (
'
( (
Eq. 1
where G
i,colocal
= G
i
if R
i
> 0 and G
i,colocal
= 0 if R
i
= 0. R
i
and G
i
are the intensity values of the red
and green fluorophore in individual pixels, respectively.
2.3.5 Flow cytometry
Internalization of cell surface receptors was assayed by flow cytometry. Cells transfected
with EGFR-ELPs were serum starved for overnight and surface stained with Alexa Fluor 488-
labelled EGFR antibody at 4 °C for 30 min. After washing, cells were ramped at either 22 °C or
37 °C for indicated durations to allow microdomain assembly and receptor internalization. The
cells were then stripped with glycine buffer (0.2 M, pH 2.5, 1mg/ml BSA) to remove surface-
bound antibodies and trypsinized into single-cell suspensions for further analysis with LSRII
Flow Cytometer (BD Biosciences, San Jose, CA).
19
2.3.6 Immunoblot analysis
293T cells were seeded in 35mm dishes and transfected the following day with EGFR-
ELPs. Cells were starved 24 hours before temperature stimulation. Whole cell lysates were
prepared at different time points post temperature stimulations using RIPA buffer containing
protease/phosphatase inhibitor (#5872, Cell Signaling Technology, Danvers, MA). 100µg of
total protein was separated on a PAGEr EX 4-12% gradient gel (#59722, Lonza, Morristown, NJ)
and then transferred onto a nitrocellulose membrane using iBlot2 dry blotting system (Thermo
Fisher Scientific, Waltham, MA). The membrane was first immunoblotted with primary
antibodies, including rabbit anti-phospho-ERK1/2 (#9101, Cell Signaling Technology, Danvers,
MA), rabbit anti-ERK1/2 (#9102, Cell Signaling Technology, Danvers, MA), rabbit anti-
phospho-STAT3 (#9145, Cell Signaling Technology, Danvers, MA), rabbit anti-GAPDH (#5174,
Cell Signaling Technology, Danvers, MA), then incubated with secondary antibody anti-rabbit
IgG (HRP-linked) (#7074, Cell Signaling Technology, Danvers, MA). Protein bands were
visualized using ProSignal Dura Chemiluminescent Substrate (#20-301, Genesee Scientific, San
Diego, CA) and imaged on a ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Hercules,
CA).
2.3.7 Live cell imaging
Prepared cells were grown on 35mm glass bottom dishes (MatTek Corporation, Ashland,
MA) and starved overnight before imaging. A DIAPHOT epifluorescent microscope equipped
20
with a DS digital camera (Nikon Instruments, Melville, NY) was used to examine GFP-V60 vs.
GFP-V60 coassembly with EGFR-V96 while the temperature was increased from 4 °C to 60 °C
at a rate of 4 °C/min on a temperature controlled microscope stage (Linkam Scientific). The
actual medium temperature was monitored using a thermometer coupled with a probe from Sper
Scientific (Scottsdale, AZ). To calculate the mean particle size and particle number, each single
cell in the last image of the time-lapse video was analyzed using ImageJ (US NIH, Bethesda,
MD). Briefly, 12-bit images were thresholded and converted to a binary black and white 8-bit
image. The “Analyze Particles” function was used to count the particle number and measure the
total area, the mean particle size was then calculated by dividing the total area by the number of
particles.
2.3.8 Statistics
All experiments were replicated at least three times and data were presented as Mean ±
SD. The significance of changes in receptor internalization and signal activation were
determined using t-tests. The upregulation of pSTAT3 after a repetitive heat stimulation was
analyzed using a one-way ANOVA followed by Tukey’s post-hoc test using Prism (GraphPad
Software, La Jolla, CA). A Wilcox rank sum test was performed with R to examine the effect of
EGFR-V96 coassembly on GFP-V60 microdomain formation. A p value less than 0.05 was
considered statistically significant.
2.4 Results
2.4.1 Generation of thermo-responsive EGFR-ELP fusion proteins
21
A comprehensive library of chimeric receptors containing full-length EGFR and ELP was
designed (Figure 2). An ELP is a repetitive polypeptide with a sequence of (Val-Pro-Gly-X
aa
-
Gly)
n
, where X
aa
and n represents a guest residue and the number of repeats, respectively. ELPs
phase separate above a transition temperature that can be easily tuned by changing its chain
length or guest residue. Based on prior characterization (Pastuszka et al. 2012), a panel of ELPs
was genetically fused to EGFR, including V96, V72, 2VA96 as well as A96 as a negative control
(Figure 2b). These fusions vary in their intracellular transition temperature and composition
(Table 1); therefore, it was expected that they could be used to tune microdomain assembly to
relevant physiological temperatures. Expression of fusion proteins in mammalian cells was
confirmed to demonstrate the correct molecular weight and expected glycosylation when
assessed using a western blot (Figure 2c).
22
Table 1. ELP protein polymers fused with EGFR.
ELP
Nomenclature
Amino acid sequence
a
Intracellular
T
t
[°C]
b
Molecular
weight [kD]
c
EGFR-A96 EGFR-myc-G(VPGAG)
96
Y >42 173.5
EGFR-V96 EGFR-myc-G(VPGVG)
96
Y 32 176.2
EGFR-V72 EGFR-myc-G(VPGVG)
72
Y 37 166.4
EGFR-2VA96 EGFR-myc-G(VPGVGVPGVGVPGAG)
32
Y 42 175.3
a)
EGFR-myc sequence:
“MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNC
EVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYA
LAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMS
MDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQ
CAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVK
KCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSI
NATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPEN
RTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTI
NWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGR
ECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVK
TCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMV
GALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEF
KKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEILDEAYVMASVDN
PHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLED
RRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILH
RIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVK
CWMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEEDMD
DVVDADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYS
SDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHS
TAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKG
STAENAEYLRVAPQSSEFIGA”
b)
Observed transition temperature yielding EGFR-ELP microdomains in more than 50% of 293T
cells using fixed cell imaging.
c)
Estimated molecular weight from amino acid sequence.
23
Figure 2. Design and function of EGFR-ELPs. a) A schematic illustration of ligand-
induced vs. ELP-mediated EGFR signaling. b) Genes encoding four ELPs (V72, V96,
2VA96, A96) were fused to the carboxyl terminus of EGFR with a myc tag for indirect
immuno-detection of expressed proteins (Table 1). c) EGFR-ELPs were expressed in 293T
cells and detected using an anti-myc antibody (red). GAPDH (green) was also included as a
protein loading control, suggesting different expression levels of recombinant proteins. Lane:
1) No transfection; 2) EGFR-myc; 3) EGFR-V72; 4) EGFR-V96; 5) EGFR-2VA96; 6)
EGFR-A96.
24
2.4.2 Temperature-triggered clustering of cell surface EGFR
To characterize the behavior of EGFR-ELPs upon temperature stimulation, 293T cells
expressing EGFR-ELPs were incubated at various temperatures (22, 27, 32, or 37 °C) for 30
minutes (Figure 3a). Prior to assay, the extracellular domain of EGFR was pre-stained at 4 °C
cells with an AlexaFluor-488 conjugated antibody that specifically assess cell surface receptor
(Figure 4). Even in the absence of ligand EGF, EGFR-V96 assembled small clusters with strong
green fluorescence after incubation at 32 °C and nearly all of the transfected cells showed
evidence of receptor internalization at 37 °C (Figure 3b). In contrast, EGFR-V96 remained
evenly distributed on the cell surface when incubated at or below 27 °C. EGFR-V72 and EGFR-
2VA96 also phase separate near physiological temperatures; however, they required a slightly
higher temperature to reach their maximal assembly. In contrast, for non-switchable EGFR-myc
and thermo-insensitive EGFR-A96, no receptor internalization was detected at any temperature.
(Figure 3a, b). As our lead construct, the kinetics of EGFR-V96 internalization were followed at
37 °C, which show that within 15 minutes more than half of transfected cells show clear
evidence of receptor internalization (Figure 3c). This temperature-dependent clustering of
EGFR-ELPs were reconfirmed using live cell imaging in 293T cells (Figure 5), and similar
results were observed in CHO (Chinese hamster ovary) cells (Figure 6). Among the
temperature-sensitive EGFR fusion receptors, EGFR-V96 was selected for all follow-up studies
since it reaches maximal microdomain assembly at 37 °C.
25
Figure 3. Temperature dependent clustering of surface EGFR-ELP is fast and tunable.
a) 293T cells expressing EGFR-ELPs were pre-stained with an anti-EGFR antibody at 4 ºC
prior to incubation (30 min) at indicated temperatures. After fixation, images were acquired
using confocal laser scanning microscopy. Green: AlexaFluor-488 conjugated anti-EGFR
antibody; Blue: DAPI; Scale bar: 10 µm. b) The temperature of microdomain assembly can
be tuned by changing ELP hydrophobicity and length. The majority of cells transfected with
EGFR-V96, EGFR-V72, and EGFR-2VA96 clustered the label above 32, 37, and 42 ºC
respectively. A temperature insensitive control, EGFR-A96 did not assemble microdomains at
the temperatures evaluated. c) The kinetics of EGFR-V96 microdomain assembly were
characterized at 37 ºC. Phase separation occurs rapidly with more than 50% of cells showing
microdomains within 15 min. Mean ± SD (n=3).
26
Figure 4. Immunofluorescence of total EGFR vs. cell surface EGFR in 293T cells
transiently transfected with EGFR fusions. Cells were pre-incubated with anti-EGFR
antibody (green) at 4 °C and cultured at a) 22 °C and b) 37 °C for 30 minutes. Cells were then
fixed, permeabilized and stained for total EGFR using an anti-myc antibody (red). In addition
to the receptors expressed on the plasma membrane, a significant amount of intracellular
EGFR was also observed in the transfected cells. Blue: DAPI. Scale bar: 20 µm.
27
Figure 5. Live cell imaging of temperature-dependent EGFR assembly. 293T cells
expressing EGFR-V96/A96 were stained with an AlexaFluor 488 conjugated anti-EGFR
antibody (green) for 30 min at 4 °C and then heated to different temperatures. Live cell
images were taken using an epifluorescent microscope equipped with a temperature
controlled stage. Medium temperatures were measured by a temperature probe. Scale bar: 20
µm.
28
Figure 6. EGFR-ELP microdomain assembly is retained across cell lines and polymer
species. Chinese hamster ovary (CHO) cells were transfected with EGFR-myc / EGFR-ELPs
and the cell surface receptors were assessed using an antibody (green) targets the extracellular
domain of EGFR. Cells were then incubated at different temperatures, fixed and imaged using
confocal microscope. a) Representative images of different constructs in CHO cells at 22 °C
and 37 °C. Blue: DAPI. Scale bar: 10 µm. b) The percentage of cells had puncta formation
was quantified and plotted against temperature. Different constructs behave similarly in CHO
cells as in 293T cells (Figure 2).
29
2.4.3 Temperature-triggered internalization of cell surface EGFR
Having shown that cell-surface EGFR-ELPs can be clustered in response to heating
(Figure 3), EGFR internalization was probed using flow cytometry on cells that were stripped of
surface antibody (Figure 7). As shown in Figure 7a, at 22 °C, cell-surface EGFR-V96 pre-
labeled with an AlexaFluor-488 conjugated EGFR antibody was slowly endocytosed over time in
a similar pattern to the control EGFR-A96, which could potentially result from non-specific fluid
phase endocytosis. However, at 37 °C, internalization of EGFR-V96 increased significantly,
which suggested that ELP-mediated receptor clustering induced a rapid and effective
internalization of EGFR. Moreover, the subcellular localization of internalized EGFRs was
confirmed using confocal microscopy (Figure 8). To further determine whether the endocytosed
fusion proteins sort through early endosomes as the wild type EGFR, EEA1 (early endosome
antigen 1), a protein involved in vesicular transport through early endosomes (Mu et al. 1995),
was assessed for colocalization with microdomains. When soluble at room temperature, most of
the EGFR-V96 remained on the plasma membrane and their colocalization with EEA1 was low,
whereas significantly more EGFR clustered and colocalized with EEA1 after microdomain
formation (Figure 7b, c). EGFR-A96, which does not assemble microdomains at physiological
temperatures, did not colocalize with EEA-1, suggesting the important role of receptor clustering
in internalization.
30
Figure 7. EGFR-ELP assembly initiates receptor internalization to early endosomes.
293T cells were transiently transfected with EGFR fusions and the surface receptors were
labeled by an anti-EGFR antibody (green) at 4 ºC prior to incubation above (37 ºC) or below
(22 ºC) the transition temperature. a) EGFR internalization was quantified using flow
cytometry. After incubation, the cell surface was stripped with acidified glycine buffer (0.2
M, pH 2.5, 1 mg/ml BSA). By 30 min at 37 ºC, there was a significant increase in receptor
internalization for cells expressing EGFR-V96 compared to those with EGFR-A96, which
does not phase separate at 37 ºC (**** p<0.0001, ** p<0.01). Mean ± SD (n=3). b) and c)
Alternatively, to follow the internalization pathway of surface-stained EGFR, 293T cells
transfected with EGFR-V96 or EGFR-A96 surface-stained (green), incubated for 30 min,
fixed, and stained for early endosomes (red). Immunofluorescence was observed using
confocal laser scanning microscopy (Scale bar: 10 µm). At room temperature, EGFR-V96
remained on the cell surface. Once induced to phase separate at 37 ºC, EGFR-V96
internalized to puncta that colocalize significantly with EEA1, a marker of early endosomes.
*** p=0.0001, Mean ± SD (n=11-23).
31
Figure 8. Subcellular localization of internalized EGFR-V96 in 293T cells. Cells
expressing EGFR-V96 were pre-incubated with anti-EGFR antibody (green) at 4 °C and
cultured at 37 °C, 22 °C or 4 °C for 30 minutes. Antibodies bound to the cell-surface were
then stripped by acidified glycine buffer. Blue: DAPI. Scale bar: 10 µm.
32
2.4.4 Temperature-triggered activation of EGFR signaling pathways
Having characterized the phase behavior and fate of EGFR-ELPs, their ability to activate
known signaling pathways was explored. Using indirect immunofluorescence and colocalization
analysis, the phosphorylation state of cell-surface EGFR was monitored. This method allows us
to specifically quantify the percentage of cell surface receptors phosphorylated. At 22 °C, some
of cells transfected with EGFR-V96 showed a strong intracellular red fluorescence signal arising
from the auto-phosphorylation of receptors in the cytoplasm, which may be due to their
overexpression through transient transfection. After 20 min incubation at 37 °C, in addition to
pre-existing larger red fluorescent puncta, a pattern of smaller phosphorylation-positive puncta
appeared to colocalize with the clustered cell surface receptors (Figure 9a). Colocalization
analysis revealed that the activation of cell surface EGFR-V96 increased significantly post-
stimulation (Figure 9b). Subsequent activation of ERK1/2, indicated by a phosphorylation signal,
was also observed in cells transfected with EGFR-V96. While a certain level of auto-activation
was detected at 22 °C, there remained a remarkable upregulation of phospho-ERK1/2 after
heating up the cells to 37 °C for 20 min. Importantly, this response was completely blocked with
the treatment of AG1478, a selective inhibitor of EGFR tyrosine kinase activity and PD0325901,
a MEK inhibitor (Figure 9c and 10). These results indicate that temperature-induced ERK
phosphorylation is mediated through the Ras-Raf-MEK signaling cascade following EGFR
activation. As expected, similar activation was not observed when EGFR-A96 heated. ERK1/2,
known to be involved in the activation of many transcription factors, is strongly amplified by
translocation to the nucleus (Chen, Sarnecki, and Blenis 1992). The occurrence of nuclear
translocation was confirmed by immunofluorescent staining of phosphorylated ERK1/2 (Figure
11).
33
Figure 9. EGFR-V96 assembly specifically activates EGFR and the ERK1/2 pathway. a)
293T cells transfected with EGFR-V96 were pre-chilled at 4 ºC and surface EGFR was
labeled by immunofluorescence (green). Cells were then incubated either below (22 ºC) or
above (37 ºC) the transition temperature for 30 min, fixed, permeabilized, and the activation
of EGFR was detected using an anti-pEGFR antibody (red). Scale bar, 20 µm. b)
Colocalization coefficient of each individual cells representing the percentage of surface
EGFR phosphorylated was determined from the pixels in the overlapped region to the total
green pixel intensity using Eq. 1. Temperature-dependent activation of surface EGFR was
detected in cells expressing EGFR-V96 not EGFR-A96. *p = 1e-16. Mean ± SD (n=26-39). c)
Using a western blot, temperature-dependent phosphorylation of ERK1/2 was observed in
cells transfected with EGFR-V96 while same levels of activation were not observed in EGFR-
A96; furthermore, an EGFR kinase inhibitor AG1478 (0.5 µM) specifically blocked this ELP-
mediated activation.
34
Figure 10. Inhibition of ELP-mediated ERK1/2 activation. 293T cells transfected with
EGFR-V96 (temperature-sensitive) or EGFR-A96 (control) were incubated at either 22 or 37
°C for 20 min with or without a 30-min pre-incubation with 500 nM AG1478 or 100 nM
PD0325901. AG1478 specifically blocks the temperature-dependent ERK1/2 phosphorylation
in cells expressing EGFR-V96, whereas PD0325901, a MEK inhibitor, completely abolished
ERK1/2 activation in any conditions.
35
Figure 11. Subcellular localization of pERK1/2 in 293T cells. 293T cells expressing
EGFR-V96 were incubated below/above their transition temperature for 20 min and the
activation of downstream ERK1/2 was observed using an anti-pERK1/2 antibody (red). Blue:
DAPI. Scale bar: 20 µm.
36
To compare the microdomain-mediated signaling with the ligand-triggered responses, the
phosphorylation state of ERK1/2 was monitored over time (0, 10, 20, 60, 120, and 240 min) after
applying heat (37 °C) or EGF (5 ng/mL). Both stimuli resulted in rapid, but transient activation
of ERK1/2 (Figure 12a), consistent with previously published results (Marshall 1995).
Interestingly, the phosphorylation state of ERK1/2 in the presence of EGF is stronger relative to
the temperature-triggered activation, which may result from promiscuous binding of EGF to
endogenous receptor tyrosine kinases. Taken together, these observations demonstrate that
EGFR-V96 enables transient activation of canonical ERK signaling in a temperature-dependent
manner.
2.4.5 Dynamic and quantitative control of microdomain-mediated signaling activation
In comparison to small molecule inhibitors and inducible promoters, the potential to
reversibly assemble microdomains in a biological environment is a strong advantage of ELP
systems. To assess the reversibility of ELP-mediated EGFR signaling, 293T cells expressing
EGFR-V96 were exposed to three rounds of heating (20 min) and cooling (40 min) and the
activity of endogenous ERK1/2 was monitored. Western blots revealed that exposure of cells to
37 °C yielded approximately a four-fold increase in pEKR1/2 and this activation was fully
reversed upon cooling cells down to room temperature. Similar changes were observed after
repetitively applying a second and third stimulation, demonstrating that this process can be
reversibly controlled without losing activity (Figure 12b).
37
Figure 12. ELP-mediated ERK signaling is transient and reversible. a) To verify that the
construct retains activity similar to that obtained upon the addition of free ligand (EGF), 293T
cells expressing EGFR-V96 were either heated to 37 ºC or treated with 3 ng/mL EGF at room
temperature for the indicated time periods and monitored by western blot. Both yielded a
transient elevation in activated pERK1/2 with a peak near 20 min after activation. b) In
EGFR-V96 transfected 293T cells, phosphorylation of ERK1/2 was monitored and quantified
after three cycles (C1, C2, C3) of heating (20 min) and cooling (40 min), indicating the
reversibility of the switch. **** p=0.00004, *** p=0.0003, ** p=0.005. Mean ± SD (n=3).
38
It has been shown that dynamic changes of signaling in response to distinct stimuli can
confer specificity to biological outcomes, especially for the ERK pathway (Marshall 1995). For
example, in PC12 cells, EGF induces transient ERK1/2 activation that results in cell proliferation,
whereas NGF (nerve growth factor) stimulates a sustained ERK1/2 activation and cell
differentiation. Taking advantage of their reversible phase behavior, I hypothesized that ELP
fusions can serve as a powerful platform for the dynamic control of signals by manipulating the
temporal patterns of cell stimulation. To test this, various heating/cooling patterns were applied
to 293T cells expressing EGFR-V96, and the changes in signaling outputs were assessed using
pERK1/2 as a simple readout (Figure 13). Low-frequency stimulation, such as one round of
heating and cooling per hour, yielded transient and pulsatile patterns of ERK1/2 activation,
which corresponded with its pattern of stimulation. By increasing the frequency of stimulation, it
was possible to generate robust and sustained elevations in pERK1/2 over the entire study
(Figure 13a). To examine the effect of high frequency stimulation of EGFR-ELP, a downstream
module of ERK signaling was assessed. STAT3 has been reported to selectively respond to
sustained- rather than transient- ERK1/2 activation (Toettcher, Weiner, and Lim 2013). Using
western blot, the phosphorylation of STAT3 was observed to increase after 1 hr of repetitive
simulation whereas no significant change was detected following a single round of stimulation
(Figure 13b). In addition, the effect of stimulation duration was also characterized by heating
cells at 37 °C for different periods of time (5, 10, 15, and 20 min). The magnitude of ERK1/2
phosphorylation showed a proportional response to the heat duration (Figure 13c), suggesting
the quantitative control of ERK1/2 activity by EGFR-ELPs. Collectively, these results suggest
that EGFR-ELPs can be used to study distinct temporal patterns of dynamic and quantitative
39
stimulation through the ERK pathway, which will be essential for studying and manipulating
diverse physiological responses.
40
Figure 13. EGFR-V96 mediated ERK signaling can be temporally modulated. The
change of ERK1/2 activity was monitored by western blot according to frequency and
duration of heating (37 ºC, red boxes) and cooling (22 ºC, baseline) in 293T cells expressing
EGFR-V96. a) Cells were heated for 10 min repetitively at 5, 10, 20, and 50 min intervals.
High-frequency heating led to sustained ERK1/2 activation, whereas low-frequency heating
showed pulsatile ERK1/2 activation. b) Cells were exposed either to constant heating (37 ºC)
or repetitive heating (10 min 37 ºC, 5 min 22 ºC). STAT3 is a transcription factor downstream
of EGFR/ERK; furthermore, phosphorylation of STAT3 responds to repetitive heating and
not to continuous heating after 2-hr treatment. GAPDH was included as a protein loading
control. *p = 0.04. Mean ± SD (n=4). c) Cells were heated at 37 ºC for 5, 10, 15, or 20 min
over a total period of 30 min. pERK1/2 intensity increased as the duration increased to 20
min.
41
2.4.6 Single-cell characterization of EGFR microdomain assembly
Within individual cells the assembly of ELP microdomains is expected to occur within a
small change in temperature; however, different cells have variability in expression of EGFR-
V96, which could influence their transition temperature (Figure 3b). To examine this possibility,
the exact transition temperature of each cell was monitored using live cell imaging. Our prior
studies have shown that co-expressed ELPs co-assemble different proteins within cytosol (Shi et
al. 2014). Given this and the fact that the GFP-ELPs can be visualized in real time, a cytosolic
GFP-ELP was selected as a surrogate to follow the phase separation of EGFR-V96 (Figure 14a).
To validate co-assembly of EGFR and GFP, 293T cells expressing both EGFR-V96 and GFP-
V60 were incubated at 37 °C to induce intracellular microdomains, and their colocalization was
confirmed in fixed cells using indirect immunofluorescence (Figure 15). This approach has
significant applications during time-lapse imaging over temperature gradients, which can refine
estimates of the exact phase transition temperature for individual cells. The results showed that
individual cells transitioned at slightly different temperatures/time points. However, each cell
consistently formed puncta above its own transition temperature (Figure 14b). In general, 293T
cells dually transfected with GFP-V60 and EGFR-V96 exhibit microdomains that assemble at a
lower temperature (Figure 14b), are larger, and fewer in number, than microdomains from cells
transfected with GFP-V60 alone (Figure 14c). Notably, the median transition temperature of
EGFR-V96 is 32.8 °C, which is consistent with our previous observation using fixed cells
(Figure 3b). Moreover, co-assembly with GFP-V60 was confirmed to have no effects on the
temperature-dependent activation of ERK by EGFR-V96 (Figure 16). These results reinforce the
hypothesis that a GFP-ELP can serve as a proxy for intracellular assembly of ELP fusion
proteins.
42
Figure 14. GFP-ELP microdomains reveal intracellular phase separation of EGFR-V96
using live cell imaging. a) Co-transfection with EGFR-V96 and GFP-V60 enables live cell
imaging of microdomain assembly in individual 293T cells. b) Epifluorescence imaging was
conducted over a temperature ramp of 10 to 40 °C at a rate of 4 °C per min. Co-transfection
with EGFR-V96 and GFP-V60 significantly (p = 0.002) decreased intracellular T
t
32.8 °C
[31.0, 34.0, n=19] compared to cells transfected with GFP-V60 alone 35.9 °C [33.0, 37.0,
n=19]. Values are reported as Median [15
th
percentile, 85
th
percentile, sample size]. c) In
addition to lowering the T
t
, co-transfection alters the morphology of GFP-V60 microdomains.
When compared to GFP-V60 alone, co-transfection significantly increased the puncta size (p
= 7e-06) and reduced the puncta number (p = 2e-04) of GFP-V60.
43
Figure 15. EGFR-ELPs co-expressed with cytosolic GFP-ELPs co-assemble above the
ELP phase transition temperature. 293T cells were transiently transfected with both
EGFR-V96 and GFP-V60 constructs. After microdomain assembly, the colocalization of
EGFR (anti-myc, red) and GFP-V60 (green) was verified by indirect immunofluorescence.
Scale bar: 20 µm.
44
Figure 16. Co-expression of GFP-V60 with EGFR-V96 does not affect the ELP-mediated
ERK1/2 activation. 293T cells were either transfected with GFP-V60 alone or co-transfected
with EGFR-V96 and GFP-V60. After 48 hours, cells were incubated at different temperatures
(22 vs. 37 °C) for 20 min and the activation of downstream ERK1/2 was detected using an
anti-pERK1/2 antibody.
45
2.5 Discussion
Controlled cell-signaling plays an undeniable role in diverse biological processes, and
better tools to manipulate these signaling pathways will provide insight into how the temporal
dynamics of signaling components regulate cellular function. Towards this end this manuscript
demonstrates how to design a protein switch that provides ON-OFF regulation of a specific cell-
surface receptor, which enables dynamic control of downstream signaling pathways. The switch
is designed using a thermally responsive ELP protein-polymer to drive reversible-assembly of a
receptor tyrosine kinase expressed at the cell surface. A temperature sensitive ELP, V96, was
genetically fused to EGFR, which is inactive in its default monomeric state and active in its
clustered state. When heated above 32 °C EGFR-V96 forms microdomains and activates the
ERK signaling cascade, which demonstrates that ELPs confer their phase behavior without
altering signaling targets of the native receptors. In comparison, a temperature-insensitive
EGFR-A96 failed to show these effects at the same temperatures, which supports the hypothesis
that ELP-mediated EGFR clustering is responsible for the receptor activation and signal
transduction. While EGFR is known to activate Akt and PLC pathways in many cell lines, in the
293T cell line I only observed significant upregulation in pERK1/2 stimulated by either heating
of EGFR-V96 or treatment with its endogenous ligand EGF. This suggests that signal sensitivity
of different pathways is highly cell type-dependent and may vary across different cell lines.
As a genetically encoded protein, an ELP fusion only requires a single transgene to
introduce temperature-dependent signal activation. The activation is limited to receptors that are
genetically engineered, thus providing specificity beyond that of natural ligands. In this study,
fusion receptors were delivered through transient transfection, which can result in variability in
46
levels of overexpression. Total intracellular EGFR-V96 was visualized using confocal
fluorescence microscopy, which revealed significant clusters of fusion proteins forming inside
cells (Figure 4). This likely resulted from culturing transfected cells at 37 °C prior to assay,
above their phase transition temperature. Multiple experiments suggest that EGFR-V96 phase
separates in cells above 32 °C by either fixed cell imaging (Figure 3b) or by live cell imaging
(Figures 14b and 5). To visualize only the behavior of cell-surface EGFR-V96, an antibody was
used to specifically label external EGFR-V96, which shows the clearest temperature-dependent
internalization and phosphorylation (Figures 7 and 9). One challenge with transient expression
is that receptor overexpression can elevate signaling above the physiologically basal state. Even
though a certain level of phosphorylation of both EGFR and ERK was detected below the
transition temperature, there remained a remarkable increase in their activity at 37 °C, which far
exceeded that of a temperature-insensitive EGFR-A96 control (Figure 9c). Further optimization
of viral gene vectors or zinc finger nucleases may offer longer-term expression for ELP fusion
proteins in stable cell lines or transduction of primary cells and tissues, which may make this
switch even more powerful.
While ELP mediates similar responses as EGF at 22 °C (Figures 12), it showed slower
activation kinetics compared to that at 37 ºC, which was likely due to slow transfer of convective
heat from air within the incubator. In contrast, signaling from EGF occurred instantaneously
once mixed with the cell-culture medium. To quantify this possibility, the temperature was
monitored by a probe immersed in media above cells cultivated in a 37 °C incubator. As in the
Figure 17a, the temperature increase took 15 mins to reach the median T
t
for EGFR-V96
(Figures 3b, 14), which corresponds to the observed delay in signal activation (Figure 17b).
47
However, changing the heat source from air to conductive heating using a hot plate easily
increased the rate of heating. The activation kinetics of pERK1/2 accelerated as the heat transfer
rate increased, and the magnitude of activation remained the same. With the optimized system, I
detected ERK phosphorylation within 2 minutes, which reached its maximum at 5 minutes. The
rapid change of signaling pathways clearly demonstrates the fast response of EGFR-V96, which
was now even faster than for exogenous EGF (Figure 17b).
48
Figure 17. Optimization of signaling kinetics using different heating systems. a)
Temperature changes following different heating sources were monitored over time using a
temperature probe. b) 293T cells expressing EGFR-V96 were either treated with EGF at
37 °C or heated to 37 °C using different heating systems. The activation of ERK1/2 was then
monitored over time using western blot. Although an air incubator induced slower activation
in pERK1/2 compared to EGF, the heat transfer using a hot plate significantly sped up
activation, which reached maximum levels within 5 mins.
49
In addition, ELP-mediated assembly offers benefits compared with alternative
magnetogenetic or optogenetic strategies. Molecular switches using magnetic nanoparticles were
previously developed for manipulation of cell signals, however, this method can only be used
with transmembrane proteins unless single-cell microinjection is applied. In contrast, ELPs can
be easily adapted to a broad range of signaling molecules including intracellular proteins. An
advantage of ELPs over optogenetics is that it allows the use of green or blue light emitting
fluorescent proteins for time-lapse imaging of receptor clustering in living cells. For example,
GFP-V60, an ELP with a higher phase transition temperature than EGFR-V96, was explored as a
live cell marker for microdomain formation. Co-assembly with EGFR-V96 not only lowered the
apparent transition temperature of GFP-V60, but also changed the morphology of microdomains
(Figure 14). GFP-V60 thus enables real-time characterization of EGFR-V96 at a single-cell
level without the need to directly link GFP to the EGFR gene. Furthermore, it should be noted
that ELP phase separation results in receptor oligomerization, which may differ significantly
from ligand-induced dimerization. Although the dimerization of receptor tyrosine kinases has
been reported for decades (Yarden and Schlessinger 1987), there is increasing evidence that
higher-order oligomers may also form in response to EGF binding (Saffarian et al. 2007; Clayton
et al. 2008). In these regards, the EGFR-ELP system offers multiple potential advantages to
explore receptor clustering and its potential mechanisms in cell signaling and human disease.
2.6 Conclusion
This chapter demonstrates that clustering of a receptor tyrosine kinase, EGFR, through
thermo-responsive microdomain assembly can modulate signaling activities in a dynamic
manner, offering an approach to investigate the complex dynamic properties of signal
50
transduction, as well as for the guidance of biomedical applications. With RTKs being one of the
main cancer research targets (Gschwind, Fischer, and Ullrich 2004), the discovery of inhibitors
requires cellular models of engineered receptors without the need for ligand-induced activation
and here our switch can serve as a selective system. From a synthetic biology perspective, I
provide a new strategy for versatile control of protein clustering which can be extended to
numerous signaling molecules and pathways.
51
CHAPTER 3
Tunable assembly of protein-microdomains in living vertebrate embryos
3.1 Abstract
Subcellular events such as trafficking and signaling are regulated self-assembled protein
complexes inside the cell. The ability to rapidly and reversibly manipulate these protein
complexes would likely enhance studies of their mechanisms and their roles in biological
function and disease manifestation (Papapostolou and Howorka 2009; Ng et al. 2017). To
explore a new way to modulate protein assemblies, this manuscript reports that thermally-
responsive elastin-like polypeptides (ELPs) linked to fluorescent proteins can regulate the self-
assembly and disassembly of protein microdomains within the individual cells of zebrafish
embryos. By exploring a library of fluorescent ELP proteins, this reports demonstrates that ELPs
can co-assemble different fluorescent proteins inside of embryos. By tuning ELP length and
sequence, fluorescent protein microdomains can be assembled at different temperatures, in
varying sizes, or for desired periods of time. For the first time in a multicellular living embryo,
these studies demonstrate that temperature-mediated ELP assembly can reversibly manipulate
assembly of subcellular protein complexes, which may have applications in the study and
manipulation of in vivo biological functions.
3.2 Introduction
A major goal of synthetic biology is the design of biocompatible materials that emulate
the dynamic structures formed within cells (Benner and Sismour 2005; Cameron, Bashor, and
Collins 2014; Rawls 2000). Subcellular structures (e.g. transcription machinery, the nuclear
52
envelope, signaling complexes, endosomes) must assemble and disassemble in a regulated
fashion for proper function. Since this fine-tuned assembly is so pervasive in biological systems,
synthetic biology strategies to regulate and manipulate self-assembly promise greater control,
and therefore understanding, of a wide range of cellular pathways and their consequent roles in
physiology and disease.
In previous work, our group has shown that thermally responsive elastin-like
polypeptides (ELPs) can be fused to effector proteins to reversibly assemble fluorescent
cytosolic protein microdomains and to reversibly inhibit clathrin mediated endocytosis (Shi et al.
2014; Pastuszka et al. 2012; Pastuszka et al. 2014). ELPs are biocompatible, biodegradable
polypeptides consisting of a pentameric repeat of (Val-Pro-Gly-X
aa
-Gly)
n
, where X
aa
determines
the ELP hydrophobicity and n determines the molecular weight (Shah et al. 2012a; Despanie et
al. 2016). ELPs abruptly self-assemble in response to heating, whereby they form a secondary
aqueous phase known as a coacervate. A thermodynamically reversible process, ELP coacervates
quickly resolubilize in bulk water upon cooling. The temperature of self-assembly, termed the
transition temperature (T
t
), is specified by the hydrophobicity of the guest residue and the
number of repeats. When fused to an effector protein, such as green fluorescent protein (GFP) or
clathrin light chain (CLC), this temperature sensitive behavior is retained, producing a
temperature sensitive mutant that rapidly cycles between self-assembly and disassembly in
response to temperature, which enables the reversible control of protein clustering. While this
system has been proven robust in cell culture, it remains unknown whether the same strategy will
work in a multicellular organism.
53
Zebrafish (Danio rerio) is a small freshwater fish that is commonly used for the study of
vertebrate developmental biology (Dooley and Zon 2000; Lieschke and Currie 2007). Several
features make zebrafish an attractive model organism, including low cost of husbandry, high
reproductive rates, and the ex vivo development of optically clear embryos. Importantly,
zebrafish can tolerate a reasonable range of temperatures found in their native environments,
allowing specimens to be maintained above and below the T
t
of optimized ELP fusion proteins
(Lahiri et al. 2005). This chapter shows that GFP-ELPs (GFP cloned to amino-terminus of ELP)
can be easily expressed in zebrafish, where they undergo temperature-dependent self-assembly
and disassembly of protein microdomains (Figure 18b). Moreover, by tuning the ELP sequence
it is possible to design microdomains that form at different temperatures, have distinct sizes, or
remain assembled for extended durations. These findings suggest ELPs are potentially a
powerful strategy for controlled protein assembly and disassembly in zebrafish.
3.3 Materials and methods
3.3.1 Zebrafish husbandry and care
Zebrafish were raised and maintained at 28.5 °C in a circulating system according to
standard protocols that are in accordance with Children’s Hospital Los Angeles IACUC animal
care protocol (Brand, Granato, and Nüsslein-Volhard 2002). Wild-type AB fish were set up for
breeding the day prior to obtaining embryos for microinjection.
3.3.2 Plasmid construction
GFP-ELPs in mammalian expression vector pcDNA3.1 were cloned as previously
described (Pastuszka et al. 2012). To engineer GFP-ELPs for bacterial expression, a GFP gene
54
was amplified using PCR and inserted to the N terminus of ELPs in pET25b (+) vectors using
the BseRI digestion site. The sequences were verified by DNA sequencing (Retrogen, San Diego,
CA).
3.3.3 ELP purification and physicochemical characterization
pET25b (+) vectors encoding GFP-ELPs were transformed into BLR (DE3) Escherichia
coli competent cells (Novagen Inc., Milwaukee, WI) for protein expression. Inverse transition
cycling (ITC) was used to purify ELP samples from the bacteria lysates as previously published
(Meyer and Chilkoti 1999). To characterize the phase behaviors of GFP-ELPs, optical density
(OD) at 310 nm was monitored using DU800 UV-Vis spectrometer while the temperature was
ramped from 15 °C to 45 °C at a rate of 1 °C/min, and then plotted as a function of temperature.
The maximum first derivative of the curve was defined as the transition temperature.
3.3.4 mRNA preparation and microinjection
To linearize the plasmids before in vitro transcription, the GFP plasmid was cut with
XbaI and the GFP-ELP plasmids were cut with EcoRI. Linearized GFP and GFP-ELP plasmids
were then transcribed into full length capped mRNAs with the T7 mMESSAGE mMACHINE
Ultra Transcription Kit (AM1345, Life technologies, Carlsbad, CA) using the manufacturer’s
protocol. This mRNA was then purified with the MEGAclear™ Transcription Clean-Up Kit
(AM1908, Life technologies, Carlsbad, CA). I followed this kit’s protocol with one major caveat:
in the final step, mRNA was eluted from the silica filters using room temperature elution buffer,
instead of heated elution buffer as suggested in the protocol, which may degrade the mRNA.
mRNA purity was determined by measuring 260/280 and 260/230 values on a NanoDrop 2000
55
(ThermoFisher Scientific Inc., Rockford, IL). mRNAs with 260/280 and 260/230 values near 2.0
were selected for microinjection. mRNA degradation was assessed by running aliquots on a
denaturing bleach gel (Aranda, LaJoie, and Jorcyk 2012). Purified in vitro transcribed mRNA
was then injected into the cytoplasm of one cell stage zebrafish embryos following standard
injection protocols.
3.3.5 Immunoblotting
Zebrafish embryos injected with GFP-ELPs were lysed with RIPA buffer containing
protease/phosphatase inhibitor (#5872, Cell Signaling Technology, Danvers, MA) at 24 hpf and
electronically separated on a PAGEr EX 4-12% gradient gel (#59722, Lonza, Walkersville, MD).
Proteins were then transferred onto a nitrocellulose membrane using iBlot2 dry blotting system
(Life Technologies, Carlsbad, CA) and probed for GFP (ab290, 1:5000 dilution, Abcam,
Cambridge, MA).
3.3.6 Zebrafish imaging
Embryos at early time points (4~5 hpf) were placed on a 35-mm glass bottom dish
(MatTek, Ashland, MA) and imaged using a LSM800 confocal microscope (Carl Zeiss
Microscopy, Thornwood, NY) with a 40x 1.3 NA oil objective. For live fish imaging, embryos
were incubated on an HCS60 microscope hot and cold stage (Instec, Boulder, CO) attached to a
mK2000 high precision temperature controller (Instec, Boulder, CO) and imaged using a
Fluoview FV3000RS confocal microscope (Olympus, Waltham, MA) equipped with a 40x 1.25
NA oil objective. For fixed-fish imaging, embryos were incubated at different temperatures and
fixed overnight with 2% paraformaldehyde (Alfa Aesar, Tewksbury, MA) at the same incubation
56
temperature before imaging. Fish at late time points (24, 48 hpf) were imaged alive on a Leica
MZFLIII Stereo/Dissection Microscope (Buffalo Grove, IL).
3.3.7 Image processing
16 bit grayscale images with a resolution of 1024 by 1024 (4~5 hpf) or 1344 by 1024 (24
hpf and 48 hpf) were analyzed with FIJI (Schindelin et al. 2012). The FIJI macros used to
calculate microdomain area and number can be found on github: https://github.com/d-
tear/Image-Analysis-Pipeline-for-Tunable-assembly-of-protein-microdomains-in-living-
vertebrate-embryos-.
3.3.8 Statistical analysis
Data presented are representative curves or mean ± 95% confidence interval. Statistical
analysis was performed in R. The wilcox.test() function, which performs a Wilcox rank sum test,
was used to determine statistically significant differences of in median particle size per embryo
between the different treatments (Figure 22b). The t.test(var=FALSE) function was used to
determine statistically significant differences in particle number per embryo between the
different treatments at 24 hpf and 48 hpf (Figure 22d).
3.4 Results
3.4.1 ELP expression and microdomain assembly in vivo
A library of GFP-ELPs were first expressed and characterized within zebrafish embryos
(Table 2, Figure 18a, c). The optimal temperature used for zebrafish husbandry is 28.5 °C;
however, larvae can survive for at least several days at temperatures ranging from 21 to 33 °C
57
(Kimmel et al. 1995; Avdesh et al. 2012). Therefore, GFP-V96 was selected for study as its in
vitro T
t
is within this range (Figure 18d). To visualize the temperature-dependent microdomain
assembly, an embryo injected with mRNAs encoding GFP-V96 was imaged at late blastula
stages (4~5 hpf, hour post fertilization) while the temperature was increased from 20 to 40 °C
(Figure 18e). At 20 °C, which is below the T
t
, GFP-V96 remains soluble throughout the embryo.
Within 5 min of ramping the temperature, microdomain formation was observed in every single
cell. Immediately after heating, these fluorescent microdomains were completely resolubilized
by cooling to 15 °C. These results demonstrate the efficient temperature-mediated self-assembly
and disassembly of proteins within the single cells of a zebrafish embryo. Short durations of
heating (~5 min) suggest the high temporal resolution of this system.
58
Table 2. Nomenclature, amino acid sequence and phase behavior of expressed proteins.
Protein
Label
Amino acid
sequence
a
MW
[kD]
b
T
t
[°C]
in PBS
c
T
t
[°C]
in zebrafish
d
Intercept, b
[°C]
e
Slope, m
[°C] [log
10
(µM)]
-1
GFP GFP 30.0 NA NA NA NA
GFP-V60 GFP-(VPGVG)
60
Y 52.4 36 38
43.8
[42.7 to 44.8]
5.3
[4.5 to 6.2]
GFP-V96 GFP-(VPGVG)
96
Y 67.1 30 28
35.42
[34.8 to 36.1]
3.4
[2.8 to 4.0]
GFP-SI
GFP-
(VPGSG)
48
(VPGIG)
48
Y
67.2 22 23
27.0
[26.3 to 27.6]
3.4
[2.8 to 3.9]
Not applicable (NA)
a)
GFP indicates the amino acid sequence
“MASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPW
PTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGNYKTRAEVKFE
GDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGS
VQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGM
DELYK”
b)
Estimated molecular weight from open reading frame confirmed by western blot (Figure 18c).
c)
Transition temperature of purified GFP-ELPs suspended in phosphate buffered saline at 25 µM.
d)
Observed transition temperature in zebrafish embryos (molar amount of mRNA equal to 400pg
GFP-V96 mRNA injected at one-cell stage) using fixed-zebrafish imaging.
e)
The Intercept b and slope m, were derived from the log-linear regression analysis for transition
temperature vs. concentration (Figure 18d) fit to the equation T
t
= b - m Log
10
[C
ELP
]. Data
represent the mean [95% confidence interval].
59
Figure 18. ELP expression and microdomain assembly in a zebrafish embryo. a) Genes
encoding three ELPs (V60, V96, SI, Table 2) were fused to the carboxy-terminus of a GFP
gene for direct visualization. b) Schematic representation of reversible microdomain assembly
in a zebrafish embryo. Below their T
t
, GFP-ELPs are uniformly distributed inside the single
cells of the embryo. Upon heat stimulation, the protein polymers reversibly phase separate
and assemble microdomains. c) Expression of different GFP-ELPs in zebrafish embryos were
confirmed using an anti-GFP antibody. d) The in vitro temperature-concentration phase
diagrams for all purified GFP-ELPs follow a log-linear relationship (Table 2). e) ELP
microdomain assembly in a zebrafish embryo is rapid and reversible. Live embryo imaging
was conducted at late blastula stages (4~5 hpf) to visualize the temperature-dependent
microdomain assembly from 20 to 40 °C. These polypeptide microdomains can be rapidly
resolubilized by decreasing the temperature to 15 °C.
60
3.4.2 ELPs can co-assemble different proteins in vivo
Signaling and trafficking events inside living cells require the co-assembly of protein
complexes composed of distinct proteins. Accordingly, I examined whether ELPs fused with
different fluorescent proteins may co-assemble when induced to phase separate (Figure 19a).
mRNAs encoding for RFP-V96 and GFP-V96 were co-injected into single-cell zebrafish
embryos. After 4 to 5 hours, blastula embryos were incubated above and below the T
t
of V96,
fixed, and then imaged. Figure 19b reveals that at 23 °C, both RFP-V96 and GFP-V96 remain
soluble within the single cells of the embryo. However, at 30 °C both constructs form distinctive
microdomains which readily colocalize with each other. This result is consistent with previous in
vitro results from the MacKay group and serves as proof-of-principle that ELP mediated self-
assembly can be used to co-assemble different proteins within a multicellular organism.
61
Figure 19. ELPs can co-assemble different proteins in vivo. a) Schematic of triggered co-
assembly of fluorescent ELP fusions in a zebrafish embryo. GFP-V96 and RFP-V96 are
soluble throughout the cytosol below T
t
and co-assemble into mixed microdomains above the
T
t
. b) Confocal microscopy imaging of GFP-V96 and RFP-V96 in zebrafish embryos. Fish
embryos were co-injected with mRNAs encoding GFP-V96 and RFP-V96 at the one-cell
stage and then incubated either below (23 °C) or above (30 °C) T
t
until fixation at blastula
stages (4~5 hpf).
62
3.4.3 ELP self-assembly temperature can be tuned in vivo
Prior studies have shown that the in vitro T
t
can be fine-tuned by varying ELP length and
concentration (Janib et al. 2014). To examine this effect in vivo, GFP-V60, which contains 60
repeats of VPGVG, was compared with GFP-V96. GFP alone served as a non-switchable
negative control. Given that mRNA injection amount was the only significant predictor of
protein expression level, fish embryos were injected with equimolar amounts of mRNA
(GFP:150 pg, GFP-V60: 300 pg, GFP-V96: 400 pg) and incubated at various temperatures for 30
mins prior to overnight fixation at the same temperature. At 23 °C, nearly all GFP-V96 remains
soluble, while at temperatures above 28 °C there is extensive GFP-V96 self-assembly (Figure
20). In contrast, fish embryos injected with GFP-V60 mRNA didn’t show any microdomain
assembly until heated up to 38 °C, suggesting that the in vivo T
t
increased with decreased
polymer length. In addition, the effect of ELP concentration on the T
t
was examined by injecting
different amounts of mRNAs encoding GFP-V96 (Figure 21a). The change of in vitro T
t
as a
function of ELP concentration was modeled in Figures 18d and 21b, showing that increased
ELP concentrations result in a decreased transition temperature. Similarly, the in vivo T
t
increased ~ 5 °C by 5-fold dilution in mRNA concentrations (Figure 21c).
63
Figure 20. ELP self-assembly temperature can be tuned by varying molecular weight
both in vitro and in vivo. Temperature-dependent microdomain assembly was characterized
for GFP-V96 (400 pg mRNAs) and GFP-V60 (300 pg mRNAs). GFP (150 pg mRNAs)
served as a non-switchable control. a) Zebrafish embryos expressing different proteins at late
blastula stages (4~5 hfp) were incubated at different temperatures, fixed and imaged using a
Zeiss confocal microscope. *indicates microdomain assembly observed. b) Representative in
vitro optical density profiles for GFP-V60 and GFP-V96 at 25 µM as a function of
temperature. c) Quantification of the in vivo particle number per field as a function of
temperature for GFP-V60 and GFP-V96. Mean ± 95% confidence interval (n³3).
64
Figure 21. ELP self-assembly temperature can be tuned by varying concentrations of
ELPs both in vitro and in vivo. Temperature-dependent microdomain assembly was
characterized by injecting different amounts (80pg, 400pg, 2000pg) of mRNAs encoding
GFP-V96. a) Zebrafish embryos at late blastula stages (4~5 hfp) were incubated at different
temperatures, fixed and imaged using a Zeiss confocal microscope. *indicates microdomain
assembly observed. b) Representative in vitro optical density profiles for GFP-V96 at various
concentrations (5 µM, 25 µM, 75 µM) as a function of temperature. f) Quantification of the in
vivo particle number per field as a function of temperature for embryos injected with different
amounts (80pg, 400pg, 2000pg) of mRNAs encoding GFP-V96. Mean ± 95% confidence
interval (n³3).
65
3.4.4 ELPs can be tuned for short- or long-term microdomain assembly in vivo
Having demonstrated the temperature-dependent tunability of ELP-mediated
microdomain assembly in zebrafish embryos, I next explored the duration of assembly for
mRNA-encoded ELPs. Since mRNA is injected into the embryo immediately after fertilization,
the levels of mRNA are expected to decrease during the development. Eventually, this will
reduce protein expression too low to promote assembly at a physiological temperature. Based on
this, fusions with phase transition temperatures below 28 °C (Figure 18d) GFP-V96 and GFP-SI
were compared. GFP-SI has a similar molecular weight to GFP-V96; however, it phase separates
at lower temperature (Figure 18d). In vivo both GFP-SI and GFP-V96 exhibit significant
microdomain assembly at physiological temperatures at late blastula stages (4~5 hpf), with GFP-
SI forming microdomains (median area = 0.146 µm
2
) slightly smaller than GFP-V96 (median
area = 0.341 µm
2
) (Figure 22a, b). Once fish developed to prim-5 (24 hpf) or long-pec (48 hpf)
stages, microdomain assembly at 28 °C was only observed in fish expressing GFP-SI, but not
GFP-V96 (Figure 22c, d). These results are consistent with GFP-SI having a lower in vivo T
t
than GFP-V96. They also suggest that by selecting constructs with low transition temperatures,
microdomain assembly can be retained in zebrafish embryos over periods of at least days despite
the loss and dilution of the encoding mRNA. Finally, all constructs were compared for their
effect on embryo survival at 48 hours, which show that ELP-mediated phase separation alone
induces no significant loss of viability (Figure 23). In addition, none of the ELP fusions
produced gross developmental changes, which suggests that ELP fusions are well tolerated in
zebrafish embryos.
66
Figure 22. ELPs can be tuned for short- or long-term microdomain assembly. a) Short-
term microdomain assembly at physiological temperatures was observed in both GFP-SI and
GFP-V96. Fish embryos injected with 1000 pg of mRNAs encoding either GFP-SI or GFP-
V96 were fixed at late blastula stages (4~5 hpf) and imaged using a confocal microscope. b)
Quantification of microdomain areas inside of fixed embryos at late blastula stages. GFP-SI
(23 embryos, 6692 total particles, median area = 0.146 µm
2
). GFP-V96 (25 embryos, 12078
total particles, median area = 0.316 µm
2
). p value for difference in median particle area = 3e-
07. c) Long-term microdomain assembly at physiological temperatures was observed in GFP-
SI, but not GFP-V96. Live fish embryos expressing GFP-SI or GFP-V96 were visualized
using a dissecting scope at 24 hpf. d) Quantification of particle number per fish at 24 and 48
hpf, showing that GFP-SI assembled significantly more particles than GFP-V96 at longer
time periods. Mean ± 95% confidence interval (n=3 to 6): 24 hpf p-value = 0.03, 48 hpf p-
value = 0.009.
67
Figure 23. ELPs do not affect embryonic survival and development of zebrafish. a)
Representative images of zebrafish embryos expressing different ELPs at 24 and 48 hpf. b)
Survival rate of zebrafish embryos with different treatments at 48 hpf. Mean ± 95%
confidence interval (n=3, 20 to 50 fish embryos were evaluated within in each experiment).
68
3.5 Conclusion
In summary, for the first time I report a temperature-dependent strategy for controlled
protein assembly in a vertebrate embryo. This approach is rapid, reversible, and highly tunable.
In addition, it is biocompatible and can be used to assemble one or more functional protein
species together inside the single cells of a multicellular organism. In future work, this approach
may aid developmental biology studies by allowing tunable assembly of functional proteins
inside of zebrafish embryos.
69
CHAPTER 4
Multimeric peptide-polymer enables protection of RPE cells against oxidative
stress
4.1 Abstract
Humanin (HN), a 24-amino acid peptide derived from mitochondria, has been shown to
have strong cytoprotective actions against various stress and diseases models. For example, our
recent studies demonstrated that humanin protects human retinal pigment epithelium (RPE) cells
from oxidative stress-induced cell death, which may have therapeutic potential in age-related
macular degeneration (AMD). However, as a small peptide, it is poorly suited for drug
development due to its rapid clearance and degradation in vivo. Herein, I describe a genetically
encodable peptide delivery system that seeks to prolong drug retention by increasing the
hydrodynamic radius and molecular weight without altering the peptide activity. In this system,
the peptide was recombinantly fused with two elastin-like polypeptides (ELPs) that assemble
distinct structures (soluble vs insoluble) at body temperature. Dynamic light scattering revealed
that the soluble fusion self-assembled into multimers with a hydrodynamic radius of 39.1 nm at
37°C whereas the insoluble fusion formed large coacervates at the same temperature. The
potential pharmacology of these fusions was further evaluated in vitro with RPE cells under
oxidative stress, which showed reduced cell death and caspase-3 activation compared to cells
treated with carrier alone. These findings suggest that humanin peptide fused to ELPs may be
useful treatment for age-related diseases such as AMD.
70
4.2 Introduction
Mitochondria are key players in aging and in the pathogenesis of age-related diseases
(Hur, Cho, and Walker 2010; Edgar and Trifunovic 2009; Marcinek and Siegel 2013; Nunnari
and Suomalainen 2012). Recent studies on the mitochondrial genome revealed the existence of a
family of polypeptides encoded in distinct open reading frames within the mitochondria DNA
(Mercer et al. 2011). Humanin, a 24-amino acid peptide, was the first mitochondrial-derived
peptide (MDP) to be discovered and has been found to play a diverse role in many biological
processes, such as apoptosis, substrate metabolism, inflammatory and cellular stress responses
(Cobb et al. 2016; Yen et al. 2013; Muzumdar et al. 2009). Therefore, humanin has become an
attractive target for development as a therapeutic. More recently, our team showed that humanin
and its receptors are expressed in human retinal pigment epithelium (RPE) cells (Sreekumar et al.
2016). Exogenous treatment of humanin protects RPE cells from oxidative stress-induced cell
death, a possible cause of age-related macular degeneration (AMD), suggesting its therapeutic
potential for AMD, especially dry AMD. However, a major barrier to its therapeutic
development is the rapid clearance and degradation in vivo.
To circumvent his problem, I proposed a delivery system by fusing small peptide onto a
large molecular weight protein polymer to provide prolonged retention. Composed of repeated
motifs inspired by natural polypeptide sequences, protein polymers offer several advantages of
being biocompatible, biodegradable and genetically encodable (Shah et al. 2012b). Elastin-like
polypeptides (ELPs) are one class of protein polymers that have been shown to increase the half-
life of peptides both systemically and locally (Gilroy, Roberts, and Chilkoti 2018; Sreekumar et
al. 2018). The canonical ELP unit consists of a pentameric repeat of (Val-Pro-Gly-X
aa
-Gly)
n,
71
where X
aa
can be any amino acid and n determines the molecular weight. A primary aspect of
ELPs involves their ability to phase separate above a transition temperature (T
t
), which can be
tuned through the selection of guest residue X
aa
and the number of repeats n. Taking advantage of
this property, humanin peptide has been engineered onto two ELPs that assemble distinct
structures (soluble vs. insoluble) at physiological temperature. Similar to their parent ELPs, the
addition of humanin retains temperature-dependent phase separation and mediates the assembly
of nanoparticles even below the phase transition temperature. Furthermore, exogenous humanin
fusions confer a protective effect on RPE cells against oxidative stress-induced apoptosis by
inhibiting caspase-3 activation.
72
Table 3. Recombinant protein polymers evaluated in the study.
Protein
label
Amino acid
sequence
T
t
[°C]
a
Property
at 37 °C
Expected
MW
[kD]
b
Observed
MW
[kD]
c
Intercept, b
[°C]
d
Slope, m
[°C] [log
10
(µM)]
-1
S96
G(VPGSG)
96
Y
57.7 Soluble 38.39 38.35
62.8
[61.6 to 64.0]
3.886
[3.0 to 4.73]
HN-
S96
GMAPRGFSCLLL
LTSEIDLPVKRRA
G(VPGSG)
96
Y
81.1 Nanoparticle 41.12 41.09
87.6
[81.9 to 93.4]
6.097
[2.1 to 10.1]
V96
G(VPGVG)
96
Y
30.8 Coacervate 39.55 39.53
35.7
[35.2 to 36.2]
3.5
[3.1 to 3.8]
HN-
V96
GMAPRGFSCLLL
LTSEIDLPVKRRA
G(VPGVG)
96
Y
22.4 Coacervate 42.28 42.31
23.6
[23.5 to 23.8]
0.9
[0.8 to 1.0]
a)
The observed transition temperature at 25 µM in PBS.
b)
Estimated from open reading frame excluding start codon.
c)
Measured by MALDI-TOF.
d)
The Intercept b and slope m, were derived from the log-linear regression analysis for transition
temperature vs. concentration (Figure 25) fit to the Eq. 3. Data represent the mean [95%
confidence interval]
73
4.3 Materials and Methods
4.3.1 ELP gene design and construction
The pET25b(+) vectors containing genes for ELPs (V96/S96) were constructed by
recursive directional ligation as described previously. A DNA sequence encoding humanin
peptide was optimized using the best E.coli codons in EditSeq (DNAStar Lasergene) and cloned
into the pET25b(+) vector via the BseRI restriction site. Success cloning of the fusion proteins
was confirmed by DNA sequencing (Retrogen, San Diego, CA).
4.3.2 ELP expression and purification
Plain ELPs and the humanin ELPs were expressed in ClearColi BL21(DE3)
electrocompetent cells (60810, Lucigen) following manufacture’s protocol and purified using the
inverse transition cycling (ITC). To determine the concentration, purified protein in PBS was
mixed with the same volume of 6M Guanidine hydrochloride and assayed for absorbance using
Nanodrop (Thermofisher Scientific Inc., Rockford, IL). The final concentration is calculated
using Beer Lambert’s law:
𝑀 =
.
/01
2.
341
× 789:;8<% =>?;<#
@AB × 9
Eq. 2
where M is the molar concentration, A
280
and A
350
are absorbance at 280 and 350 nm
respectively, l is the light path length in centimeters and MEC (molecular extinction coefficient)
is the estimated molar extinction coefficient at 280nm (1285 M
-1
cm
-1
for plain ELPs and 1410
M
-1
cm
-1
for fusion ELPs). Protein purity and identity were further determined by SDS-PAGE gel
(Lonza) stained with GelCode Blue Safe Protein Stain (Thermofisher Scientific Inc., Rockford,
IL) and western blot probed with a humanin antibody. Protein molecular weight was confirmed
74
by MALDI-TOF analysis. The endotoxicity was also verified to be less than 6 EU/mL using
HEK-Blue hTLR4 cell line from InvivoGen (San Diego, CA)
4.3.3 Characterization of ELP phase behavior
The phase behaviors of ELPs were characterized by measuring the optical density as a
function of temperature. ELPs in PBS with concentrations ranging from 5 to 100 µM were
loaded in thermal mount microcells and measured for absorbance at 350 nm using a DU800 UV
visible spectrometer (Beckman Coulter) while the temperature was ramped from 10 °C to 80 °C
at a constant rate of 1 °C/min. The maximum first derivative of the optical density vs.
temperature curve was defined as the transition temperature T
t
. The data points were then fit to
the following equation:
𝑇
;
=𝑏− 𝑚 Log
JK
𝐶
MNO
Eq. 3
where C
ELP
is the ELP concentration, m is the slope and b is the intercept transition temperature
at 1 µM.
4.3.4 Characterization of ELP assembly
To characterize the self-assembly of ELPs, hydrodynamic radius (R
h
) was monitored
using dynamic light scattering. Briefly, 25 µM ELPs in PBS were filtered through 0.2 µm Supor
membrane filters (Pall laboratory) and measured using a DynaPro plate reader (Wyatt
Technology Inc., Santa Barbara, CA) over a range of temperatures from 10 °C to 40 °C in 1 °C
increment. The results were analyzed using a Rayleigh sphere model and fitted into a
regularization algorithm.
75
4.3.5 Transmission electron microscopy (TEM) imaging
The TEM imaging was carried out on a JEM-2100 Transmission Electron Microscope
(JEOL USA, Peabody, MA) at 80 kV. Samples were prepared by mixing a 25µM HN-S96
solution with same volume of 2% uranyl acetate and then deposited on a gold grid. Excess
amount of solution was removed by filter paper. Grids were dried at room temperature for 30
min prior to imaging.
4.3.6 Protection of RPE cells from oxidative stress
The anti-apoptotic activity of HN-ELPs were evaluated using TUNEL staining and
immunofluorescent detection of cleaved caspase-3 in human RPE cells challenged with tert-
Butyl Hydroperoxide (tBH) as previously reported (Sreekumar et al. 2016). Briefly, confluent
human RPE cells grown on 4-well chamber slides were co-treated with varying doses (0.5-10
µM) of HN-ELPs and 150 µM tBH or 150 µM tBH alone for 24 hours in serum-free medium.
For TUNEL staining, cell death was accessed using an In Situ Cell Detection Kit (Roche Applied
Science, Indianapolis, IN) and nucleus was stained with DAPI. The TUNEL-positive cells were
then counted and data were presented as percent of total cells undergoing cell death. Instead,
activated caspase-3 was detected using a rabbit antibody against cleaved caspase-3 (#9661, Cell
Signaling Technology, Danvers, MA) followed by a fluorescein-conjugated anti-rabbit
secondary antibody. Images were obtained using a Keyence fluorescence digital microscope
(Keyence, Itasca, IL).
76
4.3.7 Statistics
Data presented are representative curves or mean ± SD. All experiments were replicated
at least three times. Statistical analysis was performed using either a non-paired t-test or one-way
ANOVA followed by Tukey’s post hoc test. A p value less than 0.05 was considered statistically
significant.
4.4 Results
4.4.1 Purification of ELPs fused to humanin peptide
Two ELP carriers were evaluated in this study for the humanin peptide (Table 3, Figure
24a). Both V96 and S96 have 96 pentameric repeat units, however, vary in their guest residue X.
Therefore, it is expected that these two fusions result in different transition temperatures (Janib et
al. 2014). Purification of all four ELPs were conducted using inverse transition cycling (ITC),
yielding ~40 mg proteins per liter culture. Purified materials were further characterized for their
purity and identity using SDS-PAGE and western blot (Figure 24b, c). Each of the fusion
proteins appears as a major band around 40 kDa, which corresponds to the predicted and
observed molecular weight as measured by MALDI-TOF (Table 3).
77
Figure 24. Construction and purification of Humanin (HN) ELP fusions. a) Schematic
showing the plain ELPs and fusion ELPs. HN peptide was expressed at the N-terminus of
ELPs. b) SDS-PAGE stained with coomassie blue was used to determine the purity and
molecular weight of purified protein polymers (Table 3). c) Western blot probed with an anti-
HN antibody was used to further confirm the identity of HN fusions.
78
4.4.2 Humanin peptide shifts the phase diagram differently for V96 and S96
To characterize the phase behavior of ELP fusions, optical density was measured as a
function of temperature over a concentration range (5, 10, 25, 50, 100 µM) (Figure 25 a-d).
ELPs are soluble and have low optical density when below their transition temperature (T
t
).
Once heated above their T
t
, ELPs become insoluble and form coacervates. As observed before,
concentration-temperature phase diagrams for all four constructs follow a log-linear relationship
(Figure 25e, f, Table 3). V96, which contains a relatively hydrophobic guest residue valine,
phase separates above 30 °C. The addition of humanin decreases the transition temperature to
~22 °C. In contrast, S96 phase separates between 50 °C and 63 °C but humanin fusion increases
the transition temperature to above 70 °C. Although the fusion proteins behave differently in
terms of shifting the phase diagram, HN-S96 and HN-V96 remain good candidates as soluble
and insoluble formulations during intra-vitreal administration or in vitro characterization at
physiological temperatures.
79
Figure 25. Fusion of HN peptide changes the ELP transition temperature. Two ELPs
with same molecular weight but varied transition temperature (V96 and S96) were fused to
HN and characterized for optical density as a function of temperature and concentration. a)
Plain V96 phase separated below physiological temperature (~30 °C). b) Fusion of HN to
V96 decreased the phase transition temperature to ~22 °C. d) Plain S96 phase separated
above physiological temperature (~60 °C). e) In contrast, addition of HN to S96 increased its
transition temperature to ~80 °C. c) and f) Concentration-temperature phase diagrams for all
proteins followed a log-linear relationship. Lines indicate the fit of T
t
to Eq.3 (Table 3) with
95% prediction bands.
80
4.4.3 Humanin peptide mediates the assembly of ELP-stabilized nanoparticles
While optical density is useful in determining phase separation temperature, dynamic
light scattering (DLS) is necessary to confirm the size of ELPs before and after transition. As
shown in Figure 26, both V96 and S96 remained monomeric and showed a hydrodynamic radius
of 5 nm when below their transition temperatures, whereas the addition of humanin significantly
increased their radius to ~40 nm. Further investigation using regularization analysis confirmed
that both HN-V96 and HN-S96 assembled a significant population of multimers with relatively
high polydispersity at 15 °C (23.8 %) and 37 °C (20.9 %) , respectively. Since V96 and S96
themselves do not assemble structure until above T
t
, the increase in hydrodynamic radius suggest
that humanin peptide alone mediates the multimeric assembly.
The morphology of these nanostructures was next investigated using transmission
electron microscopy (TEM) (Figure 27). Negative contrast images of HN-S96 confirmed the
polydispersity and revealed the presence of spherical structures, which are in agreement with the
DLS data (Figure 26). Taken together, our results suggest that humanin peptide promotes the
assembly of the fused ELPs into spherical vehicles.
81
Figure 26. Fusion to HN peptide mediates the assembly of ELP nanoparticles. The
hydrodynamic radius (R
h
) of ELPs were measured as a function of temperature at a
concentration of 25 µM. a) and b) Cumulative fit suggests both HN-V96 and HN-S96 have a
larger radius than their parent ELP V96 and S96 even below transition temperature. c) and d)
Regularization analysis indicates both HN-V96 and HN-S96 assemble a significant
population of multimers when below transition temperature (c, 20 °C; d, 37 °C).
82
Figure 27. HN-ELP fusions assemble nanoparticles below transition temperature. TEM
imaging was used to characterize the morphology of assembled HN-S96 multimers.
Representative images of negatively stained samples revealed the evidence of nanoparticles
with an average radius of 26 ± 6 nm. Scale bar: 50 nm.
83
4.4.4 Exogenous HN-V96 protects RPE cells from oxidative-stress induced apoptosis
Having established that the HN-ELP fusions assemble into multimeric particles, it was
unclear whether the peptide would remain accessible to cellular targets. To evaluate this
possibility, the anti-apoptotic properties were assessed using human retinal pigment epithelium
(RPE) cells as an in vitro model. RPE cells are particularly sensitive to oxidative stress and
cumulative oxidative stress-induced RPE apoptosis is known to be a major factor in the etiology
of age-related macular degeneration (AMD) (Cai et al. 2000; Plafker, O'Mealey, and Szweda
2012). Therefore, confluent RPE cultures were challenged with 150 µM tert-Butyl
Hydroperoxide (tBH) and co-treated with varying concentrations of HN-V96 for 24 hours
(Figure 28). The TUNEL assay was used to identify apoptotic cells. The data revealed a dose-
dependent cell protection; furthermore, only a high concentration of HN-V96 (10 µM)
significantly protected RPE cells against oxidative stress induced cell death (p<0.001). The effect
of plain ELP was also examined in another study showing that same amount of V96 failed to
show protective activities (Figure 29). In addition, oxidative stress induced activation of
caspase-3 was reduced in HN-V96 compared to V96 alone (Figure 30). Collectively, these
results demonstrate that ELPs fused to humanin peptide retain cytoprotective activities and
protect RPE cells from oxidative-stress induced apoptosis.
84
Figure 28. Exogenous HN-V96 protects RPE cells from oxidative stress-induced cell
death in a dose dependent manner. Human RPE cells were challenged with 150 µM tBH
and treated with varying doses of HN-V96 (0.5, 5, 10µM) for 24 h. Cell death was assessed
using TUNEL stain. a) tBH challenge resulted in cell death and only high dose (10 µM) of
HN-V96 offered protection to cells. Blue: DAPI. Scale bar: 50 µm. b) Quantification of the
TUNEL-positive cells. *p<0.015, **p<0.01, ***p<0.001.
85
Figure 29. Exogenous HN-V96, not V96 protects RPE cells from oxidative stress-induced
cell death. Human RPE cells were challenged with 150 µM tBH and treated with or without
ELPs for 24 h. Cell death was assessed using TUNEL stain. Representative confocal images
showed that TUNEL-positive cells (red) were reduced when cells were co-treated with 10 µM
HN-V96. However, V96 failed to protect cells from apoptosis. Blue: DAPI. Scale bar: 50 µm.
86
Figure 30. Exogenous HN-V96, not V96 inhibits oxidative stress-induced caspase-3
activation. Human RPE cells were challenged with 150 µM tBH and treated with or without
ELPs for 24 h. The activation of caspase-3 (green) was assessed using indirect
immunofluorescence. Representative confocal images showed that tBH induced caspase-3
activation was only inhibited in cells treated with 10 µM HN-V96. Blue: DAPI. Scale bar: 50
µm.
87
4.5 Discussion and future directions
Age-related macular degeneration (AMD) is the leading cause of severe and irreversible
vision loss among aging populations worldwide. Thought to be a primary cause of AMD,
oxidative stress is associated with RPE cell loss and photoreceptor degeneration. Recent
discovery of humanin, a mitochondrial-derived peptide, has created a new category of
biologically active peptides with protective effects against oxidative stress. As more research
elucidates its physiological role, humanin has emerged as a potential therapeutic for treating
human diseases.
To date, studies in multiple disease models reveal that administration of humanin
prevents from polyglutamine toxicity (Kariya et al. 2005), stroke (Xu et al. 2006), myocardial
ischemia-reperfusion injury (Muzumdar et al. 2010) and Alzheimer’s disease-related stress
(Mamiya and Ukai 2001; Tajima et al. 2005; Niikura et al. 2011; Zhang et al. 2012). More
recently, Sreekumar et al. reported that humanin is also sufficient to protect human RPE cells
from oxidative stress-induced apoptosis, suggesting its possible use as a cytoprotective agent
against AMD. Unfortunately, the main drawback of peptide drug is its rapid clearance and
biodegradation in vivo (Chin et al. 2013). Multiple and frequent dosing of humanin is required to
achieve substantial clinical effects, however, is not clinically applicable for intravitreal
administration and may cause low compliance.
Therefore, in this chapter I genetically engineered the humanin peptide with high
molecular weight ELP protein polymers that have the potential to modulate its retention and
bioactivity. Two ELPs were evaluated in this study, including a temperature sensitive V96 which
88
forms coacervates at 37 °C and a soluble S96 with no significant assembly at physiological
temperatures. Similar to their parent ELPs, both HN-S96 and HN-V96 exhibit temperature
dependent phase separation (Figure 25). Although the addition of humanin shifts the phase
diagram differently for V96 and S96, their proposed properties (soluble vs insoluble) at body
temperature remain the same. Surprisingly, humanin, a 24-amino acid peptide, mediates the self-
assembly of ELP nanoparticles even below transition temperature. DLS measurements indicate
that both HN-ELP fusions form multimers that are stable up to their own T
t
(Figure 26). The
structure of these multimers was further confirmed by TEM (Figure 27), however, additional
studies are needed to investigate the nature of the assembly.
These observations also raised the question of whether the assembly influences its
biological activity. Thus, the cytoprotective function of HN-V96 was assessed using an in vitro
cell model. Human RPE cells undergo apoptosis with mild oxidative stress when tBH reaches a
toxic dose. Similar to the previous studies of humanin, treatment of HN-V96 shows a dose-
dependent cell protection by inhibiting caspase-3 activation (Figures 28, 29, 30) (Sreekumar et
al. 2016). It should be noted that the minimum effective dose of HN-V96 is 10 µM, which is ~3
fold higher than what has been reported before for humanin (10 µg/mL, equivalent to 3.7 µM).
This indicates that the involvement of humanin peptide in the particle assembly affects its ability
to act on the biological targets responsible for the anti-apoptotic activity.
Humanin is known to exert its neuroprotective function through different pathways by
binding to either extracellular receptors or intracellular targets. (Lee, Yen, and Cohen 2013;
Gong, Tas, and Muzumdar 2014). Sreekumar et al. demonstrated that humanin protection of RPE
89
cells from oxidative stress is mediated via dual mechanisms: 1) enhancing mitochondria function
through an intracellular pathway; 2) activating humanin receptors through an extracellular
pathway (Sreekumar et al. 2016). Given the fact that HN-ELP fusions assemble into multimeric
particles, they may work differently from the free peptide. Therefore, investigation of the
intracellular trafficking and signaling activation are necessary for understanding their
mechanisms of action and further evaluation in vivo.
90
CHAPTER 5
Pharmacokinetics of intra-vitreal αB crystallin fragment fused to an elastin-
like polypeptide
5.1 Abstract
The purpose of this study was to characterize the ocular pharmacokinetics of a
neuroprotective peptide fused to elastin-like polypeptide (ELP) following intra-vitreal
administration to mice. αB crystallin is an abundant ocular protein that maintains ocular clarity
and retinal homeostasis, and a small peptide from this protein (mini cry) displays neuroprotective
properties which may have applications as therapeutics for age-related macular degeneration
(AMD). To retain this peptide for longer in the vitreous, mini cry was fused to an elastin-like
polypeptide (crySI). While mini cry is cleared from the eye with a mean residence time of 0.4
days, crySI was retained with a mean residence time of 3.0 days; furthermore, fundus
photography revealed evidence of retention at two weeks. Given these and our previous in vitro
characterization that crySI protects RPE cells from oxidative stress induced apoptosis, I envision
that intravitreal administration of crySI may have the potential to prevent RPE atrophy and
progressive retinal degeneration in AMD.
5.2 Introduction
Age-related macular degeneration (AMD), the leading cause of irreversible visual loss in
the elderly, is complicated by two blinding late forms of the disease: choroidal
neovascularization ((CNV) and geographic atrophy (GA) or atrophic AMD. GA is an advanced
form of dry AMD with extensive atrophy and loss of the retinal pigment epithelium (RPE) and
91
overlying photoreceptors and is responsible for 10–20% of cases of legal blindness from AMD
(Bird 2010; Klein et al. 2008). There are effective treatments for complications developing from
neovascular AMD, but there is neither a treatment of the atrophic form of AMD nor effective
preventive strategies against progression to the neovascular form of AMD (Wright and Ambati
2016). Therefore, development of effective protective drugs against atrophic AMD is greatly
needed.
Within the family of small heat shock proteins (sHSPs), the α-crystallins possess multiple
functions (Kannan, Sreekumar, and Hinton 2012). Among the α-crystallins, αB crystallin is
highly expressed in RPE cells (Yaung et al. 2007). αB crystallin acts as a molecular chaperone,
preventing aggregation of proteins and inhibiting oxidative stress-induced cell death and
disruption of cytoskeletal assembly. It also inhibits inflammation and provides neuroprotection
(Kannan, Sreekumar, and Hinton 2012). Exogenous administration of full length αB crystallin to
mice ameliorates neuroinflammation (Masilamoni et al. 2006; van Noort et al. 2013),
autoimmune encephalomyelitis (Ousman et al. 2007), optic neuropathy (Pangratz-Fuehrer et al.
2011), ischemia-reperfusion injury (Velotta et al. 2011), stroke (Arac et al. 2011), and acute
spinal cord injury (Klopstein et al. 2012). The fundamental properties of several individual
peptides of α-crystallin have been the extensive work of the laboratories of Sharma (Raju,
Santhoshkumar, and Sharma 2016; Bhattacharyya et al. 2006) and Clark (Clark 2016; Ghosh,
Estrada, and Clark 2005; Ghosh, Houck, and Clark 2007). These studies reveal that short
peptide fragments from the intact protein have anti-apoptotic activity and several binding
partners (Clark 2016). In particular, a 20-mer peptide (mini cry) derived from the amino acid
residues 73-92 of αB crystallin protects RPE cells from oxidative stress induced cell death by
92
inhibiting caspase-3 activation (Sreekumar et al. 2013). Kurnellas et al. showed that mini cry
binds inflammatory mediators in plasma thereby reducing paralysis in experimental autoimmune
encephalomyelitis (Kurnellas et al. 2012). Compared to the full-length protein, the mini cry
peptide not only maintains its bioactivity but also offers advantages including the relative ease of
crossing tissue barriers, providing active doses, lower production complexity and less side effects
(Fosgerau and Hoffmann 2015). An intrinsic weakness, however, of using low molecular weight
peptides is that they tend to be cleared rapidly from the ocular compartments, demanding
repeated injections which could have serious complications such as vitreous hemorrhage, and
retinal detachment (Kim and Csaky 2010; Ghosh et al. 2017).
To avoid difficulties encountered with intra-ocular delivery and rapid turnover of low
molecular weight peptides (Li et al. 2006; Borhani et al. 1995; Bisht and Rupenthal 2016), in our
previous study mini cry was recombinantly fused with a high molecular weight elastin-like
polypeptide (ELP) called SI, which assembles multivalent nanoparticles at physiological
temperature (Table 4) (Wang et al. 2014). ELPs are biopolymers derived from a structural motif
found in the mammalian tropoelastin protein, which phase separate above tunable transition
temperatures. Below this temperature they maintain high solubility and tissue permeability.
Above the transition temperature, they form a viscous secondary aqueous phase that can enhance
tissue retention (Roberts, Dzuricky, and Chilkoti 2015; Gilroy, Luginbuhl, and Chilkoti 2016).
ELPs are genetically encoded; therefore, their DNA coding sequence was first modified to also
encode residues 73−92 of the αB crystallin sequence. Thus, a genetically modified cytoprotective
polypeptide composed of ELP and the mini cry peptide (crySI) was expressed and purified
from Escherichia coli. Our earlier in vitro work established that crySI ELPs have chaperone
93
activity, enter human RPE cells and protect them from oxidative stress induced cell death by
inhibiting caspase-3 activation (Wang et al. 2014). To further examine the validity of this drug-
delivery strategy, this chapter characterizes the prolonged retention of crySI in the ocular
compartment using ocular pharmacokinetics and fundus photography.
94
Table 4. Nomenclature, sequence, molecular weight and purity of mini cry and ELP fusion
proteins.
Peptide
label
Amino acid Sequence M.W. [kD] Purity [%]
mini cry DRFSVNLDVKHFSPEELKVK 2.4
a
98.1
c
SI G(VPGSG)
48
(VPGIG)
48
Y 39.7
b
99.2
d
crySI GDRFSVNLDVKHFSPEELKVKG(VPGSG)
48
(VPGIG)
48
Y 42.1
b
88.3
d
a)
Chemically synthesized from amino acids 73-92 of the human αB crystallin protein.
b)
Expected M.W. based on the open reading frame, excluding a methionine start codon.
c)
Purity estimated from RP-HPLC.
d)
Purity estimated from SDS-PAGE.
95
5.3 Materials and Methods
5.3.1 Materials and reagents
Fluorescein-labeled mini cry (NH2-DRFSVNLDVKHFSPEELKVK(FITC)-COOH,
M.W.=2.777 kD, 98.6% purity) were chemically synthesized by Neo-peptide (Cambridge, MA).
Purified ELP was labeled with NHS-fluorescein (#46410, ThermoFisher Scientific Inc.,
Rockford, IL).
5.3.2 ELP expression and purification
ELPs were expressed in BLR (DE3) Escherichia coli chemically competent cells
(Novagen Inc., Milwaukee, WI) and purified via inverse transition cycling (ITC) as previously
reported (Wang et al. 2014). Purity was assessed by running 10 µg of polymer on a 4-20% SDS-
PAGE gel stained with 10% copper chloride. Unlabeled ELP concentrations were determined by
UV-Visible spectroscopy at 280 nm (e
ELP
= 1,285 M
-1
cm
-1
). For preparations intended for in vivo
evaluation, additional removal of endotoxin was required. Detoxi-gel chromatography
(ThermoFisher Scientific Inc., Rockford, IL) was used to reduce endotoxin on a small scale for
volumes of 0.5 mL, and the Mustang Acrodisc syringe filter (Pall Corporation, Port Washington,
NY) were used to clear protein samples on a larger scale of volumes of 10 mL. Endotoxin burden
was estimated using a Pryogent Gel Clot assay (N289-06, Lonza, Walkersville, MD). The
endotoxin level of SI and crySI injected into the mouse vitreous was confirmed to be lower than
60 EU/mL (lower than 0.1 EU/eye) based on a negative test result for a 1:1000 dilution into PBS
(sensitivity = 0.06 EU/mL).
96
5.3.3 Fluorescent labeling of ELPs
For fluorescent visualization, SI (control ELP) and crySI were covalently modified with
NHS-Fluorescein (Thermo Fisher Scientific Inc., Rockford, IL) by conjugation to free amines.
Briefly, crySI was mixed with a 3-fold molar excess of NHS-Fluorescein in phosphate buffered
saline (PBS) and incubated overnight at 4 °C. Free fluorophore was removed by size exclusion
chromatography using a PD-10 desalting column (GE Healthcare, Piscataway, NJ). The
concentration of label after the purification, C
FL-crySI
, was estimated as follows:
𝐶
PQ2?#RST
=
.
UV3 WX
Y
Z,[+\
Eq. 4
where the molar extinction coefficient of fluorescein, e
fluor
, was assumed to be 70,000 M
-1
cm
-1
.
Due to the low molar extinction coefficient of the crySI relative to the contribution of fluorescein
at an optical absorbance of 280 nm, the degree of labeling, N
labeling
, was estimated as follows:
𝑁
9>^$98%"
=
%
_`a*\bcd,e[\(Z(fg
%
*\bcd,\f-*hfg
Eq. 5
where n
crySI,reacted
and n
FL-crySI,purified
are the moles of crySI reacted and fluorescein recovered after
purification respectively. Fluorescein labeled FL-crySI had a labeling efficiency ~0.6. To
determine the purity of labeled materials, proteins were separated on SDS-PAGE gels and
imaged on a ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Hercules, CA).
5.3.4 Color fundus photography
Mice were anesthetized by administration of ketamine and xylazine. For all animals,
pupils were dilated with one to two drops of Tropicamide Ophthalmic Solution 1% (Bausch &
Lomb, Tampa, FL) 5 minutes prior to imaging. A hydroxypropyl methylcellulose ophthalmic
demulcent solution (Gonak, Akorn Lake Forest, IL) was applied to the eye to provide a uniform
optically transparent interface between the tip of the endoscope and the cornea of the subject.
97
Images were captured using a 35 mm Kowa hand-held color fundus camera (Genesis, Tokyo,
Japan). 4-6 images were taken from each eye.
5.3.5 Quantitative analysis of fluorescence in color fundus images
To compare the intra-ocular disposition of SI, crySI and the free mini cry peptide,
fluorescein-labeled samples in PBS (2 µL, 70 µM) were injected into the vitreous as described
above and observed by color fundus photography using a bright white illumination source. The
area specifically emitting a green fluorescent signal was selected by defining the region of
interest (ROI). The fluorescent intensities within these regions were analyzed using ImageJ (US
NIH, Bethesda, MD). Briefly, captured color images were converted to RGB stacks and the
integrated intensity of each channel was measured. To correct the green signal for nonspecific
scattering of white light in each image, the intensity of the green channel was corrected for
background by the blue channel. From this corrected value, the background signal observed in
PBS-treated eyes was subtracted. This background-subtracted intensity was normalized to signal
obtained immediately after the injection of crySI (t=0) as follows:
𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑 𝐹𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑐𝑒 =
(T
w\ffW
2T
x,[f)
h
2(T
w\ffW
2T
x,[f)
z{c
(T
w\ffW
2T
x,[f)
h|1,*\bcd
2(T
w\ffW
2T
x,[f)
z{c
Eq. 6
where 𝐼
"#$$%
and 𝐼
^9:$
represents the integrated intensity of the green and blue channel,
respectively.
5.3.6 Intra-vitreal pharmacokinetics
To evaluate the pharmacokinetics of crySI relative to that of mini cry and SI, mice were
administered 100 pmol (1 µL, 100 µM) fluorescein-labeled peptide through intra-vitreal injection.
The mice were euthanized at selected time points (0.25, 1, 2.7, 8 hours, 1, 3, 6, 9 and 13 days).
98
Eyes were enucleated and immediately frozen at -80 °C with 100 µL RIPA buffer containing
protease/phosphatase inhibitor. On the last day of experiment, all samples collected previously
were thawed, homogenized using a PRO200 homogenizer (PRO Scientific Inc., Oxford, CT),
and centrifuged. Fluorescence concentration in the supernatant was determined using a calibrated
fluorescence microplate assay run on a Synergy H1 Hybrid Multi Mode Microplate Reader
(BioTek, Winooski, VT). The average background fluorescence of eyeballs injected with only
PBS was measured and subtracted from the sample data.
5.3.7 Pharmacokinetic analysis
Since both crySI and SI followed a bi-exponential elimination from the eye, the total
amount [pmoles], X
t
, of recovered fluorescence for mini cry, crySI, and SI as a function of time
[days], t, was fit to the following decay curve:
𝑋
;
=𝑋
K
𝑓
=>;
𝑒
2
Z-h
;
+𝑋
K
(1−𝑓
=>;
)𝑒
2
,+
;
Eq. 7
where f
fast
is the fraction of the dose that cleared rapidly, X
0
is the initial amount of dose detected
in the eye at time based on the fit, k
fast
is a first order rate constant fitting rapid elimination, and
k
slow
is a first order rate constant fitting slower elimination. Only one rate of decay was observed
for mini cry; therefore, f
fast
was fixed at 1. As a measure of relative ocular exposure, the AUC
(Area under the curve) and AUMC (Area under the moment curve) were estimated using the
trapezoidal rule, which was used to determine the mean residence time (MRT).
5.3.8 Data analysis
Data presented are representative curves or mean ± SD. All experiments were repeated at
least three times. Statistical analyses were performed by a student t-test and a one-way ANOVA
99
followed by Tukey’s post-hoc test using Prism (GraphPad Software, La Jolla, CA). A p value of
less than 0.05 was considered statistically significant.
5.4 Results
Unlike free mini cry, crySI assembles coacervates above ~30 °C, which raised the
possibility that it has a different disposition in the eye (Wang et al. 2014). Therefore, it is
hypothesized that ELP-mediated assembly of crySI may extend retention near the retina. To test
this hypothesis, the pharmacokinetics of crySI relative to mini cry and SI was evaluated by
injecting fluorescently-labeled samples (Table 4, Figure 31a) into the mouse vitreous and
monitoring the total fluorescence in whole, enucleated eyeballs for up to 2 weeks. Consistent
with peptides of low molecular weight (2.8 kD), labeled mini cry was undetectable in the ocular
compartment within 3 days (Figure 31b); furthermore, quantification of its removal suggests
that it cleared from the eye by a mono-exponential decay and has a mean residence time of 0.4
days (Table 5). In contrast, both crySI and SI were cleared from the eye by bi-exponential decay,
with a much slower mean residence times of 3.0 and 3.4 days, respectively. Ocular exposure to
crySI as measured by the area under curve (AUC) of the fluorescein amount was increased by 4
times from 14.5 pmol*days for mini cry to 56.2 pmol*days for crySI. To confirm differences in
ocular pharmacokinetics using a non-invasive approach, color fundus imaging was used to excite
the fluorescent dye and photograph each mouse retina over time (Figure 32a). Most of the mini
cry signal was cleared from the vitreous within one week after injection, while crySI and SI
remained easily visible in the fundus after 2 weeks (Figure 32b). Furthermore, the crySI
microparticles were observed in the vitreous above the retina using confocal imaging (Figure
100
33). Taken together, these studies indicate that ELP-mediated peptide assembly significantly
increases the duration of crySI exposure to the retina after intra-vitreal administration.
101
Figure 31. Pharmacokinetics of mini cry and crySI in mice. a) SDS-PAGE shows the
identity and purity of fluorescently labeled mini cry, crySI, and SI along with a molecular
weight marker. While the fluorescein labeled mini cry peptide electrophoreses at a higher
molecular weight than expected; its exact mass (2.78 kD) was confirmed independently by
electrospray mass spectrometry. b) The total amount of dye-labeled mini cry, crySI and SI in
the mouse eye was quantified over a 2-week window post intra-vitreal administration. Data
for mini cry (0-1 day) were fit by a single exponential decay due to its fast clearance within 3
days. Data for crySI and SI (0-13 days) were fit to a bi-exponential decay (Eq. 7). Fusion to
the ELP improves the ocular pharmacokinetics compared to mini cry. Data are expressed as
mean ± SD (n=4).
102
Figure 32. Intra-vitreal crySI is retained for long durations near the retina. a) The
retention of mini cry, crySI and SI was monitored by fundus photography following intra-
vitreal injection. The appearance of signal (green) in representative fundus images
demonstrates the prolonged ocular retention of crySI. b) The normalized fluorescence (Eq. 6)
was plotted for indicated time points. CrySI exhibited longer ocular retention with
significantly more signal remaining than mini cry after two weeks. Data are expressed as
mean ± SD (n=3-5, **p < 0.01).
103
Table 5. Ocular pharmacokinetics of mini cry and ELPs following intra-vitreal
administration to mice.
Parameter Unit
mini cry [n=4]
a
crySI [n=4]
b
SI [n=4]
b
Mean [95% Cl] Mean [95% Cl] Mean [95% Cl]
X
0
[pmol] 38.5 [29.7 to 47.3] 76.0 [49.6 to 102.5] 45.1 [27.1 to 63.0]
f
fast
1 na 0.86 [0.79 to 0.93] 0.83 [0.74 to 0.93]
k
fast
[day
-1
] 2.7 [2.2 to 3.1] 6.6 [2.7 to 10.6] 8.5 [1.9 to 15.1]
k
slow
[day
-1
] na na 0.24 [0.19 to 0.30] 0.23 [0.17 to 0.29]
t
half, fast
[days] 0.26 [0.22 to 0.32] 0.10 [0.07 to 0.26] 0.08 [0.05 to 0.37]
t
half, slow
[days] na na 2.8 [2.3 to 3.7] 3.0 [2.4 to 4.0]
AUC
c
[pmol days] 14.5 na 56.2 na 38.4 na
AUMC
c
[pmol days
2
]
6.2 na 169.0 na 130.5 na
MRT
c
days 0.4 na 3.0 na 3.4 na
a)
mini cry data (0-1 day) were fit to a mono-exponential decay equation.
b)
crySI and SI data (0-13 days) were fit to a bi-exponential decay equation.
c)
AUC, AUMC, and MRT were estimated using a non-compartmental method.
na: not applicable.
104
5.5 Discussion
This study presents a new strategy for intravitreal delivery of small peptides. Recent
therapeutic strategies for the treatment of dry AMD target inflammation and complement
activation, suppress oxidative stress, or provide neuroprotection. Since αB crystallin elicits all
these functions under multiple in vitro and in vivo patho-physiological conditions, it may be an
ideal candidate for therapy. Unfortunately, frequent dosing was required for previous in vivo pre-
clinical studies owing to the low retention time and relative bioavailability of recombinant αB
crystallin (Kurnellas et al. 2012), which is not clinically applicable for intra-vitreal
administration and may cause poor patient compliance. To overcome this critical limitation,
Hinton group previously used a synthetic polymer polycaprolactone (PCL) to encapsulate mini
cry and deliver it to cultured RPE cells, where low doses efficiently resulted in cellular
protection (Sreekumar et al. 2013). PCL has been widely used as a biomaterial due to its low
immunogenicity and high biocompatibility. However, drawbacks related to polydispersity,
encapsulation stability, burst release, and limited degradation limit the application of this
platform (Ulery, Nair, and Laurencin 2011). To overcome these limitations, our team developed
a self-assembling mini cry-ELP fusion containing the hydrophobic ELP (Val-Pro-Gly-Ile-Gly)
48
,
which modulates its nanoassembly, cellular uptake, and bioactivity (Wang et al. 2014).
Compared to PCL, ELPs are derived from human tropoelastin and can be generated via genetic
engineering and recombinant biosynthesis, which offers an exquisite level of precision and
tunability with regards to the length, molecular weight, sequence and monodispersity of the
resulting material. Most importantly, ELPs have the advantage of being thermo-responsive and
can be tailored to assemble coacervates at body temperature while maintaining the activity of
fusion proteins (Gilroy, Luginbuhl, and Chilkoti 2016; Gilroy, Roberts, and Chilkoti 2018; Janib
105
et al. 2014; Despanie et al. 2016). At physiological temperatures, the hydrophobic ELP phase
separates and forms a depot in the vitreous capsule (Figure 33), which correlates with an
increased exposure (Area Under the Curve, AUC) for the fused αB crystallin peptide compared
to that of 20-mer αB crystallin (Table 5). While the 20-mer αB crystallin alone was undetectable
after 3 days (Figure 31b), crySI was easily detectable even after 2 weeks (Figures 31b, 32). It
has been reported that the serological blood half-life for intact αB crystallin is on the order of 6
hours; however, the systemic half-life for free mini cry peptide would be much shorter and
would not guarantee access to the retina (Kurnellas et al. 2012). Concurrent with the assembly of
a nanostructure, crySI remains bioactive and retains both chaperone and anti-apoptotic functions
(Wang et al. 2014), which suggests its potential for treatment of retinal degeneration and other
inflammatory disorders. These findings corroborate other reports such as for SynB1-ELP1-Dox,
which has a longer systemic circulation than free drug (Moktan et al. 2012). In short, the
cumulative effect of improved intra-ocular pharmacokinetics, and long-term retention of crySI
compared to mini cry may support the possible efficacy of crySI to protect the retina from
oxidative stress in vivo.
106
Figure 33. Morphological characterization of crySI in phosphate buffered solution and
the mouse vitreous. a) The hydrodynamic radius of fluorescein-labeled crySI was
characterized using dynamic light scattering at 10 ºC and 37 ºC. b) Regularization analysis
enabled two populations of particles to be observed for crySI at 37 ºC with the majority
forming micron sized coacervates. c) Confocal microscopy was performed for retinal cryo-
sections obtained day 7 after mice were given intravitreal fluorescein-labeled crySI (green).
Consistent with the hydrodynamic radius observed for crySI coacervates in buffer at
physiological temperature, micron-sized fluorescent structures were observed above the
retina. Blue: DAPI. Scale bar: 100 µm.
107
5.6 Conclusion
In summary, this chapter characterizes the ocular pharmacokinetics of mini cry, SI and
crySI via intra-vitreous administration using a fluorescence-based method. Unlike the mini cry
peptide that was cleared from the ocular compartment within 3 days, crySI were retained in the
vitreous capsule for at least 2 weeks, suggesting its potential to protect against the progression of
AMD.
108
CONCLUSION AND FUTURE DIRECTIONS
In summary, the four studies described in Chapter 2 through 5 demonstrate the two major
applications of using ELPs to modulate protein/peptide assembly. As intracellular switches,
ELPs can assemble, activate and deactivate various targeted cellular processes. Future expansion
of the toolkit will not only include the application of ELPs to more cellular targets, but also the
fine tuning of key parameters in the system such as the transition temperature. The ultimate goal
would be to generate a “toolbox” in which ELPs with appropriate parameters can be selected for
individual experiments. Currently, ELP-mediated switching requires efficient gene delivery by
transient transfection, which leads to the overexpression and auto-activation of the protein
components above physiologically basal state. Recent advancements in genomic modification
using CRISPR may offer long-term expression for ELP fusion proteins at their endogenous
levels. The further adaption of these functional switches into a live animal such as zebrafish may
make this toolkit even more powerful. As peptide delivery systems, ELPs can prolong the drug
retention by modulating its hydrodynamic radius and molecular weight, yet maintaining its
biological activity. Similarly, ELPs with distinct physiochemical parameters can serve as a
“toolbox” and the suitable system can be adapted to other peptide drugs according to their
biological characteristics and optimal pharmacological profile. However, a thorough
understanding of the mechanisms of action underlying ELP-peptide fusions is necessary for their
further development as potential therapeutics for treating diseases.
109
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Abstract (if available)
Abstract
This research project primarily focuses on the development of a new temperature-dependent strategy to modulate protein/peptide assembly using a thermo-responsive elastin-like polypeptide (ELP), with applications in manipulating signal transduction and peptide-drug delivery. ELPs are biocompatible, biodegradable polypeptides consisting of a pentameric repeat of (Val-Pro-Gly-Xₐₐ-Gly)ₙ, where Xₐₐ specifies the ELP hydrophobicity and n determines the number of the repeats. ELPs are emerging as a platform to manipulate protein/peptide assembly owing to their ability to phase separate above a transition temperature, Tₜ, which can be easily tuned through the selection of Xₐₐ and n. When expressed in mammalian cells, ELPs rapidly assemble and disassemble microdomains in response to temperatures, suggesting its potential for biological tools in synthetic biology. In previous work our group has shown that ELPs fused to effector proteins such as clathrin light chain (CLC) can reversibly inhibit clathrin mediated endocytosis (CME). Extending the reach of this technology, Chapter 1 summarizes the current developments in the design of tools for manipulating protein assembly in cells and Chapter 2 presents a new platform using ELPs as regulatory component to specifically switch on/off signaling pathways. As a proof of concept, I engineered chimeric receptors containing epidermal growth factor receptor (EGFR) and ELPs and demonstrated their tunable modulation of intracellular signaling pathways through receptor clustering. Prior to thermal stimulation, EGFR-ELPs freely distributed on the cell surface and remain ‘OFF’. Once induced to phase separate, the EGFR-ELPs bring their catalytic domains into proximity, thereby switching downstream signals ‘ON’. Unlike natural ligands or synthetic agonists, the use of ELPs has high temporal resolution and reversibility, which enables dynamic control of ERK1/2 and provides an approach to quantitatively investigate signal transduction dynamics. To further pursue their application in vivo, Chapter 3, for the first time, characterizes the tunable assembly of ELPs in a vertebrate embryo. It supports the hypothesis that ELPs retains the ability to regulate the self-assembly and disassembly of protein microdomains when expressed in a multicellular organism. By tuning ELP length and sequence, protein microdomains can be induced to assemble at different temperatures, in varying sizes, or for desired periods of time within individual cells of zebrafish embryos, which may have applications in the study and manipulation of in vivo biological functions. In addition to applications in protein switching, ELP mediated protein/peptide assembly has also been explored for the delivery of therapeutic peptides. Earlier work from our group has established that fusion of a 20-mer αB crystallin (mini cry) peptide to a nanoparticle ELP scaffold called SI retains the chaperone activity of the peptide and protects human retinal pigmented epithelium (RPE) cells from oxidative stress induced cell death. Similarly, Chapter 4 evaluated the potential of ELPs to deliver a mitochondria-derived peptide (MDP) for protecting the RPE cells against oxidative stress. Furthermore, it is hypothesized that ELP mediated peptide assembly at body temperature may provide a local depot and extend its effect over longer periods. Therefore, Chapter 5 compares the ocular pharmacokinetic profiles of mini cry and crySI. While mini cry is cleared form the eye with a mean residence time of 0.4 days, crySI is retained with a mean residence time of 3.0 days, suggesting that intra-ocular crySI may provide prolonged protection against aged-related macular degeneration (AMD).
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Li, Zhe
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Temperature triggered protein assembly enables signaling switching and peptide drug delivery
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School of Pharmacy
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Doctor of Philosophy
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Pharmaceutical Sciences
Publication Date
08/06/2020
Defense Date
06/14/2018
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cell signaling,drug delivery,elastin-like polypeptides,OAI-PMH Harvest,synthetic biology,zebrafish
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cell signaling
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elastin-like polypeptides
synthetic biology
zebrafish