Understanding the site-specific effects of post-translational modifications of alpha-synuclein by using native and non-native linkages

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Content UNDERSTANDING THE SITE-SPECIFIC EFFECTS
OF POST-TRANSLATIONAL MODIFICATIONS OF
ALPHA-SYNUCLEIN BY USING NATIVE AND
NON-NATIVE LINKAGES
By

YUKA ENDO

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
MASTER OF SCIENCE
(CHEMISTRY)

December 2016

Copyright 2016 Yuka Endo

I

Acknowledgement
I would like to express my deep appreciation to my parents, Akiko and
Tamio, for believing in me, accepting who I am, being on my side, and guiding
me when I am lost. To my husband, Robbie, thank you for caring about me,
understanding me, and making me a better person. To my sister and brother,
thank you for being who you are. I am lucky to have both of you as my siblings.
To parents in law, brother and sisters in law, and my lovely nephew and niece,
thank you for being a part of my life. You are the marshmallows in my cocoa.
To Mrs. and Mr. Horiuchi, thank you for always cheering for me and giving me
courage. To Dr. Patricia Lorenzo, thank you for being my role model of a female
scientist. To the previous and current members of Pratt lab, the collection of
SUPER-talented and motivated scientists, and very cool people, thank you all
for teaching me the greater things in life: science, drinking, and friendship.
Special thanks to Anna Batt for being my colleague, being nice to me, and
feeding me with lots of tasty food. Most importantly, to my mentor, Dr.
Matthew Pratt, thank you for allowing me to work in your lab. It is my honor to
learn science from you, and to be part of your science.

II

Abstract
We are interested in elucidating the site-specific effects of post-
translational modifications (PTMs) on neurodegeneration associating protein
called α-synuclein. Under normal physiological conditions, α-synuclein is a
soluble cytosolic protein in a random coil structure. It can form an extended α-
helical structure upon contacting plasma membranes. This adaptation is
important for α-synuclein’s physiological role in synapse regulation. However,
under pathological conditions, it forms stable beta-sheet rich aggregates, which
are the main component of cellular inclusion bodies found in some
neurodegenerative diseases. α-Synuclein is a substrate of various PTMs, and
each modifier modifies α-synuclein at multiple sites. One PTM is
ubiquitination.
Previously, we ligated ubiquitin protein at 9 different physiologically
relevant lysine sites of α-synuclein using disulfide nonnative linkage. With the
site-specifically ubiquitinated α-synuclein, we showed that ubiquitination
affects α-synuclein aggregation and turn-over in a site-specific manner. To
investigate the cytotoxicity of ubiquitianted α-synuclein, we developed a non-
hydrolyzable linkage as describes in Chapter 2. Given the pioneering work done
by Wilkinson and coworkers, we improved the bis-thiol-acetone (BTA) ligation
reaction conditions by using 1,3-dibromoacetone and guanidine HBr. BTA-
linked ubiquitinated α-synuclein at 4 representing lysine sites showed that
ubiquitination inhibits α-synuclein aggregation and cellular toxicity. However,
BTA-linked and disuflide-linked ubiquitinated α-synuclein at lysine 23 showed

III

that slight difference in aggregation kinetics under an aggressive aggregation
condition, which indicated that non-native linkage may interfere with
biochemical analysis, and the use of appropriate linkages is important to
elucidate the precise biochemical effects of PTMs.
In addition to ubiquitination, we are interested in investigating the site-
specific effects of O-GlcNAcylation on α-synuclein. O-GlcNAcylation is an
intracellular addition of monosaccharide on a serine and threonine residues of
a substrate protein. Previously, we showed that O-GlcNAc modification at T72
of α-synuclein inhibits aggregation and toxicity. The work describes in Chapter
3 aimed to elucidate the effects of O-GlcNAcylation at serine 87, which can be
also phosphorylated. By taking advantage of expressed chemical ligation, we
site-specifically synthesized S87 O-GlcNAc modified α-synuclein. Biochemical
and biophysical investigations revealed that the O-GlcNAc modification at this
site did not interfere with the membrane binding of α-synuclein, and slightly
inhibited aggregation of α-synuclein. Thus, the O-GlcNAcylation of α-synuclein
has a cytoprotective effect by inhibiting aggregation without altering its
physiological function.

IV

Table of Contents
Acknowledgement .............................................................................................. I
Abstract ............................................................................................................. II

Table of Figures ............................................................................................... IX

Chapter 1.
PARKINSON’S DISEASE AND ALPHA-SYNUCLEIN .............. 1

1.1
Introduction .......................................................................................... 1

1.2
α-Synuclein .......................................................................................... 2

1.2.1
Physiological function of α-synuclein ....................................... 2

1.2.2
Physiological structure of α-synuclein ...................................... 3

1.2.3
Pathological structure of α-synuclein ........................................ 5

1.2.4
Cellular toxicity and α-synuclein species .................................. 8

1.2.5
Post translational modification ................................................. 8

1.2.5.1
Ubiquitination ............................................................ 9

1.2.5.2
SUMOylation ............................................................ 12

1.2.5.3
O-GlcNAc modification ............................................ 13

1.2.5.4
Nitration .................................................................... 15

1.2.5.5
Phosphorylation ....................................................... 16

1.3
Reference ............................................................................................ 19

Chapter 2.
Development of Bis-Thiol-Acetone (BTA) linkage to study
toxicity of site-specific ubiquitinated alpha-synuclein .................................. 28

2.1
Introduction ....................................................................................... 28

2.2
Result ................................................................................................. 33

2.2.1
Generation of BTA linkage with 1,3-dichloroacetone using
Wilkinson method .................................................................... 33

2.2.2
Generation of BTA linkage with 1,3-dichloroacetone and
ubiquitin-aminoethanethiol .................................................... 34

V

2.2.3
Generation of BTA linkage with 1,3-dibromoacetone and
ubiquitin-aminoethanethiol .................................................... 35

2.2.4
Characterisation of BTA-linked ubiquitinated α-synuclein .... 38

2.2.5
Aggregation assay of BTA-linked ubiquitinated α-synuclein . 42

2.2.6
Cellular toxicity of ubiquitinated α-synucleins ....................... 46

2.3
Discussion .......................................................................................... 49

2.4
Material and methods ........................................................................ 52

2.4.1
General ..................................................................................... 52

2.4.2
Plasmids ................................................................................... 52

2.4.3
Preparation of Ubiquitin(1-75)-aminoethanethiol .................. 53

2.4.4
Preparation of Ubiquitin(G76C) .............................................. 53

2.4.5
Reaction of 1,3-dichloroacetone and Ubiquitin(G76C) .......... 54

2.4.6
Coupling reaction of activated Ubiqutin(G76C) and α-
synuclein(k23C) ....................................................................... 55

2.4.7
Coupling reaction of activated Ubiqutin(G76C) and α-
synuclein(k23C) in 3 M guanidine-HCl buffer ........................ 55

2.4.8
Reaction of ubiquitin C-terminal thiol and 1,3-dichloroacetone
................................................................................................ 55

2.4.9
Reaction of 1,3-dichloroacetone and Ubiquitin C-terminal thiol
with 5 equivalent TCEP ............................................................ 56

2.4.10
Reaction of 1,3-dichloroacetone and Ubiquitin C-terminal thiol
with 0.5 equivalent TCEP ........................................................ 56

2.4.11
Reaction of 1,3-dichloroacetone and Ubiquitin C-terminal thiol
with 0.5 equivalent TCEP in basic pH ...................................... 57

2.4.12
Reaction of 1,3-dichloroacetone and Ubiquitin C-terminal thiol
with ascorbic acid ...................................................................... 57

2.4.13
Coupling reaction of activated ubiquitin C-terminal thiol and α-
synuclein(K23C) ...................................................................... 58

VI

2.4.14
Coupling reaction of activated ubiquitin C-terminal thiol and α-
synuclein(K23C) in 3 M guanidine-HCl borate buffer ............ 58

2.4.15
Synthesis of 1,3-dibromoacetone ............................................. 59

2.4.16
Reaction of 1,3-dibromoacetone and ubiquitin C-terminal thiol
................................................................................................ 59

2.4.17
Protein thermostability measurements by Circular Dichroism
60

2.4.18
Coupling reaction of activated ubiquitin C-terminal thiol and α-
synuclein(K6C) ........................................................................ 60

2.4.19
Coupling reaction of activated ubiquitin C-terminal thiol and α-
synuclein(K23C) in 3 M guanidine-HCl borate buffer at pH 8.3
................................................................................................ 60

2.4.20
Coupling reaction of activated ubiquitin C-terminal thiol and α-
synuclein(K23C) in 3 M guanidine-HCl borate buffer at pH 7.5
61

2.4.21
Synthesis of guanidine-HBr ...................................................... 61

2.4.22
Coupling reaction of activated ubiquitin C-terminal thiol and α-
synuclein(K23C) in 3 M guanidine-HBr borate buffer at pH 7.5
62

2.4.23
Circular Dichroism .................................................................. 62

2.4.24
Dynamic Light Scattering ....................................................... 63

2.4.25

Aggregation assay .................................................................... 63

2.4.26
Aggressive aggregation assay .................................................. 64

2.4.27

ThT fluorescence measurement .............................................. 64

2.4.28
Transmission Electron Mscroscope ....................................... 64

2.4.29
Cell culture .............................................................................. 65

2.4.30
Cellular toxicity assay ............................................................. 65

2.5
Reference ............................................................................................ 66

VII

Chapter 3.
Understanding the effects of O-GlcNAc modification
at serine 87 of α-synuclein by semi-synthesis ............................................... 72

3.1
Introduction ....................................................................................... 72

3.2
Results ................................................................................................ 74

3.2.1
Mutation of α-synuclein at Serine87 alter aggregation kinetic74

3.2.2
Semisynthesis and characterization of unmodified α-synuclein
................................................................................................. 75

3.2.3
Semisynthesis and characterization of O-GlcNAcylated α-
synuclein at S87. ...................................................................... 79

3.2.4
Membrane binding of O-GlcNAcylated α-synuclein at S87 ..... 81

3.2.5
Aggregation of O-GlcNAcylated α-synuclein at S87 ............... 82

3.3
Discussion .......................................................................................... 84

3.4
Method ............................................................................................... 86

3.4.1
General ..................................................................................... 86

3.4.2
Expression of recombinant wild type α-synuclein and α-
synuclein mutants .................................................................... 87

3.4.3
Expression and purification of α-synuclein C-terminal fragment
................................................................................................ 88

3.4.4
Expression and purification of α-synuclein N-terminal thioester
................................................................................................ 88

3.4.5
Solid phase synthesis of peptide thioester .............................. 89

3.4.6
Unmodified α-synuclein synthesis .......................................... 90

3.4.7
Synthesis of PFP-activated O-GlcNAc modified Fmoc-Serine 91

3.4.8
Synthesis of O-GlcNAc modified α-synuclein .......................... 91

3.4.9
Aggregation reaction ................................................................ 92

3.4.10
Circular dichroism ................................................................... 93

3.4.11
Dynamic light scattering .......................................................... 93

VIII

3.4.12
SDS-PAGE Analysis ................................................................. 93

3.4.13
Circular dichorism of α-synuclein with lipids ........................ 94

3.4.14
Thioflavin T fluorescence ......................................................... 94

3.4.15
Transmission electron microscope .......................................... 95

3.5
Reference ............................................................................................ 95
Bibliography .................................................................................................... 98

IX

Table of Figures
Figure 1-1. Nucleation dependent kinetics of α-synuclein ............................... 6

Figure 1-2. Post translational modification of α-synuclein .............................. 9
Figure 2-1. Structure of native ubiquitination
and the corresponding bis-thiol-acetone analogs .......................................... 32

Figure 2-2. Synthesis of BTA-ubiquitinated α-synuclein
using 1,3-dichloroacetone and UbG76C ......................................................... 34

Figure 2-3. Synthesis of BTA-ubiquitinated α-synuclein
with 1,3-dichloroacetone and ubiquitin amino-ethane-thiol ........................ 35

Figure 2-4. Thermostability of Ubiquitin(AET) and Ubiquitin(BTA)
using CD spectroscopy .................................................................................... 36
Figure 2-5. Synthesis of BTA-ubiquitinated α-synuclein
using 1,3-dibromoacetone and ubiquitin-amino-ethane-thiol ...................... 37
Figure 2-6. SDS-PAGE analysis of
BTA-ubiquitinated α-synuclein proteins.. ...................................................... 39

Figure 2-7. Characterization of BTA-ubiquitinated α-synuclein proteins .... 40

Figure 2-8. Circular Dichroism (CD) analysis of
BTA-ubiquitinated α-synuclein proteins ........................................................ 41

Figure 2-9. Dynamic Light Scattering (DLS) analysis
BTA-ubiquitinated α-synuclein proteins ........................................................ 41

Figure 2-10. Aggregation of BTA-ubiquitinated α-synuclein proteins .......... 44

Figure 2-11. Transmission Electron Microscope (TEM) images of
BTA-ubiquitinated α-synuclein proteins ....................................................... 44

Figure 2-12. Characterization of pre- and post-aggregation assay
BTA-ubiquitinated α-synuclein samples ....................................................... 45

Figure 2-13. Aggregation of BTA- and disulfide-ubiquitinated
α-synuclein protens under normal and aggressive condition ....................... 46
Figure 2-14. Comparing the stability of disulfide-ubiquitinated
and BTA-ubiquitinated α-synuclein in cell culture medium.. ....................... 49

X

Figure 2-15. Extracellular toxicity of BTA-ubiquitinated
α-synuclein on SH-SY5Y neuroblastoma cells ............................................... 49
Figure 3-1. Aggregation of α-synuclein mutants ............................................. 75

Figure 3-2. Synthetic scheme of α-synuclein ................................................. 76

Figure 3-3. Characterization of synthetic unmodified α-synuclein ................ 77

Figure 3-4. Dynamic light scattering analysis of recombinant and
synthetic α-synuclein ...................................................................................... 78

Figure 3-5. Circular dichroism spectra of recombinant and
synthetic α-synuclein ...................................................................................... 78

Figure 3-6. Aggregation reaction of recombinant and synthetic α-synuclein 79
Figure 3-7. Transmission electron microscope images of
recombinant and synthetic α-synuclein ......................................................... 79

Figure 3-8. Characterization of O-GlcNAc modified α-synuclein .................. 80

Figure 3-9. Dynamic light scattering analysis of
O-GlcNAc modified α-synuclein and S87E mutated α-synuclein ................. 80
Figure 3-10. CD analysis of O-GlcNAc modified α-synuclein
and S87E mutated α-synuclein ....................................................................... 81

Figure 3-11. CD analysis of O-GlcNAc modified α-synuclein
and S87E mutated α-synuclein with lipids .................................................... 82

Figure 3-12. Aggregation reaction of O-GlcNAc
and S87E mutated α-synuclein ...................................................................... 83

Figure 3-13. TEM images of O-GlcNAc and S87E mutated α-synuclein ........ 84

Figure 3-14. SDS-PAGE analysis of O-GlcNAc
and S87E mutated α-synuclein ...................................................................... 84

Figure 3-15. Aggregation reaction of O-GlcNAc
and S87E mutated α-synuclein at 25 µM ....................................................... 84

1

Chapter 1. PARKINSON’S DISEASE AND
ALPHA-SYNUCLEIN
1.1 Introduction
Parkinson’s disease (PD) is named after British physician James
Parkinson, who first reported its symptoms in 1817 (Schnabel, 2010). PD is the
second most common neurodegenerative disease after Alzheimer’s disease
(AD). Approximately 1% of the world’s population aged over 60 years old has
been diagnosed with PD. Patients with PD suffer from muscle tremors,
stiffness, bradykinesia and postural instability (Schnabel, 2010; Sherer et al.,
2012). Loss of olfactory sense, sleep disorders, depression, and a form of
dementia occur with progressed PD (Schnabel, 2010). Current medical
treatments only delay the disease progression. In most PD cases, etiology is not
fully understood, which hampers the development of effective treatments.
There are two main pathological features of PD. First, 50-70% of
dopaminergic neurons located at substantia nigra near brainstem of a PD
patient brain are diminished (Schnabel, 2010). Second, cellular inclusions
called Lewy Bodies or Lewy Neutrites (LBs or LNs) are accumulated in
cytoplasm of neurons in the substantia nigra (Spillantini et al., 1997; Schnabel,
2010). These cellular inclusions are composed of α-synuclein fibrils (Spillantini
et al., 1997). Consequently, PD is categorized as a member of the Lewy body
dieases (LBD), or synucleinopathies.
In addition to abnormal deposition of α-synuclein as a PD hallmark,
genetic alteration of α-synuclein is an etiologic factor of PD. Mis-sense

2

mutations (A30P, A53T, E46K, G51D and H50Q) are found in familiar early-
onset PDs (Maries et al., 2003; Lashuel et al., 2013; Khalaf et al., 2014), where
polymorphisms of α-synuclein encoding gene are a risk factor for sporadic PD
(Bendor et al., 2013; Lashuel et al., 2013). Triplication causes early onset PD
with severe symptoms compared to idiopathic PDs. This pathological and
genetical evidence supports an important role of α-synuclein in PD.
1.2 α-Synuclein
α-Synuclein protein consists of 140 amino acids in three domains: a lysine
rich N-terminus (1-60), a hydrophobic Non-Amyloidal Component (NAC, 61-
95), and a highly charged C-terminus (96-140). The N-terminus includes
conserved seven 11 amino acid imperfect repeats (Goedert, 2001), which are
indispensable to the formation of an extended α-helical structure when α-
synuclein associates with plasma membrane. The NAC region was first
identified as deposits in senile plaques in AD (Bendor et al., 2013). This region
is essential for pathological 𝛽-sheet structure conversion, since truncation of
this region abolishes fibrillation (Giasson et al., 2001) and point-mutations in
this region greatly affect aggregation kinetics (Waxman and Giasson, 2010).
The proline-rich C-terminus regulates protein-protein and protein-molecule
interactions (Eliezer, 2013).
1.2.1 Physiological function of α-synuclein
The physiological function of α-synuclein is not fully elucidated. α-
Synuclein is not an essential protein, since α-synuclein knockout (KO) mice
survive without any distinct phenotypical changes to wild-type (WT) mice

3

(Abeliovich et al., 2000; Chandra et al., 2004). Early KO mice studies show that
α-synuclein regulates neurotransmitter release and replenishment of vesicle
reservoir (Abeliovich et al., 2000; Cabin et al., 2002). Abundance of α-
synuclein in pre-synaptic termini also supports its regulatory function on
synapse and vesicle homeostasis. Association of the C-terminus of α-synuclein
and the N-terminus of synaptobrevin2, an integral membrane protein for
vesicles, was reported to induce the formation of SNARE complex assembly
(Burré et al., 2010). Assembly of SNARE complex is important for membrane
fusion of synpatic vesicles during neurotransmitter release. KO mice of all three
synuclein isoforms (α-, 𝛽-, and 𝛾-) experienced a decrease in SNARE complex
assemblies over their lifespan (Greten-Harrison et al., 2010). α-Synuclein
overexpression inhibits spontaneous neurodegeneration of cysteine-string
protein α (CSPα) KO mice (Chandra et al., 2005; Burré et al., 2010). This
evidence shows that though α-synuclein is not an essential protein for survival,
its physiological function becomes crucial under stress and aging.
1.2.2 Physiological structure of α-synuclein
α-Synuclein is classified as an intrinsically disordered protein.
Recombinantly expressed α-synuclein was characterized as an unfolded
monomer because of a larger Stokes radius and slower sedimentation rate
when compared to globular proteins with similar molecular size (Weinreb et
al., 1996). Subsequent Circular Dichroism (CD) spectroscopy and Fourier
Transfer Infrared (FTIR) spectroscopy studies on recombinant α-synuclein
agreed with the previous findings (Uversky et al., 2001). In contrast, Selkoe and

4

coworkers demonstrated formation of a helical tetramer from endogenous α-
synuclein purified from various human cells (Bartels et al., 2012). α-Synuclein
purified without denaturants migrated around 58 kD on a native gel, whose
mass was also confirmed by the sedimentation equilibrium analytical
ultracentrifugation. This tetramer consisted of an α-helical structure which
resisted aggregation, even after extended incubation. Aggregation-resistant
recombinant α-synuclein oligomers purified under non-denaturing conditions
with cross-linking reagents were also characterized with electron microscopy
(EM) and nuclear magnetic resonance (NMR) experiments (Wang et al., 2011).
However, follow-up studies on α-synuclein extraction from bacteria,
human brain, rat and mice brains could not isolate a tetrameric structure.
(Fauvet et al., 2012; Burré et al., 2013). Additionally, the cross-linking reagent
purportedly used to stabilize tetramers was shown to cause nonspecific
linkages, resulting in mainly dimers (Shaikh and Nicholson, 2008). Recent
whole cell NMR studies with exogenously labeled α-synuclein introduced by
electroporation also confirmed the native random coiled structure (Theillet et
al., 2016). To explain the contradictory findings, α-synuclein may exist in
equilibrium of multiple structures and may favor one conformation when
exposed to exogenous stimuli such as post-translational modification,
denaturants, or proteolysis. On the whole, the true α-synuclein physiological
structure remains controversial, and it has been termed a “folding chameleon”.
(Dettmer et al., 2016)

5

1.2.3 Pathological structure of α-synuclein
Under pathological conditions, α-synuclein adapts a stable 𝛽-structure
composed of fibrils. LBs isolated from postmortem human brains are
composed primarily of α-synuclein fibrils, between 5 and 10 nm in diameter
with heterogenous morphologies (Spillantini et al., 1998). Based on X-ray,
electron paramagnetic resonance (EPR) (Serpell et al., 2000; Chen et al.,
2007), NMR (Vilar et al., 2008) and solid-phase NMR studies (Tuttle et al.,
2016), the middle region (residues 35-96) of α-synuclein fibrils exhibit a cross
beta structure and parallel stacking perpendicular to the axis of fibril.
Additionally, both the N-terminal and the C-terminal ends were reported to be
flexible and not participating in the fibril structure. Further work is needed to
elucidate fibril cross-structure, since the first solid-state NMR studies support
5-anti-parallel β-sheet stacking (Vilar et al., 2008), while the second solid-state
NMR studies support a Greek-key like structure (Tuttle et al., 2016).

6

Figure 1-1: Nucleation dependent kinetics of α-synuclein. A) α-Synuclein aggregation
starts with a nucleation dependent lag phase, followed by an exponential growth phase. Finally,
fibrillization slows down at plateau phase. B) Monomers form multiple oligomeric structures,
which exponentially elongate into pre-fibrils followed by mature fibrils, by the addition of
monomers. Fragmentation of fibrils causes the second nucleation events.

Understanding the transition from physiological monomers to
pathological fibrils is of great importance. Based on in vitro studies, α-
synuclein aggregation kinetics are a sigmoidal growth curve (Uversky et al.,
2001; Cremades et al., 2012). Monomers form oligomeric nuclei during the rate
limiting lag phase. Subsequently, the oligomers exponentially elongate into
protofillaments and fibrils by addition of monomers on each end. Elongation
eventually slows down after the depletion of monomer concentration. Many
groups have identified various oligomeric structures in vitro and in vivo, and
one is more aggregation prone or toxic to neurons compared to others

7

(Cremades et al., 2012; Chen et al., 2015; Ghosh et al., 2015). Disaggregation
of fibrils and release of multiple oligomeric species are observed in vitro
(Cremades et al., 2012). Fragmentation of fibrils, whose products act as seeds
to elongate exponentially, may dominate the kinetics of fibril growth
(Cremades et al., 2012). Thus, this secondary nucleation makes α-synuclein a
prion-like protein.
Prion-like spread of α-synuclein fibrils was first proposed by Braak, after
discovering the patterned spread of LBs in human brain during the PD
progression (Braak et al., 2002; Hawkes et al., 2007). Subsequently, inductions
of endogenous α-synuclein aggregation in SH-SY5Y neuroblastoma cells (Luk
et al., 2009) and primary neurons (Volpicelli-Daley et al., 2011) after exogenous
additions of preformed fibrils were demonstrated to support α-synuclein’s
prion like feature. In mouse models, intramuscular injection on mice
expressing human α-synuclein with familiar PD causing mutation causes
intraneuronal α-synuclein aggregation in brain and spinal cord. Additionally,
stereotaxic injection of α-synuclein preformed fibril to a WT mouse results in a
development of PD like motor dysfunction and formation of LB like cellular
inclusions (Luk et al., 2012). Patients with PD who received mesencephalic
dopaminergic neuron transplantation developed LB inclusions in these grafted
neurons (Kordower et al., 2008; Li et al., 2008). These studies and
observations support that α-synuclein fibril fragments can propagate and
spread PD associating aggregates.

8

1.2.4 Cellular toxicity and α-synuclein species
Even though 𝛽-sheet rich aggregates are the main component of LB or LN,
it is still obscure which species causes cellular toxicity and neuronal death.
Many point mutations (A53T, E46K, H50Q) found in familiar PD accelerate the
aggregation of α-synuclein (Lashuel et al., 2013; Khalaf et al., 2014). Exogenous
addition of preformed fibrils to neurons induces cellular toxicity and apoptosis.
(Peelaerts et al., 2015) In contrast, the other familiar PD point mutations, A30P
and G51D, favor the formation of oligomeric species but not mature fibrils
(Breydo et al., 2012; Fares et al., 2014). Donut-like oligomers can intercalate
itself in plasma membrane to cause cytosol leakage (Chen et al., 2015). FRET
studies show that high FRET efficiency oligomers induce the cellular toxicity to
neurons by generating reductive oxygen species (ROS) (Cremades et al., 2012).
However, due to the heterogeneity of oligomers generated from divergent
procedures, identifying a toxic oligomer has proven difficult. Recently,
exogenous addition of both preformed fibrils and monomer has been reported
to induce apoptosis on primary neurons, where treatment of either fibrils or
monomers did not (Mahul-Mellier et al., 2015). Thus, the fibrillation process
but not fibrils or oligomers could be toxic to cells.
1.2.5 Post translational modification
α-Synuclein is a substrate of various post-translational modifications
(PTMs), including acetylation, phosphorylation, ubiquitination, SUMOylation,
nitrosylation and O-GlcNAc modification. Covalent modifications of α-
synuclein influence α-synuclein’s secondary structure and biochemical

9

properties, but the precise consequence of most modifications on α-synuclein
is not fully understood.
Figure 1-2: Post translational modification of α-synuclein. α-Synuclein is highly post
translationally modified by ubiqutin, N-acetyl glucosamine (O-GlcNAc), small ubiquitin like
modifier (SUMO), nitrate and phosphate.

1.2.5.1 Ubiquitination
Protein ubiquitination, or a conjugation of a small protein called ubiquitin
on a substrate protein, plays a role in various cellular processes, including
proteasomal degradation, cellular signal transduction, and the DNA repair
response (reference). A lysine residue on a substrate can form an isopeptide
bond with the C-terminus of ubiquitin to be mono-ubiquitinated. Ubiquitin
itself contains 7 lysine residues and the N-terminus that can conjugate to other
ubiquitins to form a polyubiquitin chain. This complex ubiquitin assembly is

10

orchestrated by the activities of three different enzymes, E1, E2 and E3. With
the action of E1, or a ubiquitin activating enzyme, the C-terminus of ubiquitin
is activated in an ATP dependent manner, which results in a transient thioester
bond formation to a catalytic cysteine residue of E1. The activated ubiquitin is
then transferred to a cysteine residue of E2, or a ubiquitin conjugating enzyme.
There are two primary types of E3s, HECT and RING. A HECT domain E3
ligase has an active cysteine residue that can form a thioester bond with
ubiquitin, followed by a transfer of ubiquitin to a target substrate. Instead of
forming a transient covalent bond with ubiquitin, a RING domain E3 ligase
interacts with both a substrate protein and E2-ubiquitin complex to facilitate a
direct transfer of ubiquitin to the substrate. There are more than 600 E3 ligases
identified in mammalian cells, compared with 2 E1 and 30-40 E2 enzymes.
Consequently, E3 ligases are believed to be primarily responsible for substrate
recognition.
PD pathological transformation coincides with increased cellular
concentration of α-synuclein. Like other proteins, α-synuclein is degraded by
mainly proteasome, but also lysosome and autophagy (Xilouri et al., 2012).
Postmortem brains from patients with LBD and PD showed significant
proteasomal impairment in substantia nigra neurons, potentially leading to
higher α-synuclein cellular concentrations (Tofaris et al., 2003). Proteasomal
degradation is regulated by lysine 48 linked tetra-ubiquitin chain. However, for
small proteins with less than 150 amino acid residues, such as α-synuclein,
monoubiquitination is sufficient to be shuttled for the proteasome mediated

11

degradation (Shabek et al., 2012).
Based on in vitro ubiquitination (Rott et al., 2008), in vivo ubiquitination
of reticulocytes and fibrils (Nonaka et al., 2005), and analysis of α-synuclein
purified from LBs (Anderson et al., 2006), ubiquitin conjugates to α-synuclein
at 9 lysine sites (K6, K10, K12, K21, K23, K32, K34, K43, and K96). A large
fraction of α-synuclein isolated from LBs is either mono- or di-ubiquitinated,
and ubiquitin is the second most abundant protein in LB (Anderson et al.,
2006). Even though it is found in most LBs, not all in vivo inclusions contain
ubiquitin, which suggests that ubiquitin may not be essential for inclusion
formation (Spillantini et al., 1998). As a result, the precise relationship between
α-synuclein ubiquitination and LB formation remains inconclusive.
There are 4 E3 enzymes known to ubiquitinate α-synuclein. SIAH is a
RING E3 ligase found in LBs able to ubiquitinate α-synuclein (Rott et al.,
2008). In vitro ubiquitination of α-synuclein by SIAH isoform 1 accelerates α-
synuclein aggregation, and in vivo overexpression of SIAH1 ubiquitinates α-
synuclein to initiate its proteasome degradation (Rott et al., 2008; Engelender,
2012). As a result of proteasome inhibition, ubiquitinated α-synuclein
accumulates in cytosol, which induces apoptosis in PC12 neurons (Lee et al.,
2007; Rott et al., 2011). Additionally, USP9X, or a deubiquitinase counteracts
SIAH1 mediated ubiquitination on α-synuclein, induces degradation of α-
synuclein by autophagy since deubiquitination prevents α-synuclein from
proteosomal degradation (Rott et al., 2011). A subsequent study of α-synuclein
ubiquitination by NEDD4, which poly-ubiquitinates α-synuclein with K63

12

linkage, reported endosomal-lysosomal degradation (Tofaris et al., 2011;
Sugeno et al., 2014). Carboxyl Terminus Hsp70-Interacting Protein(CHIP) E3
ligase ubiquitinates α-synuclein oligomers preferentially over monomers to
initiate both proteosomal and lysosomal degradation (Tetzlaff et al., 2008).
Finally, parkin, a RING-between-RING E3 ligase found to be mutated in early-
onset juvenile PD, is found to specifically polyubiquitinate o-glycosylated α-
synuclein (Shimura, 2001), and overexpression of parkin prevents α-synuclein
induced toxicity in drosophila (Haywood and Staveley, 2004). This large body
of evidence suggests that the specific E3 ligase controls not only α-synuclein
aggregation kinetics, but also degradation pathways and toxicity.
1.2.5.2 SUMOylation
Small ubiquitin-related modifier (SUMO) is an ubiquitin like protein
subfamily, whose members have a similar three-dimensional structure to
ubiquitin. There are three isoforms of SUMOs in mammals, SUMO1, 2, and 3
(Abeywardana and Pratt, 2015). Similar to ubiquitin, the C-termini of SUMOs
form isopeptide bonds to lysine residues of substrates. SUMOs are expressed
as proproteins, whose C-terminal fragments are cleaved by a cysteine type
SUMO protease to be matured. As the result of cascade reactions of three
enzymes, SUMO-specific E1 enzyme and SUMO E2 conjugating enzyme and E3
ligase, a matured SUMO is ligated to a lysine residue of conserved sumoylation
motif (ψ-K-X-D/E, ψ-large hydrophobic residue, K-lysine, X-any amino acid,
D/E-either aspartic or glutamic acid) (Miura and Hasegawa, 2010). One quarter
of SUMOylation occurs on the lysine residue within this conserved motif.

13

Residue 96 of α-synuclein perfectly matches to this conserved
SUMOylation motif, where residue 102 partially matches (Dorval and Fraser,
2006). In vivo SUMOlyation in HEK293 cells showed that α-synuclein is
modified mainly with SUMO1, and to a lesser extent SUMO2 and SUMO3.
Interestingly, SUMO1 deposits in LBs are found in postmortem brains of PD
patients (Olanow and Tatton, 1999). Additionally, increases of SUMOylated α-
synuclein in aggregates are observed with proteasome impairment (Kim et al.,
2011; Oh et al., 2011). However, in vitro aggregation of SUMOylated α-
synuclein prepared by coexpressing SUMO1 and α-synuclein in bacteria did not
aggregate completely (Krumova et al., 2011). Due to these contradicting
previous results, it is still unclear whether SUMOylation of α-synuclein is
cytoprotective or cytotoxic to neurons.
Our group previously generated site-specifically SUMOylated α-
synucleins at residue 96 and 102 to show that aggregation kinetics depend on
isoform- and site-specific modification (Abeywardana and Pratt, 2015).
SUMOylation generally inhibited α-synuclein aggregation. Notably, SUMO1
inhibited aggregation greatly compared to SUMO3, where K102 modification
site had a greater inhibitory effect than K96. Thus, even with 4 amino acids
difference, K96 and K102 SUMOlyation affected α-synuclein aggregation
distinctively.
1.2.5.3 O-GlcNAc modification
O-GlcNAc modification is a conjugation of N-acetyl-glucosamine
(GlcNAc) to a serine and threonine residue of a substrate protein with the

14

action of O-GlcNAc transferase (OGT). This modification is a dynamic process,
where a removal of GlcNAc is conducted by O-GlcNAcase (OGA). Glucose
metabolized by cells is shuttled into the hexosamine biosynthetic pathway to
generate high-energy UDP-GlcNAc, which OGT uses as a substrate (Levine and
Walker, 2016). Because OGT can alter its effective K M depending on the
concentration of available UDP-GlcNAc, the physiological concentrations of
glucose and its analogues greatly influence cellular O-GlcNAc modification
levels (Levine and Walker, 2016).
Misregulation of this modification has been linked to various human
diseases including neurodegeneration. OGT and OGA are highly expressed in
neurons, and many of neurodegeneration causing proteins, including α-
synuclein, are O-GlcNAc modified (Yuzwa and Vocadlo, 2014b). Decreases in
blood glucose level in Alzheimer disease (AD) patients’ brains are reported,
while the overall O-GlcNAc level is reduced in neurons (Zhu et al., 2014; Yuzwa
and Vocadlo, 2014b). Inhibition of OGA either by a mutation or OGA inhibitor
treatment reduces hyperphosphorylation and aggregation of tau in rats, and
prevents neuronal loss in tau overexpressing mice, as tau protein aggregation
is closely associated with AD (Yuzwa et al., 2014). Neuronal specific OGT
knockout mice suffered defects in locomotion and died within 10 days after
their birth (Yuzwa and Vocadlo, 2014a). Thus, O-GlcNAc modification may
have inhibitory effects on protein aggregations and play a protective role
against neurodegeneration.
Based on proteomic studies on mice neurons and human erythrocytes, α-

15

synuclein is O-GlcNAc modified at up to 8 specific threonine and serine sites, 7
of which fall in NAC region (Wang et al., 2009; 2010; Alfaro et al., 2012; Morris
et al., 2015). Previously, our lab generated an O-GlcNAc modified α-synuclein
at threonine 72, and found that the modification inhibits aggregation and
associated cellular toxicity without disrupting α-synuclein’s plasma membrane
interaction (Marotta et al., 2015). However, with excess pre-formed fibril, this
single O-GlcNAc modification was not sufficient to prevent aggregation. The
effects of O-GlcNAc modification at other sites are still under the investigation.
1.2.5.4 Nitration
Oxidative stress is highly associated with aging and neurodegeneration.
One of cytosolic radical products from oxidative stress is perooxynitrite formed
by the reaction of superoxide and nitric oxide radicals. Perooxynitrite can
generate nitrogen dioxide radical, which can chemically modify tyrosine
residues to 3-nitrotyrosines (Burai et al., 2015). Additionally, nitrogen dioxide
radicals crosslink proteins together by inducing O,O-dityrosine bond
formation.
LBs and LNs are often immunoreactive to a nitration antibody, and α-
synuclein nitration is found in brains of LBD patients, and of aged monkeys
(McCormack et al., 2012). Nitrated α-synuclein favors oligomerization and
induces severe cellular toxicity to SHSY-5Y neuroblastoma cell culture (Liu et
al., 2011) and rat dopaminergic neurons in vivo (Yu et al., 2010). Thus,
nitration of α-synuclein is highly associated with pathogenic features of PD in
aggregation and cellular toxicity.

16

α-Synuclein has 4 tyrosine residues, which can be nitrated. Recently,
nitrated α-synucleins at Y125 and Y39 were prepared semi-synthetically to
elucidate the site-specific effects of nitration (Burai et al., 2015). Generally
nitration favors the formation of small oligomers but not matured fibrils, and
decrease α-synuclein interaction to negatively charged membranes by
inhibiting α-helix formation (Burai et al., 2015). Chemically induced nitration
on mixture of α-synuclein with tyrosine to phenylalanine mutation at different
sites revealed that the cross-linking of tyrosine residues depends on steric
hinderance and tyrosine availability, since presence of both C-terminal and N-
terminal tyrosines are required for high molecular weight oligomers formation.
Interestingly, nitration does not affect PLK3 mediated serine-129
phosphorylation (Burai et al., 2015), which is supported by the fact that both
phosphorylated and nitrated α-synuclein often found in PD patient brains
(McCormack et al., 2012).
1.2.5.5 Phosphorylation
Protein phosphorylation is conjugation of a phosphate group to a hydroxyl
side chain of serine, threoine, and tyrosine. Protein phosphorylation is
conducted by protein kinases, where the removal of phosphate is done by
phosphatases (Oueslati et al., 2010). Phosphorylation regulates major cell
signaling pathways, so misregulation or malfunction of kinases or
phosphatases causes various human diseases. Since S129 phosphorylation on
α-synuclein is found predominantly in postmortem human brains with LBs and
LNs, phosphorylation of α-synuclein has been extensively studied to

17

understand its consequence on PD pathology (Oueslati et al., 2010).
Due to structural and electrostatic similarity to phosphorylated Ser,
Asp/Glu can be introduced as a phosphomimetic mutation to understand the
consequence of phosphorylation on a protein of interest in vivo (Paleologou et
al., 2008). Additionally, a loss of function study can be conducted by
introducing Ser to Ala mutation at a phosphorylation site. Both Ser to Asp/Glu
or Ser to Ala mutations have been employed to understand effects of Ser129
phosphorylation on α-synuclein.
In Drosophila, overexpression of WT human α-synuclein caused the loss
of dopaminergic neurons, and more severe neurodegeneration and
dopeminergic neuron loss were observed when phosphomimetic (S129D)
mutant was overexpressed (Chen and Feany, 2005). Additionally,
overexpression of both WT and a S129 specific kinase Gprk2 to induce the
phosphorylation at S129 caused flies to develop severe neurodegeneration,
which supports the previous result. On the other hand, flies overexpressing
S129A mutant did not suffer neuronal loss, but developed more inclusion
bodies in neurons (Chen and Feany, 2005). Taken together, in Drosophila, the
phosphorylation at S129 of α-synuclein induces toxicity and
neurodegeneration. However, in a rat model, S129A human α-synuclein
induced death of dopaminergic neurons (Gorbatyuk et al., 2008). S129D
human α-synuclein caused the least neuronal toxicity in rats, compared to
S129A and WT human α-synucleins, but it caused the formation of numerous
inclusions in neuronal cytoplasm.

18

Because of contradictory results generated with S129D and S129A
mutants, Lashuel and coworkers compared biophysical and biochemical
property of enzymatically phosphorylated α-synuclein at S129 and α-synuclein
with phosphomimetic mutations (Paleologou et al., 2008). Based on NMR
analysis, phosphorylation disturbed the long range interactions in α-synuclein,
while Asp or Glu mutated α-synuclein retained the long range interactions.
Additionally, phosphorylation at S129 inhibits α-synuclein aggregation in vitro,
where Asp or Glu mutation did not affect aggregation kinetics (Paleologou et
al., 2008). Thus, phosphomimetic mutation does not precisely recapitulate
effects of phosphorylation on α-synuclein biochemical and biophysical
property, and phosphorylation at S129 of α-synuclein inhibits aggregation and
has a cytoprotective effect.
Other than S129, α-synuclein has 4 more other phosphorylation sites
(S87, Y125, Y133, and Y136) (Hejjaoui et al., 2012). Phosphorylation at S87 also
expands synuclein’s structure and inhibits aggregation in vivo (Paleologou et
al., 2010). S87 phosphorylation prevents plasma membrane induced α-helical
formation based on CD spectra (Paleologou et al., 2010). In contrast to
phosphorylation at S129 and S87, phosphorylation at Y125 does not affect α-
helical formation with plasma membrane or aggregation kinetics, but
phosphorylation at this site caused a slight rearrangement in helixes upon its
interaction with micelle, based on
1
H-
15
N Heteronuclear Single Quantum
Coherence (HSQC) (Hejjaoui et al., 2012). Thus, phosphorylation of α-
synuclein affects its aggregation kinetics and biophysical properties in a site-

19

specific manner.

Understanding pathological transition of α-synuclein is important to
understand the pathogenesis and progression of PD. The presence of various
post-translationally modified α-synucleins under pathological conditions
makes this mission more challenging, and it highlights the needs of intense α-
synuclein PTM studies in a site-specific manner.

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Chapter 2. Development of Bis-Thiol-Acetone
(BTA) linkage to study toxicity of site-specific
ubiquitinated alpha-synuclein
Reproduced in part with permission from Lewis, Y. E., Abeywardana, T.,
Lin, Y. H., Galesic, A., & Pratt, M. R. (2016). Synthesis of a Bis-thio-acetone
(BTA) Analogue of the Lysine Isopeptide Bond and its Application to
Investigate the Effects of Ubiquitination and SUMOylation on α-Synuclein
Aggregation and Toxicity. ACS Chem. Biol., 2016, 11 (4), pp 931–942.
Copyright 2016 American Chemical Society.
2.1 Introduction
Ubiquitin is the founding member of a family of small proteins that share
a common three-dimensional structure.(Komander and Rape, 2012; van der
Veen and Ploegh, 2012) Many of these proteins function as posttranslational
modifications (PTMs) through their addition to free amines located at the N-
terminus, or more commonly through isopeptide linkages to lysine side chains,
of substrate proteins. These complex PTMs participate in almost every
biological process within the cell. Ubiquitin alone is involved in proteasomal
degradation of proteins, the mediation of protein-protein interactions
necessary for signal transduction and the DNA damage response, and receptor
endocytosis from the cell membrane. Given the key roles that ubiquitin and
ubiquitin-like proteins play in cellular biology, understanding the site-specific
consequences of individual modifications on particular substrate proteins is an
important goal. Unfortunately, traditional enzymatic methods for the

29

modification of proteins have not risen to the challenge. Ubiquitination is both
multistep processes carried out by three classes of enzymes. (Hershko and
Ciechanover, 1998; Spasser and Brik, 2012) Exploiting this multistep
enzymatic cascade has proven challenging for the production of site-specifically
modified proteins for subsequent biochemical studies. Therefore, an ever
expanding toolbox of chemical methods have been developed for the site-
specific installation of ubiquitin. (Spasser and Brik, 2012; Abeywardana and
Pratt, 2014; Weller et al., 2014; Pham and Strieter, 2015)
Several of these chemical methods result in the formation of native
isopeptide bonds by taking advantage of the native chemical ligation reaction
in combination with an ubiquitin-thioester and cleavable auxiliaries
(Chatterjee et al., 2007; McGinty et al., 2008; Weller et al., 2014) or unnatural
amino acids, (Ajish Kumar et al., 2009; Kumar et al., 2010; Oualid et al., 2010)
both of which are site-specifically incorporated using solid phase peptide
synthesis. Alternatively, unnatural amino acid mutagenesis can be exploited for
the incorporation of selectively protected lysine residues that enable the
chemical transformations necessary to generate ubiquitinated proteins (Virdee
et al., 2010; Castañeda et al., 2011; Singh et al., 2014; Madrzak et al., 2015) or
the site-specific incorporation of lysine analogs that can undergo native
chemical ligation reactions. (Virdee et al., 2011) These methods are ideal for
understanding the consequences of the native ubiquitin linkage, particularly
for in vitro experiments where either deubiquitination is not a concern or
alternatively the substrate selectivity of a deubiquitinase is under investigation.

30

However, they may not be appropriate in experiments (e.g., cell lysates, cellular
uptake, or microinjection) where the stable effects of protein ubiquitination are
of interest. Other strategies have been developed for the generation of
isopeptide-bond analogs. These include the attachment of ubiquitin through
both triazole-(Eger et al., 2010; Weikart and Mootz, 2010) and oxime-based
linkages. (Shanmugham et al., 2010) Complementary strategies have relied on
the unique reactivity of cysteine residues as lysine replacements in the
substrate protein to perform disulfide-forming reactions, (Chatterjee et al.,
2010; Chen et al., 2010) thiol-ene couplings, (Trang et al., 2012; Valkevich et
al., 2012) and ligations with electrophiles. (Hemantha et al., 2014) While all of
these methods have been applied for the investigation of site-specific
ubiquitination, they have certain drawbacks. Solid phase peptide synthesis and
unnatural amino acid strategies require a certain level of chemical expertise.
Cysteine-targeted reactions circumvent the need for chemistry to prepare the
substrate protein, but this disulfide strategy is limited to experiments in non-
reducing conditions, and the preparation of activated ubiquitin proteins
required for thiol-ene and electrophilic methods use aminolysis reactions with
moderate yields. Additionally, these analogs may not accurately recapitulate
the consequences of the native isopeptide linkage. Therefore, significant
thought should be given to which type of strategy is most suitable for a given
experiment. For example, if chemical and enzymatic stability is a requirement,
then a isopeptide-analog would be most appropriate.
The first method used to generate ubiquitin analogs was reported by

31

Wilkinson and co-workers and required no prior chemical manipulation of
either the ubiquitin or substrate proteins. (Yin et al., 2000) Specifically, they
first used site-specific mutagenesis and recombinant expression to prepare
both ubiquitin with a cysteine in place of the last glycine residue of ubiquitin
(G76C) and a substrate protein with a cysteine at the site of modification. These
two thiol-bearing proteins were then reacted with 1,3-dichloroacetone to obtain
an isopeptide analog that was stable to chemical and enzymatic cleavage
(Figure 2-1A). (Yin et al., 2000) This approach requires fewer chemical
transformations, and is therefore less technically challenging; however, it does
result in an analog that is more divergent from the native isopeptide linkage.
We report here the novel replacement of this C-terminal cysteine with a 2-
aminoethanethiol linker for the robust synthesis of stable bis-thio-acetone
(BTA) analog of ubiquitin modification that more accurately mimics the
structure of the native isopeptide bond (Figure 2-1B). Notably, these analogs
could not be prepared in good yields using the conditions reported by
Wilkinson and instead required development of the reaction, which yielded
robust conditions that can be readily applied by the biochemical community for
the preparation of stable, site-specifically modified proteins. After optimization
of these conditions, we explored the scope of our procedure by preparing the
protein α-synuclein bearing site-specific ubiquitination at four physiologically-
relevant sites. α-Synuclein forms the toxic protein-aggregates that are closely
associated with the progression of Parkinson’s disease and is the substrate for
a variety of posttranslational modifications that have the potential to directly

32

affect this process. We previously described the consequences of site-specific
ubiquitination on α-synuclein using the previously mentioned disulfide-
linkage strategy.(Meier et al., 2012; Abeywardana et al., 2013) We found that
these modifications have interesting site-specific effects on both protein
aggregation and degradation, but due to the labile nature of the disulfide bond,
we were unable to test their effects on the well-documented extracellular
toxicity of α-synuclein when added to living cells in culture. Here, we
demonstrate that the bis-thio-acetone analogs of ubiquitinated α-synuclein are
nearly identical to the corresponding disulfide analogs in terms of their effects
on protein aggregation and that they have interesting site-specific differences
in their ability to inhibit the toxicity of α-synuclein.

Figure 2-1. Structure of native ubiquitination and the corresponding bis-thiol-
acetone analogs. A) Structure of the native ubiquitin isopeptide bond and the analog utilized
by Wilkinson and co-workers. B) Structure of the bis-thiol-acetone (BTA) linkage developed
here.

33

2.2 Result
2.2.1 Generation of BTA linkage with 1,3-dichloroacetone using Wilkinson
method
The original conditions developed by the Wilkinson lab used 1,3-
dichloroacetone in sodium borate buffer to generate di-ubiquitin analogs for
biochemical analysis.(Yin et al., 2000) To determine if these reaction
conditions were applicable to α-synuclein, we first recombinantly expressed
and purified ubiquitin(G76C) and α-synuclein with a lysine to cysteine
mutation at residue 23, α-synuclein(K23C). We then proceeded in a step-wise
fashion (Figure 2-2). Ubiquitin(G76C) was first reacted with 1,3-
dichloroacetone in 71.4 mM sodium borate, resulting in the corresponding C-
terminal chloro-thiol-acetone. Notably, the unreacted starting material and the
product co-eluted on reverse-phase HPLC (RP-HPLC). We overcame this issue
by incubating the mixture with N-(aminoethyl)malaimide, which reacted with
the starting material and caused its retention time to shift. Using this two-step
purification the product was isolated in 18% yield (Figure 2-2). This activated
ubiquitin, was then incubated with α-synuclein(K23C) in borate buffer with or
without 3M guanidine HCl (GuHCl) to prevent protein aggregation.
Unfortunately, we did not observe the formation of any detectable product by
RP-HPLC followed by ESI-MS.

34

Figure 2-2: Synthesis of BTA-ubiquitinated α-synuclein using 1,3-dichloroacetone
and UbG76C. *, isolated yield.

2.2.2 Generation of BTA linkage with 1,3-dichloroacetone and ubiquitin-
aminoethanethiol
Despite this disappointing result, we decided to move on to the
aminoethanethiol linker (Figure 2-3). Accordingly, ubiquitin(1-75) was
recombinantly expressed as a in-frame fusion to the AvaDNAE intein,(Shah et
al., 2012) followed by introduction of the C-terminal linker by treatment with
2-aminoethanethiol. The resulting ubiquitin C-terminal thiol was incubated
with 1,3-dichloroacetone in borate buffer. In contrast to ubiquitin(G76C), the
reaction was easily monitored by HPLC, and we allowed the reaction to proceed
for 24 h. These conditions gave 30% product by HPLC (Figure 2-3), suggesting
that the activation yield of ubiquitin(G76C) could be improved with longer
reaction times. Next, to avoid the potential formation of ubiquitin disulfides,
which would compete with product formation, we repeated the same reaction
conditions with different reducing reagents (Figures 2-3). Interestingly, under

35

all of these conditions, we observed a different amounts of reductive
dechlorination to give the C-terminal ketone product, which again resulted in
only moderate yields. Despite this, we chose to move onto the second step: the
coupling of ubiquitin to α-synuclein. Activated ubiquitin was incubated with α-
synuclein(K23C) under the same conditions as described above (Figures 2-3).
No detectable product was formed without the addition of GuHCl; however, in
its presence, we were pleased to observe conversion of the starting materials to
the BTA-ubiquitinated α-synuclein, which was isolated in 61% yield.

Figure 2-3: Synthesis of BTA-ubiquitinated α-synuclein with 1,3-dichloroacetone
and ubiquitin amino-ethane-thiol. *, isolated yield; #, by HPLC.

2.2.3 Generation of BTA linkage with 1,3-dibromoacetone and ubiquitin-
aminoethanethiol
Given the low overall yield (~18% over two steps) of BTA-ubiquitinated α-

36

synuclein using dichloroacetone, we next decided to explore a more
electrophilic linker, 1,3-dibromoacetone. Again, the reactions were performed
in a step-wise fashion (Figure 2-5). Ubiquitin C-terminal thiol was incubated
with 1,3-dibromoacetone in 71.4 mM sodium borate, resulting in essentially
complete conversion to the corresponding activated ubiquitin in 95% isolated
yield after only 1 h. To determine whether the reaction conditions affected the
stability of ubiquitin, we determine the melting temperature of both the C-
terminal thiol starting material and the product using circular dichroism (CD)
spectroscopy as previously described.(Greenfield, 2007) Importantly, we found
that the reaction had essentially no effect on the stability of ubiquitin in the
product (Figure 2-4).

Figure 2-4: Thermostability of Ubiquitin(AET) and Ubiquitin(BTA) using CD
spectroscopy. Ubiquitin C-terminal thiol and the corresponding product of 1,3-
dibromoacetone reaction were heated from 60 to 105 ℃ and ellipticity at 220 nm was measured
at each 0.3 ℃ change in temperature. The data were plotted against fractional change in
ellipticity, and the melting temperature (T M) was calculated using Spectra Manager software.

Next, α-synuclein(K6C) was incubated with this product in 71.4 mM
sodium borate with or without 3M GuHCl. Without GuHCl, we observed the
formation of the BTA-product (Figure 2-5). However, it was not readily

37

separable from the symmetric α-synuclein(K6C) disulfide that also formed
during the reaction. In the presence of GuHCl at two different pHs, the reaction
produced only moderate yields of the desired product due to poor solubility or
slow reaction kinetics. Interestingly, Brik and co-workers recently reported the
replacement of the bromine in a bromoacetamide-containing protein with a
chlorine directly from the chloride ions from GuHCl in solution.(Hemantha et
al., 2014) They subsequently overcame this unwanted side-reaction by the
addition of 100 equivalents of sodium iodide. We explored an alternative
approach by replacing GuHCl with GuHBr. Accordingly, GuHBr was readily
synthesized from guanidine carbonate. After replacement of the GuHCl with
GuHBr, we were delighted to see a large product peak that was isolated in 62%
yield (Figure 2-5).

Figure 2-5: Synthesis of BTA-ubiquitinated α-synuclein using 1,3-dibromoacetone
and ubiquitin-amino-ethane-thiol. #, by HPLC; *, isolated yield.

38

2.2.4 Characterisation of BTA-linked ubiquitinated α-synuclein
We next set out to synthesize ubiquitinated α-synuclein analogs at four
different residues (Figures 2-7). Mono-ubiquitination is a major modifier of α-
synuclein in the aggregates isolated from Parkinson’s disease
patients.(Hasegawa, 2002; Sampathu et al., 2003; Nonaka et al., 2005;
Anderson et al., 2006) It has been shown to be modified by ubiquitin at up to
nine different positions using a variety of experimental techniques, including
in vitro modification and cell culture experiments. We previously used disulfide
analogs of ubiquitination to demonstrate that site-specific ubiquitination at the
majority of these sites inhibits the formation of α-synuclein fibers.(Meier et al.,
2012) However, as mentioned in the introduction, the disulfide-bond will be
reversed under reducing conditions, preventing the application of these
proteins to cell culture. To test whether BTA-ubiquitinated α-synuclein is also
resistant to aggregation, we used our optimized conditions to also modify α-
synuclein at residues 23, 43, and 96 in isolated yields of 52%, 56%, and 59%
over two steps, respectively. These proteins were characterized by RP-HPLC
and ESI-MS, as well as SDS-PAGE analysis (Figure 2-6, 2-7). We next analyzed
the effect of BTA-ubiquitin on the structure of monomeric α-synuclein using
CD and dynamic light scattering (DLS). The CD spectra of all four ubiquitinated
proteins were highly similar and resembled the expected combination of of the
random-coil structure of α-synuclein and the ubiquitin fold (Figure 2-8),
indicated that the linkage does not have any consequences on either protein in
solution. Characterization by DLS showed that all four proteins had stokes radii

39

of less than 10 nm (Figure 2-9), consistent with monomeric protein in solution.

Figure 2-6: SDS-PAGE analysis of BTA-ubiquitinated α-synuclein proteins.
Unmodified α-synuclein (α-syn K#C) and modified α-synuclein proteins (Ubiquitination
residue #) were separated by SDS-PAGE and visualised by Coomassie staining.

40

Figure 2-7: Characterization of BTA-ubiquitinated α-synuclein proteins. Each
purified ubiquitinated α-synuclein protein was characterised by HPLC (Vydac® C4 analytical
RP-HPLC column, 0-60% B gradient over 30 min) and ESI-MS.

41

Figure 2-8: Circular Dichroism (CD) analysis BTA-ubiquitinated α-synuclein
proteins. Equimolar of indicated proteins were analyzed by CD spectroscopy. Signals were
normalized to amino acid numbers.

Figure 2-9: Dynamic Light Scattering (DLS) analysis BTA-ubiquitinated α-
synuclein proteins. Unmodified α-synuclein and modified α-synuclein proteins
(Ubiquitination residue #) were analyzed by light scattering, and plotted as mass percentage
against Stokes radius.

42

2.2.5 Aggregation assay of BTA-linked ubiquitinated α-synuclein
Next, we used a combination of thioflavin T (ThT) fluorescence and
transmission electron microscopy (TEM) to investigate the effects of BTA-
ubiquitination on α-synuclein aggregation. Specifically, unmodified and the
four different ubiquitinated analogs of α-synuclein were incubated at a
concentration of 50 µM at 37 °C with constant agitation. To measure the
kinetics of aggregation, aliquots of the aggregation reactions were removed
after different lengths of time, and the extent of β-sheet rich fiber formation
was measured using ThT fluorescence (Figure 2-10). Despite the clear
aggregation of unmodified α-synuclein, all four of the BTA-ubiquitinated
proteins showed essentially no formation of ThT-positive aggregates over the
course of the assay. Importantly, analysis of the proteins at the end of the
aggregation reactions by RP-HPLC and mass spectrometry showed that the
BTA-linkage remained intact throughout the experiment (Figure 2-12). At the
termination of the aggregation reactions, samples were also analyzed by TEM
to visualize any aggregates that formed (Figure 2-11). A large number of mature
fibers were observed in the unmodified α-synuclein reaction. In contrast, BTA-
ubiquitination at residues 23, 43, and 96 completely blocked the formation of
any fibrous structures and only resulted in disperse, amorphous structures.
Notably, in the TEM images from α-synuclein modified at residue 6, we found
some structures that resemble irregular, short fibers that are distinct from both
unmodified α-synuclein and the other sites of ubiquitination. We attribute the
lack of detectable ThT fluorescence to a combination of the relatively small

43

number of fiber structures that were formed and the somewhat irregular
structure of the fibers, which may provide a less-than-ideal binding site for
ThT. This is partially consistent with previous data showing that preformed α-
synuclein fibers can be enzymatically ubiquitinated at lysine 6.(Nonaka et al.,
2005) Additionally, the data from BTA-ubiquitination at residue 23 does not
recapitulate what we previously observed using the disulfide-modification
approach, where ubiquitination at residue 23 inhibited the kinetics of
aggregation but not the formation of fibers.(Meier et al., 2012) In this previous
publication, the protein was subjected to harsher aggregation conditions of 100
µM protein concentration and stir-bar agitation, which have been shown to
accelerate fiber formation.(Pronchik et al., 2010) We therefore chose to
simultaneously compare the effects of both linkages at residue 23 on the
aggregation of α-synuclein using ThT fluorescence. First, disulfide-
ubiquitinated α-synuclein was prepared as previously described and both
proteins were incubated at 50 µM concentration at 37 °C with agitation in a
thermomixer. Analysis using ThT fluorescence showed that both types of
linkages result in complete inhibition of α-synuclein aggregation (Figure 2-13).
When the same proteins were subjected to the more aggressive aggregation
conditions, the disulfide-linked material showed less inhibition (Figure 2-13),
consistent with our previous experiments. This highlights the fact that different
analogs of the isopeptide bond can result in different experimental results, so
caution should be taken in the selection of the specific chemistry that will be
used. In the case of α-synuclein aggregation, the conditions used throughout

44

this paper more closely mirror the concentration of the protein at synapses,
suggesting that ubiquitination at residue 23 will inhibit aggregation and
toxicity in Parkinson’s disease.

Figure 2-10: Aggregation of BTA-ubiquitinated α-synuclein proteins. Unmodified α-
synuclein and modified α-synuclein proteins (Ubiquitination residue #) were solubilized in
phosphate buffer at the concentration of 50 µM and incubated at 37 ℃, and at each indicated
time point, a small aliquot was analyzed by ThT fluorescence (λ
ex
= 450 nm, λ
ex
= 482 nm).

Figure 2-11: Transmission Electron Microscope (TEM) images of BTA-
ubiquitinated α-synuclein proteins. Unmodified α-synuclein and modified α-synuclein
proteins (Ubiquitination residue #) after 120 hours of aggregation reaction were deposited on
copper coated grids and visualized by TEM. Scale bar, 500 nm.

45

Figure 2-12: Characterization of pre- and post-aggregation assay BTA-
ubiquitinated α-synuclein samples. Unmodified α-synuclein and modified α-synuclein
proteins (Ubiquitination residue #) before and after aggregation assay were analyzed by both
C4 analytical RP-HPLC column (0-60% B gradient over 30 minutes), and ESI-MS.

46

Figure 2-13: Aggregation of BTA- and disulfide-ubiquitinated α-synuclein protens
under normal and aggressive condition. Unmodified α-synuclein and the indicated
ubiquitinated α-synuclein proteins were subjected to different aggregation condition and
analyzed by ThT fluorescence (λ
ex
= 450 nm, λ
ex
= 482 nm).

2.2.6 Cellular toxicity of ubiquitinated α-synucleins
These in vitro experiments demonstrate that ubiquitination has an
inhibitory effect on the formation of α-synuclein fibers. However, they do not
completely rule out the possibility that other toxic species are being formed.
One way to test the toxicity of ubiquitinated α-synuclein is the overexpression
in cell culture of mutant proteins (i.e., lysine to arginine) that cannot be
modified. Unfortunately, the overexpression of α-synuclein in primary neurons
in culture and cell-line models is not always toxic.(Tabrizi et al., 2000; Lee et
al., 2001; Ko et al., 2008; Khalaf et al., 2014) For example, the very high levels
of protein expression driven by a tetracycline-inducible promoter was
necessary for observable toxicity in a SH-SY5Y cell line.(Vekrellis et al., 2009)
One way to overcome this limitation that has been widely adopted is the
exogenous addition of α-synuclein to cells in culture.(Lee et al., 2005; Desplats

47

et al., 2009; Nonaka et al., 2010; Volpicelli-Daley et al., 2011; Freundt et al.,
2012; Khalaf et al., 2014; Volpicelli-Daley et al., 2014) Importantly, the
extracellular toxicity of α-synuclein is supported by observations in human
patients(Brettschneider et al., 2015) and has been translated to in vivo animal
models.(Mougenot et al., 2012; Luk et al., 2012a; 2012b; Masuda-Suzukake et
al., 2013) As stated above, our previous studies with disulfide-linked ubiquitin
analogs could also not be extended to these type of toxicity measurements in
cell culture due to the reducing environment. To determine if the BTA-
ubiquitinated analogs were stable to culture conditions, we incubated α-
synuclein that had been ubiquitinated at residue 43 with either a disulfide- or
BTA-linkage in cell culture media and analyzed the stability of the protein using
SDS-PAGE (Figure 2-14). The disulfide-linkage was readily reduced over the
course of 48 h, while the majority of the BTA-ubiquitinated α-synuclein
remained intact, although we did observe the formation of some new bands at
intermediate molecular weights that might be attributable to non-specific
degradation. Given this higher degree of stability, we next moved on to test the
consequences of ubiquitination on the extracellular toxicity of α-synuclein.
Aggregation reactions were again initiated with either unmodified or BTA-
ubiquitinated α-synuclein. After 168 h, any aggregates that formed were
collected by centrifugation, followed by resuspension in cell culture media by
sonication. To test for the possibility of any soluble toxic-species, the
supernatant was also concentrated by lyophilization before addition of culture
media and sonication. These preparations were then added to SH-SY5Y cells

48

for 60 h. SH-SY5Y cells are a neuroblastoma cell-line derived from
dopaminergic neurons that express endogenous α-synuclein and therefore can
serve as a reasonable surrogate for primary neurons in these toxicity
measurements.(Lopes et al., 2010; Xie et al., 2010) The toxicity of the proteins
was measured by treatment with the small-molecule ethidium homodimer
(Figure 2-15). Ethidium homodimer is excluded from healthy intact cells but
will intercalate into the DNA of damaged cells, which increases its fluorescence.
The aggregates from unmodified α-synuclein had a large effect on ethidium
homodimer signal, consistent with its toxicity previous extracellular culture
experiments. In contrast, the BTA-ubiquitinated proteins induced very little
toxicity in the cells. In general, the soluble fractions of the aggregation reactions
also did not result in a much toxicity, with only modification at residue 43
showing any signal that was statistically significant. Together, these results
indicate that ubiquitination of α-synuclein has largely an inhibitory effect on
both aggregation and toxicity.

49

Figure 2-14: Comparing the stability of disulfide-ubiquitinated and BTA-
ubiquitinated α-synuclein in cell culture medium. Disulfide- or BTA-ubiquitinated α-
synuclein(K43C) were resuspended in DMEM, and incubated at 37 ℃ for indicated time.
Samples were separated by SDS-PAGE and visualized by Coomassie staining.

Figure 2-15: Extracellular toxicity of BTA-ubiquitinated α-synuclein on SH-SY5Y
neuroblastoma cells. SH-SY5Y cells were treated for 60 h with vehicle or insoluble
aggregates or remaining soluble material (25 µM based on monomer concentration) collected
from aggregation reactions initiated with corresponding proteins. Toxicity was measured with
ethidium homodimer fluorescence (λex = 528 nm, λex = 617 nm). Results are the mean ± SEM
of three separate experiments. Statistical significance compared to vehicle treated (two-tailed,
t test): N.S. = P ≥0.05, *P <0.05, ****P <0.0001.
2.3 Discussion
Chemical synthesis and semisynthesis has enabled the site-specific
preparation and biochemical characterization of ubiquitinated proteins and
transformed the type of experiments that can be performed. However, many of
these techniques require expertise in peptide and/or small molecule synthesis,
reducing their widespread adoption by the biological community. Therefore,
the continued development of robust and simple ubiquitination methods is an
important goal. We report here the incorporation of ubiquitin into a protein
using a bis-thio-acetone (BTA) linkage, which takes advantage of only
recombinantly expressed proteins and commercially available reagents. Our
approach builds on the pioneering work by Wilkinson and co-workers (Yin et

50

al., 2000) but results in a more structurally accurate isopeptide linkage and
proceeds in a much higher overall yield. While the yield of the first step of the
Wilkinson method could undoubtedly be increased by longer incubation times,
we did not observe any appreciable product formation in the coupling to α-
synuclein. The original Wilkinson procedure produced ubiquitin dimers, and
we speculate that the high solubility of ubiquitin over α-synuclein may explain
their success by increasing the reaction kinetics. During the development of our
optimized synthetic conditions, we made several other interesting
observations. The reactions involving 1,3-dichloroacetone and the less
sterically hindered animoethanethiol-functionalized ubiquitin were sluggish,
and our attempts to optimize the reaction conditions only led to the competitive
formation of side-products. To address these issues, we took advantage of 1,3-
dibromoacetone as a more electrophilic reagent. The activation of ubiquitin
with this reagent proceeded in excellent yield to give the corresponding
product; however, the coupling of this protein to α-synuclein in GuHCl was
inefficient, which we attribute to both limited solubility of α-synuclein and
potentially to in situ replacement of the bromide with chloride. (Hemantha et
al., 2014) Switching the solubilizing agent to GuHBr solved both of these
problems and enabled the synthesis of four different ubiquitinated α-synuclein
analogs.
We previously used a disulfide-based strategy to investigate the
consequences of either ubiquitination on the aggregation of α-synuclein and
found that different modification sites all largely inhibit aggregation but to

51

different extents depending on the site of modification. (Meier et al., 2012;
Abeywardana et al., 2013) However, we were unable to test the toxicity of these
semisynthetic proteins in cell culture due to the labile nature of the disulfide
bond. Importantly, we show that BTA-ubiquitination of α-synuclein at residues
6, 43, and 96 behaves almost identically to the disulfide-linked material. All
three modification sites inhibit aggregation to mature fibers as measured by a
lack of ThT fluorescence, but modification at residue 6 is capable of forming
some small irregular fiber structures that were visible by TEM. In contrast,
modification at residue 23 generated somewhat different results under
aggressive aggregation conditions, demonstrating that different linkage-
analogs are not completely interchangeable in all experiments and that the
subsequent confirmation of any results using other biochemical or cellular
methods is key. We then used the chemical stability of the BTA-linkage to
enable the treatment of SH-SY5Y cells with the aggregation reactions to
investigate the effect of ubiquitination on α-synuclein toxicity. Notably, the
three tested ubiquitination sites inhibited the toxicity of α-synuclein, while
aggregates formed by the unmodified protein induced significant cell death.
These results add additional key evidence that indicates a protective role for
ubiquitination in Parkinson’s disease. This is somewhat counterintuitive, as a
large fraction of deposited α-synuclein in the brains of Parkinson’s disease
patients is ubiquitinated. However, we favor a model where ubiquitination of
α-synuclein occurs as an early response in the disease where it slows the rate of
protein aggregation, but this protective function is overcome during the

52

progression of neurodegeneration resulting in ubiquitinated protein becoming
“trapped” in the large neuronal aggregates.

2.4 Material and methods
2.4.1 General
Cysteamine was purchased from Tokyo Chemical Industry CO, Ltd. All
the other commonly used reagents and solvents were purchased from
commercial sources (Sigma-Aldrich, VWR, EMD, Novagen, Invitrogen, Fluka)
and used without any further purification. Ampicillin antibiotic (EMD) was
prepared as a stock solution (100 mg mL
-1
or 500 mg mL
-1
) and stored at -20 ℃.
Ethidium homodimer was resuspended in DPBS at 3 mM concentration and
stored at - 20 ℃. Growth media (Luria-Bertani Broth, Miller) was prepared,
sterilized and stored according to the manufacture instruction. Agilent
technologies 1200 series HPLC with diode array detector was used for reverse
phase high performance liquid chromatography (RP-HPLC). The buffer system
is as following: buffer A (water with 0.1 % TFA), buffer B (90% acetonitrile, 10%
water, 0.1 % TFA). Either an API 3000 LC/MS-MS system (Applied
Biosystems/MDS SCIEX) or an API 150EX system (Applied Biosystems/MDS
SCIEX) were for mass spectra.
2.4.2 Plasmids
All constructions were generated by following standard molecular cloning
techniques (Marotta et al., 2012; Meier et al., 2012; Abeywardana et al., 2013).

53

Preparation of all plasmids were described in previous publications. A
QuikChange mutagenesis kit (Tratagene) was utilized to introduce a glycine to
cysteine mutation on ubiquitin, Ubiquitin(G76C), and terminal glycine deletion
on ubiquitin, Ubiquitin(1-75).
2.4.3 Preparation of Ubiquitin(1-75)-aminoethanethiol
Ubiquitin(1-75)-aminoethanethiol was expressed and purified as
described in previous publication (Meier et al., 2012; Abeywardana et al.,
2013). Yield of the protein was 7.6 mg L
-1
of culture. Purified protein product
was characterized by ESI-MS (M+H
+
). Expected mass is 8568.15 Da. Observed
mass was 8567.7 ± 1.3 Da.
2.4.4 Preparation of Ubiquitin(G76C)
Expression and purification of ubiquitin(G76C) was done as described
previously with slight modifications (Meier et al., 2012; Abeywardana et al.,
2013). E. coli BL21(DE3) cells that had been transformed with the pTXB1-
UbG76C-AvaDnaE-6XHis plasmid were grown in 1 L of LB medium containing
ampicillin (100 µg/mL) at 37 ℃. When the OD600 nm reached 0.6, the culture
was induced by addition of 0.5 mM IPTG and was then incubated for 16 h at 25
℃. The culture was pelleted by centrifugation (5000 rpm, 30 mins, 4 ℃), and
lysed by resuspension in lysis buffer (10 mL for 1 L culture, 50 mM phosphate,
300 mM NaCl, 5 mM imidazole, pH 8.0 complete protein inhibitor cocktail
[mini-complete EDTA free, Roche]) and subsequent tip sonication (6X 30
seconds on/off cycle, 4 ℃). The cellular debris was pelleted by centrifugation
(15,000 rpm, 30 mins, 4 ℃). The supernatant was loaded on 1 mL HisTrap

54

column (GE Heathcare). The protein was bound to the column by washing 10
column volume (CV) of buffer A (50 mM phosphate, 300 mM NaCl, 20 mM
imidazole, pH 8.0), and eluted with 4.5 CV of buffer B (50 mM phosphate, 300
mM NaCl, 250 mM imidazole, pH 8.0). The elution fractions were pooled and
dialyzed against buffer C (30 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP,
pH 8.5). Hydrolysis to cleave intein was done by inducating 50 mM TCEP (in
buffer C, pH 8.5) for 16 hours at 25 ℃. Ubiquitin(G76C) was purified over a C4
semi-preparative RP-HPLC using 25-60% B linear gradient over 60 minutes.
Corresponding peak was pooled and lyophilized. Yield was 6.0 mg per 1 L of
culture. Pure ubiquitin(G76C) was characterized with C4 analytical RP-HPLC
and ESI-MS (M+H
+
). Expected mass is 8,611.1 Da. The calculated mass was
8,612.4 ± 1.4 Da.
2.4.5 Reaction of 1,3-dichloroacetone and Ubiquitin(G76C)
All reagents were chilled to 4 ℃ before the reaction. Lyophilized
ubiquitin(G76C) (2.0 mg) was dissolved in 10 mM HCl (200 µL), and sodium
borate buffer (80 µL, 250 mM, pH 8.3) was added to the mixture. Then 10 equiv
of 1,3-dichloroacetone (100 mM in DMF) were added to the reaction, and the
resulting solution was rotated at 4 ℃ for 6 hours. To increase the separation of
unreacted starting material from product on RP-HPLC, 25 equiv of N-(2-
aminoethyl)maleimide were added and the resulting mixture was rotated for 2
h. The desired product was isolated by C4 semiprep RP-HPLC (25-60% B over
60 min). The isolated yield of the activated protein product was 18%. The
activated ubiquitin product was characterized by ESI-MS (M+H
+
). Expected

55

mass is 8,701.5 Da. Observed mass was 8,701.9 ± 0.3 Da.
2.4.6 Coupling reaction of activated Ubiqutin(G76C) and α-
synuclein(k23C)
Lyophilized, activated ubiquitin(G76C) (0.5 mg, 2 equiv) and α-
synuclein(k23C) (0.5 mg, 1 equiv) were resuspended in 10 mM HCl (100 µL),
followed by addition of 40 µL of 250 mM sodium borate buffer (pH 8.3). The
reaction mixture was rotated at 4 ℃ for 48 h. Prior to the HPLC injection, 20%
v/v 𝛽-mercaptoethanol was added to the reaction solution and heated for 5 min
at 98 ℃. The reaction was monitored by C4 analytical PR-HPLC (25-60% B
linear gradient over 60 min).
2.4.7 Coupling reaction of activated Ubiqutin(G76C) and α-
synuclein(k23C) in 3 M guanidine-HCl buffer
Lyophilized, activated ubiquitin(G76C) (2 equiv, 0.5 mg) and α-
synuclein(K23C) (0.5 mg, 1 equiv) were resuspended in a reaction buffer (100
µL, 3 M guanidine-HCl, 71.4 mM borate). The resultant solution was rotated at
4 ℃ for 48 h. Before the HPLC injection, 20% v/v 𝛽-mercaptoethanol was
added to the mixture, and the resulting mixture was heated at 98 ℃ for 5 min.
The reaction was monitored by C4 analytical RP-HPLC (25-60% B linear
gradient over 60 min).
2.4.8 Reaction of ubiquitin C-terminal thiol and 1,3-dichloroacetone
All reagents were cooled to 4 ℃ before the reaction. Lyophilized
ubiquin-aminoethanethiol (1.0 mg) was solubilized in 10 mM HCl (100 µL),

56

with a subsequent addition of sodium borate buffer (40 µL, 250 mM, pH 8.3).
Then 10 equiv of 1,3-dichloroacetone (100 mM in DMF) were added to the
reaction mixture. The reaction was rotated at 4 ℃ for 24 h and then purified by
C4 analytical RP-HPLC (25-60% B over 60 min) to yield 28% of the activated
ubiquitin product that was characterized by ESI-MS (M+H
+
). Expected mass is
8,659.6 Da. Observed mass was 8,658.5 ± 0.50 Da.
2.4.9 Reaction of 1,3-dichloroacetone and Ubiquitin C-terminal thiol with
5 equivalent TCEP
All reagents were cooled to 4 ℃ before the reaction. Lyophilized
ubiquin-aminoethanethiol (4.3 mg) was solubilized in 10 mM HCl (400 µL),
with a subsequent addition of sodium borate buffer (240 µL, 250 mM, 14.6 mM
TCEP, pH 8.0). Subsequently, 10 equiv of 1,3-dichloroacetone (100 mM in
DMF) were added to the reaction mixture. The reaction was rotated at 4 ℃ for
12 h and purified by C4 analytical RP-HPLC (25-60% B over 60 min). The major
peak, ubiquitin thiopropanone, was characterized by ESI-MS (M+H
+
).
Expected mass is 8,624.1 Da. Observed mass was 8,622 ± 1.22 Da.
2.4.10 Reaction of 1,3-dichloroacetone and Ubiquitin C-terminal thiol with
0.5 equivalent TCEP
All reagents were cooled to 4 ℃ before the reaction. Lyophilized
ubiquin-aminoethanethiol (1.0 mg) was solubilized in 10 mM HCl (100 µL),
followed by the addition of sodium borate buffer (40 µL, 250 mM sodium
borate, 1.46 mM TCEP, pH 8.0). Then 10 equiv of 1,3-dichloroacetone (100 mM

57

in DMF) were added to the reaction mixture. The final reaction was rotated at
4 ℃ for 5 h and then purified by C4 analytical RP-HPLC (25-60% B over 60
min) to yield 49% of the activated ubiquitin product that was characterized by
ESI-MS (M+H
+
). Expected mass is 8,659.6 Da. Observed mass was 8,658.5 ±
0.50 Da.
2.4.11 Reaction of 1,3-dichloroacetone and Ubiquitin C-terminal thiol with
0.5 equivalent TCEP in basic pH
All reagents were cooled to 4 ℃ before the reaction. Lyophilized
ubiquin-aminoethanethiol (2.0 mg) was solubilized in 10 mM HCl (200 µL),
with a subsequent addition of sodium borate buffer (80 µL, 250 mM sodium
borate, 28 mM NaOH, 1.46 mM TCEP, pH 8.96). Subsequently, 10 equiv of 1,3-
dichloroacetone (100 mM in DMF) were added to the solution. The reaction
was rotated at 4 ℃ for 5 h and monitored by C4 analytical RP-HPLC (25-60%
B over 60 min) The RP-HPLC trace was used to calculate the activated ubiquitin
product yield, which was 41%.

2.4.12 Reaction of 1,3-dichloroacetone and Ubiquitin C-terminal thiol with
ascorbic acid
All reagents were cooled to 4 ℃ before the reaction. Lyophilized
ubiquin-aminoethanethiol (2.0 mg) was solubilized in 10 mM HCl (200 µL),
with a subsequent addition of sodium borate buffer (80 µL, 250 mM sodium
borate, 5.84 mM ascorbic acid, pH 8.0). To the mixture, 10 equiv of 1,3-

58

dichloroacetone (100 mM in DMF) were added. The reaction was rotated at 4
℃ for 16 h and monitored by C4 analytical RP-HPLC (25-60% B over 60 min).
The 51% yield of the activated ubiquitin product was calculated from the RP-
HPLC trace.
2.4.13 Coupling reaction of activated ubiquitin C-terminal thiol and α-
synuclein(K23C)
Activated ubiquitin (0.5 mg, 2 equiv) and α-synuclein(K23C) (0.5 mg, 1
equiv) were resuspended in a reaction buffer (80 mM sodium borate, pH 8.3)
to give a final protein concentration of 10 mg mL
−1
. The reaction mixture was
rocked at RT for 24 h. The reaction was monitored by C4-analytical RP-HPLC
(0− 70% B linear gradient over 60 min).
2.4.14 Coupling reaction of activated ubiquitin C-terminal thiol and α-
synuclein(K23C) in 3 M guanidine-HCl borate buffer
Activated ubiquitin (0.5 mg, 2 equiv) and α-synuclein(K23C) (0.5 mg, 1
equiv) were resuspended in 3 M guanidine-HCl with 71.4 mM sodium borate
buffer (pH 8.3) to give a final protein concentration of 10 mg mL
−1
. The reaction
mixture was rotated at 4 °C for 24 h. Before the injection to HPLC, the reaction
was mixed with 20% v/v β-mercaptoethanol and boiled for 5 min at 98 °C. The
reaction was purified by C4-analytical RP-HPLC (30−55% B linear gradient
over 60 min). The 61% yield of BTA-ubiquitinated α-synuclein was calculated
from the RP-HPLC. The product was characterized by ESI-MS (M + H
+
).
Expected mass is 23,058.1 Da. The calculated mass was 23,059.2 ± 1.5 Da.

59

2.4.15 Synthesis of 1,3-dibromoacetone
1,3-Dibromoacetone is commercially available (Santa Cruz
Biotechnology) but can also be prepared as follows. Bromine (10 mL, 194
mmol) was added slowly to an acetone−methanol (6.08 mL of acetone in 72.9
mL of methanol, 82.8 mmol acetone) mixture with continuous stirring. The
resulting mixture was stirred at RT for an additional 2 h, followed by cooling to
−20 °C for 16 h. The resulting precipitate ( ∼12 g) was collected and dissolved in
72 mL of water. Concentrated sulfuric acid (1 mL) was added dropwise, and the
mixture was stirred at 48 h at 60 °C. The resulting mixture was then cooled to
RT and extracted 3× with CH 2Cl 2. The organic fractions were dried over
Na 2SO 4, filtered, and concentrated under reduced pressure in a cool bath to
give a 12% yield of 1,3-dibromoacetone as an orange oil.
2.4.16 Reaction of 1,3-dibromoacetone and ubiquitin C-terminal thiol
All reagents were cooled to 4 °C before the reaction. In a typical reaction,
lyophilized ubiquitin C-terminal thiol (2.0 mg) was resuspended in 10 mM HCl
(200 µL). Sodium borate buffer (80 µL, 250 mM, pH 8.3) was added to the
solution. Subsequently 20 equiv of 1,3-dibromoacetione (DMF, 100 mM) were
added to the mixture, and the solution was rotated at 4 °C for 1 h. The reaction
mixture was purified over C4 semipreparative RP-HPLC (25−50%B linear
gradient over 60 min). The purified ubiquitin product was characterized by C4
analytical RP-HPLC and ESI-MS (M+H
+
). Expected mass is 8703.14 Da.
Observed mass was 8702.0 ± 1.1 Da. Yield of Ubiquitin(BTA) was 95%.

60

2.4.17 Protein thermostability measurements by Circular Dichroism
The ubiquitin-aminoethanethiol and the associated production after
reaction with 1,3-dibromoactone were solubilized at the concentration of 7.5
µM in 10 mM phosphate buffer (pH7.4). Samples in a 1 mm path length quartz
cuvette were heated from 60 to 105 °C at the rate of 1 °C per min. The ellipticity
at 220 nm was collected every 0.3 °C, with 2.0 mm slit width, and DIT of 8 s.
The transition temperature (T M) was calculated using Spectra Manager
software. The calculated TM for Ub-SH was 84.23 ± 0.14 °C. The calculated T M
for Ub-BTA was 82.44 ± 0.16 °C.
2.4.18 Coupling reaction of activated ubiquitin C-terminal thiol and α-
synuclein(K6C)
Purified activated ubiquitin (4.2 mg) and α-synuclein(K6C) (2.7 mg)
were resuspended in 10 mM HCl (700 µL) The final protein concentration was
10 mg mL
−1
. Sodium borate buffer (250 mM at pH 8.3, 280 µL) was added to
the solution. The resultant mixture was rocked for 2 h at RT and purified on C4
RP-HPLC (0−70% B linear gradient over 60 min). The purified BTA-linked
ubiquitinated α-synuclein(K6C) was characterized on SDS-PAGE.
2.4.19 Coupling reaction of activated ubiquitin C-terminal thiol and α-
synuclein(K23C) in 3 M guanidine-HCl borate buffer at pH 8.3
Purified activated ubiquitin (0.5 mg, 2 equiv) and α-synuclein(K6C) (0.5
mg, 1 equiv) were resuspended in 3 M guanidine-HCl with 71.4 mM sodium
borate buffer (pH 8.3) to give a final protein concentration of 10 mg mL
−1
. The
reaction mixture was rotated at 4 °C for 48 h. β-mercaptoethanol (20% v/v)

61

was added to the reaction mixture, and the resulting solution was heated at 98
°C for 5 min. The reaction was monitored by C4-analytical RP-HPLC (30−55%
B linear gradient over 60 min). The 36% yield of BTA-ubiquitinated α-
synuclein was calculated from the RP-HPLC and the product was analyzed on
SDS-PAGE.
2.4.20 Coupling reaction of activated ubiquitin C-terminal thiol and α-
synuclein(K23C) in 3 M guanidine-HCl borate buffer at pH 7.5
Purified activated ubiquitin (0.5 mg, 2 equiv) and α-synuclein(K6C) (0.5
mg, 1 equiv) were resuspended in 3 M guanidine-HCl with 71.4 mM sodium
borate buffer (pH 7.5) to give a final protein concentration of 10 mg mL
−1
. The
reaction mixture was rotated at 4 °C for 48 h. β-Mercaptoethanol (20% v/v)
was added to the reaction mixture, and the resulting solution was heated at 98
°C for 5 min. The reaction was monitored by C4-analytical RP-HPLC (30−55%
B linear gradient for 60 min). The 56% yield of BTA-ubiquitinated α-synuclein
was calculated from the RP-HPLC and was analyzed on SDS-PAGE.
2.4.21 Synthesis of guanidine-HBr
Guanidine HBr is commercially available (Santa Cruz Biotechnology)
but can be prepared as follows. In a round-bottom flask, guanidine carbonate
(100 g, 0.56 mol) was dissolved in 57 mL of water and then cooled to 4 °C. In a
separate beaker, concentrated hydrobromic acid (124 mL, 1.10 mol) was diluted
with 177 mL of water and the diluted HBr was added slowly to the ice-cold
guanidine carbonate solution. The mixture was removed from the ice and

62

stirred overnight at RT. The solvent was removed by a rotary evaporator,
resulting in rapid crystallization of the product, and crystals were dried on a
filter paper (mp 178−180 °C).
2.4.22 Coupling reaction of activated ubiquitin C-terminal thiol and α-
synuclein(K23C) in 3 M guanidine-HBr borate buffer at pH 7.5
Purified activated ubiquitin and α-synuclein(K#C) (at a 2:1 molar ratio)
were resuspended in a reaction buffer (3 M guanidine HBr, 71.4 mM sodium
borate, pH 7.5) to make a final protein concentration 10 mg mL
−1
. The reaction
mixture was rotated at 4 °C for 18 h. β-Mercaptoethanol (20% v/v) was added
to the reaction mixture, and the resulting solution was heated at 98 °C for 5
min. The reactions were then purified by semipreparative C4 RP- HPLC
(30−55% B linear gradient over 60 min). The proteins were characterized by
C4 analytical RP-HPLC, SDS-PAGE, and ESI-MS (M+H
+
). Expected mass of
BTA-ubiquitinated α-synuclein is 23,058.1 Da. Ubiquitination residue 6: yield
= 62%, t R = 27.92 min, observed = 23,064.7 ± 2.71 Da. Ubiquitination residue
23: yield = 55%, t R = 28.59 min, observed = 23,065.6 ± 1.61 Da. Ubiquitination
residue 43: yield = 60%, t R = 27.92 min, observed = 23,059.1 ± 2.8 Da.
Ubiquitination residue 96: yield = 63%, t R = 28.06 min, observed = 23,063.7 ±
1.78 Da.
2.4.23 Circular Dichroism
All CD spectra was collected using Jasco-J-815 CD spectrometer. Sample
protein solutions were diluted to 7.5 µM in a reaction buffer without sodium

63

azide (10 mM phosphate buffer, pH 7.4). The far UV-DC spectra (190−250 nm)
were collected at 25 °C in a 1 mm path length quartz cuvette. For every
spectrum, an average of three scans was obtained with a 0.1 nm data pitch, 1.0
m bandwidth, 50 nm min
−1
scanning speed, and data integration time of 4 s.
The backgrounds of buffers were subtracted for all spectra, and the data were
converted into mean residue ellipticity.
2.4.24 Dynamic Light Scattering
Dynapro Titan temperature controlled microsampler (Wyatt) was used
to collect dynamic light scattering data. Aliquots from aggregation reactions
(50 µM, at time = 0 h) were analyzed with ten 10 s acquisition, at 25 °C, with
laser power adjusted to give an intensity of 2.0 × 10
6
counts s
−1
. Radii were
calculated based on a Rayleigh sphere approximation.
2.4.25 Aggregation assay
Proteins were resuspended to a concentration of 50 µM in phosphate
buffer (10 mM NaH 2PO 4, pH 7.4, 0.05% NaN 3) with bath sonication for 15 min.
The supernatant was cleared by centrifugation (14000g, 15 min, 4 °C). The
supernatant was then carefully aliquoted into triplicates (typical reaction
volume was 200 µL). Samples were incubated at 37 °C under continuous
shaking (1000 rpm) in an Eppendorf Thermomixer for indicated times. ThT
signals were read as described below.

64

2.4.26 Aggressive aggregation assay
Disulfide linked ubiquitination residue 23 was prepared as previously
described (Meier et al., 2012). Proteins were resuspended to a concentration of
100 µM in phosphate buffer (10 mM phosphate, pH 7.4, 0.05% NaN 3) with bath
sonication for 15 min. The supernatant was cleared by centrifugation (14000g,
15 min, 4 °C). The supernatant was carefully aliquoted into 0.5 mL screw cap
vials (Axygen scientific). A 2 × 2 mm
2
Teflon coated stir bar was added to each
tube. All reactions were arranged symmetrically and placed on a magnetic stir
plate in a 37 °C. All reactions were incubated with constant stirring for
indicated times. ThT signals were collected as described below.
2.4.27 ThT fluorescence measurement
Samples from aggregation assay reaction were diluted in 96-well plate
to a concentration of 1.25 µM with a reaction buffer (10 mM phosphate, pH 7.4,
0.05% NaN 3) containing 10 µM Thioflavin T. The plate was read by Synergy H4
hybrid reader (BioTek). The plate was shaken at 300 rpm for 3 min, followed
by data collection (λex = 450 nm, 20 nm band path, λem = 482 nm, 9.5 nm band
path, reading from the bottom of a plate, gain = 100, read height was 5.00 mm).
All of the readings were normalized to the ThT signal of buffer.
2.4.28 Transmission Electron Mscroscope
Formvar coated copper grids (150 mesh, Electron Microscopy Sciences)
were incubated with the end of aggregation assay samples (10 µL) for 5 min.
Then the grids were negatively stained for 2 min with 1% uranyl acetate, with a

65

subsequent wash 3× with 1% uranyl acetate. Each time, the excess liquid was
removed from the grids using a filter paper. The grids were dried for 48 h in a
vacuum desiccator and then visualized using a JEOL JEM 2100 LaB6
transmission electron microscope operated at 200 kV, 60000× magnification.
2.4.29 Cell culture
SH-SY5Y cells were maintained in 1:1 DMEM/F12 medium (Corning)
supplemented with 10% fetal bovine serum and incubated at 37 °C, 5.0% CO 2.
Medium was changed every 2−3 days. Two days prior to an aggregate
treatment, SH-SY5Y cells were plated in 96-well plate at a density of 12500
cells/well.
2.4.30 Cellular toxicity assay
Lyophilized proteins were dissolved in phosphate buffer (10 mM
NaPO 4H, 0.05% NaN 3) to a concentration of 50 µM and incubated at 37 °C with
agitation (1000 rpm) in a Eppendorf Thermomixer for 7 days. On the day of a
treatment, aggregates were pelleted by the centrifugation (20000g, 1 h, 25 °C).
The supernatant was collected and lyophilized as a soluble material fraction.
Aggregates and soluble material fractions were resuspended in 1:1 DMEM/F12
media (supplemented with 10% FBS) to a concentration of 25 µM protein,
followed by bath sonication (20 min) and tip-sonication (7 × 1 s on/off cycle,
20% amp). Cells were treated with 100 µL of synuclein containing medium and
incubated for 60 h. After 60 h, 100 µL of ethidium homodimer (3 µM, in DPBS)
was added to each well, and the plate was incubated at 37 °C for 40 min.

66

Fluorescent signal was read by a plate reader (Synergy H4 hybrid reader,
BioTek). The plate was shaken for 10 s at 300 rpm, followed by the data
collection (λex = 530 nm, bandwidth 20.0 nm, λem = 620 nm, bandwidth 20.0
nm, reading from top, gain 100, read height was 5.00 mm). Signals were
normalized to a background reading.

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Chapter 3. Understanding the effects of O-
GlcNAc modification at serine 87 of α-synuclein
by semi-synthesis
3.1 Introduction
Protein O-GlcNAc modification is a post-translational modification
(PTM) where N-acetyl-glucosamine (GlcNAc) is linked to a threonine and a
serine residues of substrate proteins. The high energy UDP-GlcNAc donor,
which is the end product of hexosamine biosynthetic pathway (HBP), is
incorporated by O-GlcNAc transferase (OGT) to covalently modify the
substrates (Levine and Walker, 2016). Subsequently, GlcNAc is removed by O-
GlcNAcase (OGA). This dynamic process and substrate selection are governed
by the cellular concentration of glucose and other intermediate molecules of
HBP, because OGT changes its effective K M based on the concentration of
cellular UDP-GlcNAc (Levine and Walker, 2016). Several lines of evidence
suggest that misregulation of this PTM plays an important role in
neurodegeneration. Mice with neuro-specific OGT knockout suffered
locomotor defects and hyperphosphorylation of tau protein, and they died
within 10 days after birth (Yuzwa and Vocadlo, 2014a). Many aggregating
proteins in neurodegenerative disease are O-GlcNAc modified (Yuzwa and
Vocadlo, 2014b). The lower O-GlcNAc modification levels in neurons were also
observed in Alzheimer’s disease patients (Yuzwa and Vocadlo, 2014b). OGA
inhibitors have been shown to prevent tau protein aggregation and slow
neurodegeneration in an Alzheimer’s disease mouse model (Yuzwa et al., 2014).
These studies highlighted that maintaining a certain O-GlcNAc level in neurons

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may play a protective role against neurodegenerative diseases by preventing
protein aggregation. We are interested in determining whether O-GlcNAc
modification also prevents α-synuclein aggregation and plays a neuroprotective
role against Parkinson disease.
α-Synuclein is a 140 amino acids protein and a major component of
cellular inclusion bodies, which are a pathological feature of Parkinson disease
and other synucleinopathies. Previous proteomic studies have shown that eight
positions of α-synuclein can be O-GlcNAc modified (Wang et al., 2009; 2010;
Alfaro et al., 2012; Morris et al., 2015). We have prepared O-GlcNAc modified
α-synuclein at threonine 72 residue using a chemical semi-synthetic approach,
and revealed that O-GlcNAc modification at this site directly prevented α-
synuclein aggregation, and corresponding toxicity to both neuroblastoma and
primary rat neurons without altering membrane binding important for α-
synuclein’s physiological function (Marotta et al., 2015). Given strong evidence
that O-GlcNAc modification of α-synuclein is neuroprotective, we decided to
explore other modification sites. We were especially interested in serine 87
position for following reasons. 1) This modification site is the only site found in
human erythrocyte proteomic study (Wang et al., 2009). Notably, residue 87 is
glutamine in rodent α-synuclein isoform, therefore O-GlcNAc modification at
this site could not be found in proteomic studies on rodent models. 2) It is the
only serine in α-synuclein that is O-GlcNAc modified. 3) This modification site
can be also phosphorylated, and this phosphorylation completely inhibited
aggregation and altered membrane binding (Paleologou et al., 2010).

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Here we report a semi-synthetic preparation of O-GlcNAc modified α-
synuclein at S87 and biochemical and physical analysis to reveal that the O-
GlcNAc modification at this site slightly inhibits formation of matured fibrils
without altering physiological interaction with plasma membranes.
3.2 Results
3.2.1 Mutation of α-synuclein at Serine87 alters aggregation kinetics.
One approach to explore the effects of PTMs at a specific site is to develop
animal models with either wild-type (WT) or a mutant protein of interest whose
mutation inhibits a modification at a target modification site. However, the
previous study showed that serine 87 to glutamic acid mutation of α-synuclein
completely inhibits α-synuclein aggregation (Paleologou et al., 2010).
Therefore, we prepared recombinant WT α-synuclein and α-synuclein with
mutation at S87 to alanine (αsynS87A) and S87 to glutamic acid (αsynS87E).
These proteins were subjected to aggregation condition (at 50 µM
concentration, with constant agitation at 37 ℃), and the degree of aggregation
was determined by measuring thioflavin T (ThT) fluorescence. ThT dye
specifically intercalates itself into β-sheet structure to be fluorescent. Similar to
the previously published results, αsynS87E did not aggregate for 7 days, while
WT protein aggregated after 48 hours. Previous studies showed that S87A
mutation did not alter α-synuclein aggregation at concentration of 100 µM
(Paleologou et al., 2010), but at lower concentration (50 µM), the mutant
aggregated at slower than WT. These data proved that mutations at S87 have
inhibitory effects on α-synuclein aggregation, and loss of function experiments

75

by expressing these mutants in animal models could result in inaccurate
interpretation.

Figure 3-1: aggregation of α-synuclein mutants. Unmodified and α-synuclein mutants
at S87 were subjected to aggregation condition, and at each time point, ThT fluorescence was
taken (λ
ex
= 450 nm, λ
ex
= 482 nm).

3.2.2 Semisynthesis and characterization of unmodified α-synuclein.
Because the mutations at S87 can influence aggregation kinetics, we
turned our focus to semi-synthesis of O-GlcNAc modified α-synuclein at S87 by
using expressed protein ligation (EPL). EPL requires a cysteine residue at a
ligation site, but the α-synuclein native sequence does not contain any
cysteines. Therefore, we decided to mutate alanine residues at 76 and 91 to

76

cysteines to make them ligation sites. Retrosynthetically, α-synuclein was
divided into three fragments, N-terminal protein thioester (1, residue 1-75),
peptide thioester (2, residue C76-90), and C-terminal fragment (3, residue
C91-140) (Figure 3-2). Fragment 1 and 3 were expressed recombinantly in E.
coli. The N-terminal methionine of fragment 3 was cleaved by the endogenous
methionine aminopeptidase during the expression to yield a N-terminal
cysteine. Fragment 1 was expressed in E. Coli in an intein-AvaDnaE fusion
manner, which was cleaved to generate a C-terminal protein thioester in the
presence of an exogenous thiol. The peptide thioester, 2, was prepared by solid-
phase peptide synthesis with a Dawson resin. N-terminal cysteine of the
peptide was introduced as thioproline to prevent self-polymerization.

Figure 3-2: Synthetic scheme of α-synuclein.

Incubation of 3 and 2 in a ligation buffer (6 M Guanidine-HCl, 300 mM
Phosphate, 30 mM MPAA, 30 mM TCEP, pH 7.8) for 3 days resulted in ligation
product 4, whose thioproline was subsequently deprotected by methoxyamine
treatment to yield 5. Completion of deprotection was confirmed by ESI-MS.

77

The purified 5 was incubated with 1 for 3 days to yield full length protein 6.
Finally, cysteine mutations were converted back to alanine by radical catalyzed
desulfurization to yield wild-type synthetic α-synuclein.

Figure 3-3: Characterization of synthetic unmodified α-synuclein. Purified synthetic
α-synuclein protein was characterised by HPLC (Vydac® C4 analytical RP-HPLC column, 0-
70% B gradient over 60 min) and ESI-MS.

The resulting synthetic unmodified α-synuclein was characterized by RP-HPLC
and ESI-MS (Figure 3-3). Additionally, the synthetic protein was monomeric
structurally as determined by DLS (Figure 3-4), and it retained a similar
secondary structure to recombinant unmodified α-synuclein according to
Circular Dichroism (CD) analysis (Figure 3-5). Recombinant and synthetic α-
synucleins were then subjected aggregation conditions as described above and
after 72, 120 and 168 hours of incubation, proteins were analyzed with ThT
fluorescence (Figure 3-6). Both recombinant and synthetic α-synuclein
proteins aggregated as soon as 72 hours in a similar manner. End of assay
samples were visualized with Transmission Electron Microscope (TEM). Both
recombinant and synthetic α-synuclein proteins formed similar fibrils of ~10
nm diameter (Figure 3-7). Taken together, semi-synthetically prepared α-
synuclein is biochemically and biophysically similar to recombinant α-
synuclein.

78

Figure 3-4: Dynamic light scattering analysis of recombinant and synthetic α-
synuclein. Both recombinant and synthetic α-synuclein at 50 µM were analyzed by DLS.

Figure 3-5: Circular dichroism spectra of recombinant and synthetic α-synuclein.
CD spectra were collected for dissolved samples of recombinant and synthetic α-synuclein at
7.5 µM concentration. All samples show similar spectra that are consistent with a random-coil
secondary structure.

Figure 3-6: Aggregation reaction of recombinant and synthetic α-synuclein.
Unmodified recombinant and synthetic α-synuclein (50 µM) were subjected to aggregation
condition, and at each time point, ThT fluorescence was taken (λ
ex
= 450 nm, λ
ex
= 482 nm).

79

Figure 3-7: Transmission electron microscope images of recombinant and
synthetic α-synuclein. The indicated proteins after the aggregation reaction were analyzed
by TEM; scale bar: 500 nm.

3.2.3 Semisynthesis and characterization of O-GlcNAcylated α-synuclein
at S87.
To prepare S87 glycosylated α-synuclein, (αsynS87Glc), we followed the same
synthetic route as described above, except replacing the unmodified peptide 2
with the peptide with glycosylated serine at the corresponding position (7). The
selectively protected O-GlcNAcylated serine was synthesized as previously
described (Marotta et al., 2015), and incorporated by Fmoc-based solid phase
peptide synthesis to generate the glycosylated peptide. Multi-milligram (~6
mg) of glycosylated α-synuclein was synthesized by following the same
synthetic pathway as described above. The full length glycosylated α-synuclein
was characterized by RP-HPLC and ESI-MS (Figure 3-8). RP-HPLC confirmed
its purity and observed mass on ESI-MS closely matched to the theoretical
mass.

80

Figure 3-8: Characterization of O-GlcNAc modified α-synuclein. Purified S87 O-
GlcNAcylated α-synuclein protein was characterised by HPLC (Vydac® C4 analytical RP-
HPLC column, 0-70% B gradient over 60 min) and ESI-MS.

Unmodified α-synuclein and αsynS87Glc and αsynS87E were analyzed
with CD spectrometer and DLS. CD spectra of αsynS87Glc and αsynS87E were
similar to unmodified αsynuclein, and they were all in native random coil
structure (Figure 3-9). All proteins were in less than 10 nm stokes radii based
on analysis with DLS, which indicates that either O-GlcNAc modification or
glutamic acid mutation at S87 does not induce oligomer formation (Figure 3-
8).

Figure 3-9: Dynamic light scattering analysis of O-GlcNAc modified α-synuclein
and S87E mutated α-synuclein. The indicated proteins (50 µM) were analyzed with DLS.

81

Figure 3-10: CD analysis of O-GlcNAc modified α-synuclein and S87E mutated α-
synuclein. Proteins at 7.5 µM concentration were analyzed by CD spectrometer, and the
readings were subtracted by the buffer reading.

3.2.4 Membrane binding of O-GlcNAcylated α-synuclein at S87
It is well-known that α-synuclein forms an extended α-helical structure after
its association with negatively charged plasma membranes. To test wether
modification at S87 of α-synuclein alters this physiological feature, unmodified,
αsynS87GlcNAc, and αsynS87E were all incubated with lipid vesicles for 20
minutes, and their secondary structures were analyzed with the CD
spectrometer (Figure 3-10). Unmodified α-synuclein showed typical α-helical
spectra when it was incubated with negatively charged lipids. Under the
presence of higher percentage of neutrally charged lipids, it remained in a
random coil structure. Both αsynS87Glc and αsynS87E formed α-helical
structure as unmodified when they were incubated with negatively charged
lipids, but they were in random coil with high composition of non-charged
lipid. Therefore, O-GlcNAc modification does not alter the adaptation of helical

82

structure, which is important for α-synuclein physiological function.

Figure 3-11: CD analysis of O-GlcNAc modified α-synuclein and S87E mutated α-
synuclein with lipids. Each proteins were incubated with different lipid vesicles for 20
minutes and analyzed with CD spectrometer.

3.2.5 Aggregation of O-GlcNAcylated α-synuclein at S87
Recombinant unmodified α-synuclein, phosphomimetic α-synuclein
S87E and αsynS87Glc at concentration of 50 µM were subjected to aggregation
condition (37 ℃, continuous agitation) for 7 days. After 48, 72, 120 and 168
hrs, small aliquot was taken from the protein solutions and ThT fluorescence
was measured (Figure 3-11). While recombinant α-synuclein and O-GlcNAc
modified α-synuclein aggregated at similar manner, while αsynS87E did not
aggregate after 7 days. At 168 hours, the protein samples were also analyzed by
transmission electron microscope (Figure 3-12). Under the microscope,

83

recombinant α-synuclein formed long matured fibrils, which matches to the
ThT reading. In contrast, αsynS87Glc formed more broken shorter fibril
fragments. Phosphomimetic α-synuclein formed amorphous deposits. At each
time point, samples were also centrifuged down and the supernatants were
analyzed by SDS-PAGE (Figure 3-13). While soluble unmodified α-synuclein
decreased over the course of assay, more O-GlcNAc modified protein remained
in soluble at the end of aggregation assay. No change in soluble fraction of
αsynS87E mutant was observed. Aggregation reaction at 25 µM concentration
of proteins showed slower aggregation kinetics of O-GlcNAc modified α-
synuclein compared to recombinant unmodified α-synuclein (Figure 3-14).
Phosphomimetic α-synuclein mutant showed no aggregation. These data
showed that O-GlcNAc modification at S87 inhibits the formation of mature
fibrils at 50 µM concentration, and it blocks aggregation at 25 µM
concentration, while phosphomimetic mutation completely inhibits
aggregation at both 50 and 25 µM.

Figure 3-12: Aggregation reaction of O-GlcNAc and S87E mutated α-synuclein.
Unmodified, αsynS87Glc and αsynS87E (50 µM) were subjected to aggregation condition, and
at each time point, ThT fluorescence was taken (λ
ex
= 450 nm, λ
ex
= 482 nm).

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Figure 3-13: TEM images of O-GlcNAc and S87E mutated α-synuclein. End of
aggregation assay samples were visualized under the TEM; Scale bar: 500 nm.

Figure 3-14: SDS-PAGE analysis of O-GlcNAc and S87E mutated α-synuclein. At
each time point, aggregation reaction mixtures were centrifuged down and the supernatant was
separated by SDS-PAGE and the gel was stained with Coomassie brilliant blue.

Figure 3-15: Aggregation reaction of O-GlcNAc and S87E mutated α-synuclein at
25 µM. Unmodified, αsynS87Glc and αsynS87E (24 µM) were subjected to aggregation
condition, and at each time point, ThT fluorescence was taken (λ
ex
= 450 nm, λ
ex
= 482 nm).

3.3 Discussion
We report the semi-synthesis of serine 87 O-GlcNAc modified α-synuclein
by using EPL, for subsequent biochemical analysis. O-GlcNAc modification did
not alter native random coil structure of α-synuclein and α-helical structure

85

after the association with small lipids. This result was important since α-helical
adaptation is important for α-synuclein’s physiological function, the vesicle
trafficking. This was not surprising, since Serine 87 does not participate α-
helical structure. Additionally, O-GlcNAc modification at T72 did not interfere
the secondary structure adaptation. However, previously Paleologou showed
that phosphorylation at this site prevented α-helical structure formation and
decreased the membrane binding, so our results clearly highlight the difference
between O-GlcNAc modification and phosphorylation of S87.
O-GlcNAcylation of α-synuclein at S87 blocked the aggregation and
prevented the formation of mature fibrils. The inhibitory effect was lesser
extend compared to O-GlcNAc modified α-synuclein at T72. However, T72 is
within the core-NAC region, which is indispensable to the fibril formation,
while S87 is located on the C-terminal end of NAC region. Additionally, all
structural studies of α-synuclein using electron spin resonance (EPR) (Chen et
al., 2007) and solid-state NMR spectroscopies (Vilar et al., 2008; Tuttle et al.,
2016) showed that T72 lies on the core of fibrils and S87 was located towards
the end. On the other hand, phosphorylation of S87 completely inhibits
aggregation (Paleologou et al., 2010). Therefore, electrostatic disruption than
steric hinderance at S87 is more inhibitory to fibril formation at this site, and
O-GlcNAcylation mediated inhibition on aggregation is greatly influenced by
the modification site.
Even though O-GlcNAc modification at S87 slows α-synuclein
aggregation slightly, it still highlights that the O-GlcNAcylation has an

86

inhibitory effect on protein aggregation, and our study supports the need of
OGA inhibitors that can increase neuronal O-GlcNAc level as possible
therapeutics against neurodegeneration and synucleinopathies.

3.4 Method
3.4.1 General
All chemicals and solvents were purchased from commercial venders
(Fluka, EMD, Novagen, Sigmα-Aldrich, etc) and used without any further
purification. Growth media (Luriα-Bertani, Miller) were prepared and
sterilized according to the manufacture protocol. Antibiotics (Kanamycin
sulfate, EMD, and Ampicillin sodium salt, EMD) were prepared as stock
solution (50 mg mL
-1
and 100 mg mL
-1
respectively), and stored at -20 ℃.
Analytical thin-layer chromatography was performed on 60 Å F254 silica plates
with detection by ceric ammonium molybdate (CAM) and/or UV light. Agilent
Technologies 1200 Series HPLC with Diode Array Detector was used for reverse
phase high performance liquid chromatography (RP-HPLC). Unless otherwise
noted, the HPLC buffers were, buffer A: water with 0.1% TFA, buffer B: 90%
acetonitrile, 10% water with 0.1% TFA. Mass spectra were obtained using API
150EX (Applied Biosystems/MDS SCIEX).

87

3.4.2 Expression of recombinant wild type α-synuclein and α-synuclein
mutants
Transformed BL21(DE3) E. coli with pRK172 construct containing
human wild-type α-synuclein or α-synuclein(S87A) or α-synuclein(S87E) was
grown until its OD600 reached to 0.6. The culture was induced by addition of
0.5 mM IPTG and incubation for 20 h at room temperature. The culture was
pelleted by centrifugation at 6000 rpm. The pellet was lysed by 3 times of freeze
and thaw cycle using liquid nitrogen and 37 ℃ incubator. The resulting lysate
was resuspended in lysis buffer (500 mM NaCl, 100 mM Tris, 10 mM betα-
mercaptoethanol, 1 mM EDTA, pH 8.0), and heated at 80 ℃ for 10 minutes.
The lysate was allowed to cool down to room temperature before the addition
of protease inhibitor cocktail (mini complete EDTA free, Roche). The resulting
mixture was incubated in ice for 30 minutes, and the cell debris was pelleted by
centrifugation at 15000 rpm for 30 min at 4 ℃. The pH of the supernatant was
adjusted to 3.5 with HCl, and the resulting solution was incubated on ice for
additional 30 min. The lysate was cleared by centrifugation (15,000 rpm, 30
min, 4 ℃), and then dialyzed against 3 X 1 L of a degassed 1% acetic acid
solution. The dialyzed solution was cleared by centrifugation (6,000 rpm, 15
min, 4 ℃). α-Synuclein was purified on RP-HPLC (40-60% B over 60 min),
and lyophilized. The purified protein was characterized by RP-HPLC and ESI-
MS. Expected mass of wild type α-synuclein is 14,460 Da. The observed mass
was 14,460 ± 2.94 Da. Expected mass of α-synuclein(S87A) is 14,445 Da. The

88

observed mass was 14,446 ± 0.94 Da. Expected mass of α-synuclein(S87E) is
14,502 Da. The observed mass was 14,504 ± 1.17 Da.
3.4.3 Expression and purification of α-synuclein C-terminal fragment
Transformed BL21(DE3) E. coli with pET42b construct containing α-
synuclein(C91-140) was expressed and purified as described above for α-
synuclein mutant. α-Synuclein(C91-140) was purified on RP-HPLC (10-45% B
linear gradient over 60 min). The purified fragment was characterized by RP-
HPLC and ESI-MS. Expected mass is 5,593 Da and the observed mass was
5,594.7 ± 1.7 Da.
3.4.4 Expression and purification of α-synuclein N-terminal thioester
Transformed BL21(DE3) E. coli with pTXB1 construct containing α-
synuclein(1-75)-AvaDnaE-Histag was grown to OD600 0.6. The induction of
culture was conducted by addition of 0.5 mM IPTG and incubation for 17 h at
room temperature. The culture was centrifuged (6,000 rpm, 30 min, 4 ℃) and
resuspended with lysis buffer (50 mM phosphate, 300 mM NaCl, 5 mM
imidazole, pH 8.0). The cells were lysed by tip sonication (30s/30s ON/OFF
cycle, 6 min total, 4 ℃). The lysate was cleared by centrifugation (15000 rpm,
30 min, 4 ℃), and loaded on HisTrap column (GE healthcare). The protein was
bound to the column by washing with 5 column volumes (CVs) of buffer A (50
mM phosphate, 300 mM NaCl, 20 mM imidazole, pH 8.0), and eluted with 5
CVs of buffer B (50 mM phosphate, 300 mM NaCl, 250 mM imidazole, pH 8.0).
Elution fractions were pooled and dialyzed against 3X 1 L buffer C (100 mM

89

phosphate, 150 mM NaCl, 1 mM TCEP, 1mM EDTA pH 7.5). The dialyzed
solution was incubated with mercaptoethane sulfonate (MesNa, 200 mM final
concentration) and fresh TCEP (2 mM final concentration) for 3 days to
generate C-terminal thioester group. The resulting solution was purified on C4
semiprep RP-HPLC (35-55% B over 60 minutes). The expected mass is 7,686
Da, and the observed mass was 7,686.4 ± 1.2 Da.
3.4.5 Solid phase synthesis of peptide thioester
All peptide synthesis was performed manually using protected Dawson
linker resin (Millipore). Commercially available side chain and N-Fmoc
protected amino acids (10 eq) were activated by the incubation with HBTU (10
eq) and DIEA (20 eq) for 15 minutes, and coupled for 1 h 30 min with N2 gas
agitation. Reaction completion was confirmed using Kaiser test, which was
conducted as described previously (Marotta et al., 2015). If needed, a second
coupling was performed by incubating 10 eq amino acids, 10 eq HOBt, and 12
eq DCC for 2 h with N2 agitation. Terminal N-Fmoc protecting group was
removed by treating the resin with 20% piperidine (in DMF) for 5 minutes,
followed by 20 min incubation with flesh 20% piperidine. For O-GlcNAc
modified peptide, acyl group was removed by treatment of hydrazine hydrate
(80% in MeOH) for 30 minutes twice. Before the cleavage of peptides, Dawson
linker was activated with treatment of parα-nitrophenyl chloroformate (5 eq in
DCM) for 1 hour, followed by incubation with excess DIEA (5 eq in DMF) for
30 minutes. The peptide was cleaved from resins by incubating in cleavage
cocktail (95% TFA, 2.5% TIS, 2.5% water) for 4 hours, and precipitated in ice-

90

cold diethyl ether overnight. The pellet was centrifuged (6000 rcf, 30 min, 4
℃), and resuspended with water/acetonitrile mixture. The solution was
lyophilized, resuspended, and incubated in thiolysis buffer (6 M guanidine-
HCl, 200 mM phosphate, 100 mM MesNa, pH 7.5) for 4 h. The desired peptide-
thioester was purified on C18 semiprep RP-HPLC (0-30% B over 60 min).
Purified peptides were characterized by ESI-MS. The expected mass for
unmodified peptide is 1540 Da. The observed mass was 1540.6 Da. The
expected mass for O-GlcNAc modified peptide is 1,743 Da. The observed mass
was 1,743 Da.
3.4.6 Unmodified α-synuclein synthesis
C-terminal fragment (26 mg, 2 eq) and peptide thioester (4 mg, 1 eq, 4
mM) were resuspended in ligation buffer (6 M guanidine-HCl, 300 mM
phosphate, 30 mM TCEP, 30 mM MPAA, pH 7.5) and rocked at room
temperature. The reaction was monitored by RP-HPLC (10-45% B over 60
min). Once the reaction completion was confirmed by RP-HPLC and ESI-MS,
the reaction mixture was diluted to 2 mM, and acidified to pH 4 with HCl.
Methoxyamine (100 mM final concentration) was added, and the resulting
solution was incubated at room temperature for additional 4 h. The
deprotection of thioproline was confirmed by ESI-MS. The product was
purified on C18 semiprep RP-HPLC, and lyophilized. Subsequently, purified
and lyophilized product (1 eq, 2 mM) and N-terminal thioester (2 eq) were
resuspended in ligation buffer as above. The reaction was rocked at room
temperature and monitored by RP-HPLC (25-60% B over 60 min). Once the

91

reaction is completed, the product was purified by C4 semiprep RP-HPLC and
lyophilized. Radical catalyzed desulfurization was performed by resuspending
the full length protein at 0.75 mg ml
-1
in a buffer (6 M guanidine-HCl, 300 mM
phosphate, 300 mM TCEP, 2.5% v/v ethanethiol, 10% v/v tertbutylthiol, pH
7.0) and addition of radical initiator, VΑ-061 (200 mM in MeOH, 2 mM final
concentration). The reaction was heated at 37 ℃ with constant agitation for 16
h. The reaction was worked up by C4 analytical RP-HPLC (25-60% B over 60
min). The purified unmodified α-synuclein was characterized by RP-HPLC (0-
70% B over 60 min), ESI-MS. The expected mass is 14,460 Da, and the
observed mass was 14,461 ± 1.5 Da.
3.4.7 Synthesis of PFP-activated O-GlcNAc modified Fmoc-Serine
Pentafluorophenyl (PFP) activated O-GlcNAc Fmoc-Serine was prepared
and purified as described previously. (Marotta et al., 2012)
3.4.8 Synthesis of O-GlcNAc modified α-synuclein
Purified O-GlcNAc modified peptide thioester (4 mM, 1 eq) and α-
synuclein C-terminal fragment (2 eq) were solubilized in ligation buffer (6 M
guanidine-HCl, 300 mM phosphate, 30 mM TCEP, 30 mM MPAA, pH 7.5). The
resulting solution was rocked for 24 h at room temperature. The reaction was
monitored by RP-HPLC. Once the disappearance of peptide was observed on
RP-HPLC, the reaction was diluted to twice the volume, and acidified to pH 4
by addition of HCl. Methoxyamine (100 mM final concentration) was added to
the solution, and incubated for additional 4 h. Removal of methyl group on

92

thioproline was observed by ESI-MS. The product was purified by RP-HPLC
(10-45% B over 60 min) and lyophilized. Subsequently, lyophilized product (1
eq, 2 mM) was dissolved in ligation buffer with N-terminal thioester (2 eq), and
rocked at room temperature for 24 h. The reaction was monitored by RP-HPLC.
Once the reaction was completed, the product was purified on C4 analytical RP-
HPLC (25-60% B over 60 min). The product fractions were pooled and
lyophilized. Finally, radical catalyzed desulfurization was performed by
resuspending full length O-GlcNAc modified α-synuclein with cysteines in
desulfurization buffer (6 M guanidine-HCl, 200 mM phosphate, 300 mM
TCEP, 2.5% ethanethiol, 10% tertbutylthiol, pH 7.0). The reaction was initiated
with the addition of radical initiator, VΑ-061 (200 mM in MeOH, 2 mM final
concentration), and the solution was incubated at 37 ℃ with constant agitation
in inert gas for 16 h. Desulfurized product was purified on C4 analytical RP-
HPLC (25-60% B over 60 min). The purified O-GlcNAc modified α-synuclein
was characterized by RP-HPLC and ESI-MS. The expected mass is 14,663 Da
and the observed mass was 14,666 ± 2.1 Da.
3.4.9 Aggregation reaction
Recombinant or synthetic protein was dissolved with bath sonication in
a reaction buffer (10 mM phosphate, 0.05% sodium azide, pH 7.4) to make its
final concentration at either 50 µM or 25 µM. The solution was centrifuged at
15,000 rpm for 15 min to remove any debris, and the supernatant was aliquoted
into triplicate reactions. The samples were incubated at 37 ℃ with constant

93

agitation (1000 rpm) in a Thermomixer F1.5 (Eppendorf) for 7 d. At each time
point, small aliquot was taken for ThT analysis.
3.4.10 Circular dichroism
All circular dichorism (CD) spectra were collected with Jasco-J-815
spectrometer at room temperature. Sample aliquots were diluted to 7.5 µM
with a reaction buffer without NaN 3 in a 1 mm path length quartz cuvette at 25
℃. The far UV spectra (195-250) were obtained by averaging three scans with
50 nm min
-1
scanning speed, a 0.1 nm step size, 1 nm bandwidth, data integral
speed of 4. The buffer readings were subtracted for all samples, and the data
were converted into mean residue ellipticity.
3.4.11 Dynamic light scattering
Dynamic light scattering data were obtained on Wyatt Technologies
Dynastar. All samples were time = 0 h of aggregation reaction (50 µM). For all
data, an average of 10 scans at 25 ℃ was obtained with laser power adjusted to
intensity of 2.6E
6
counts sec
-1
. Raleigh sphere approximation was used to
calculate radii.
3.4.12 SDS-PAGE Analysis
At each time point, 10 µL of aggregation reaction sample was
aliquoted, centrifuged at 20,000 x g for 1 hour at 25 ℃. The supernatant was
carefully transferred into a new tube and lyophilized. The lyophilized sample
was solubilized in 8M urea 20 mM HEPES buffer (pH 8.0) and subsequent bath

94

sonication for 20 minutes. The sample was boiled for 10 minutes with 4X SDS
loading buffer and loaded on 4-20% Criterion precast gel (BioRad) and
separated by SDS-PAGE at 195V. The gel was stained with Coomassie brilliant
blue for 30 minutes, and destained with 1:4:5 acetic acid/water/methanol
solution overnight.
3.4.13 Circular dichroism of α-synuclein with lipids
All circular dichroism (CD) spectra were collected with Jasco-J-815
spectrometer at room temperature. Samples were prepared by mixing 1:100
ratio of a protein and desired lipid mixture and incubated at room temperature
for 20 min. Lipid vesicles were prepared with 1-palmitoyl-2-oleoyl-sn-glycero-
3-[phospho-RAC-(1-glycerol)] (POPG), or by mixing different ratio of 1-
palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and 1-palmitoyl-2-
oleoyl-sn-glycero-3-phosphocholine (POPC). Dried lipid films were solubilized
in 10 mM phosphate buffer at pH 7.4 by vortexing. All spectra (190-250 nm)
were collected with scan rate of 50 nm/min, band width of 1 nm, data
integration time of 8 sec, and 1 nm step resolution. Appropriate buffer spectra
were subtracted from the final spectra.
3.4.14 Thioflavin T fluorescence
α-Synuclein aggregation progression was quantified by Thioflavin T
fluorescence. Samples from the aggregation reaction were diluted to 1.25 µM
protein concentration with 20 µM Thioflavin T dye in the reaction buffer,
followed by brief vortex and incubation for 2 min. Samples in 10 mm path

95

length quartz cuvette were analyzed using NanoLog spectroflourometer
(Horiba), λex at 450 nm with 4 nm slit, λem at 482 nm with 4 nm slit, data
integration time 0.1 sec, 3 averaged scans. Data were measured in triplicate for
all aggregation reaction conditions.
3.4.15 Transmission electron microscope
At the end of aggregation reaction, protein solution was diluted to 15 µM
by adding the reaction buffer, and 10 µL of the diluted solution was incubated
with a formvar coated copper grid (150 mesh, Electron Microscopy Science) for
5 min and the excess liquid was removed with filter paper. Subsequently, the
grid was negatively stained with 1% uranyl acetate for 2 min, and washed three
times with 1% uranyl acetate. Each time excess liquid was removed with filter
paper. The grid was dried for 48 h. Grids were visualized with a JOEL JEM-
2100F transmission electron microscope operated at 200 kV, 600,000x
magnification and an Orius Pre-GIF CCD.

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Asset Metadata

Creator Endo, Yuka (author)

Core Title Understanding the site-specific effects of post-translational modifications of alpha-synuclein by using native and non-native linkages

Contributor Electronically uploaded by the author (provenance)

School College of Letters, Arts and Sciences

Degree Master of Science

Degree Program Chemistry

Publication Date 09/29/2016

Defense Date 09/29/2016

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag alpha-synuclein,OAI-PMH Harvest,post-translational modification

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Language English

Advisor Pratt, Matthew Robert (committee chair)

Creator Email yendo@usc.edu,yuka_ukauka@icloud.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-310448

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Abstract (if available)

Abstract We are interested in elucidating the site-specific effects of post-translational modifications (PTMs) on neurodegeneration associating protein called α-synuclein. Under normal physiological conditions, α-synuclein is a soluble cytosolic protein in a random coil structure. It can form an extended α-helical structure upon contacting plasma membranes. This adaptation is important for α-synuclein’s physiological role in synapse regulation. However, under pathological conditions, it forms stable beta-sheet rich aggregates, which are the main component of cellular inclusion bodies found in some neurodegenerative diseases. α-Synuclein is a substrate of various PTMs, and each modifier modifies α-synuclein at multiple sites. One PTM is ubiquitination. ❧ Previously, we ligated ubiquitin protein at 9 different physiologically relevant lysine sites of α-synuclein using disulfide nonnative linkage. With the site-specifically ubiquitinated α-synuclein, we showed that ubiquitination affects α-synuclein aggregation and turn-over in a site-specific manner. To investigate the cytotoxicity of ubiquitianted α-synuclein, we developed a non-hydrolyzable linkage as described in Chapter 2. Given the pioneering work done by Wilkinson and coworkers, we improved the bis-thiol-acetone (BTA) ligation reaction conditions by using 1,3-dibromoacetone and guanidine HBr. BTA-linked ubiquitinated α-synuclein at 4 representing lysine sites showed that ubiquitination inhibits α-synuclein aggregation and cellular toxicity. However, BTA-linked and disuflide-linked ubiquitinated α-synuclein at lysine 23 showed that slight difference in aggregation kinetics under an aggressive aggregation condition, which indicated that non-native linkage may interfere with biochemical analysis, and the use of appropriate linkages is important to elucidate the precise biochemical effects of PTMs. ❧ In addition to ubiquitination, we are interested in investigating the site-specific effects of O-GlcNAcylation on α-synuclein. O-GlcNAcylation is an intracellular addition of monosaccharide on a serine and threonine residues of a substrate protein. Previously, we showed that O-GlcNAc modification at T72 of α-synuclein inhibits aggregation and toxicity. The work describes in Chapter 3 aimed to elucidate the effects of O-GlcNAcylation at serine 87, which can be also phosphorylated. By taking advantage of expressed chemical ligation, we site-specifically synthesized S87 O-GlcNAc modified α-synuclein. Biochemical and biophysical investigations revealed that the O-GlcNAc modification at this site did not interfere with the membrane binding of α-synuclein, and slightly inhibited aggregation of α-synuclein. Thus, the O-GlcNAcylation of α-synuclein has a cytoprotective effect by inhibiting aggregation without altering its physiological function.