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Uncovering the influence of N-terminal phosphorylation on conformational dynamics of huntingtin exon 1 monomer
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Uncovering the influence of N-terminal phosphorylation on conformational dynamics of huntingtin exon 1 monomer
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Content
Uncovering the Influence of N-terminal Phosphorylation on Conformational
Dynamics of Huntingtin Exon 1 Monomer
By
Sean Seungjoon Chung
A thesis presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fullfillment of the
Requirement for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Biology)
August 2016
1
Table of Contents
Acknowledgements 2
List of Figures 3
Abstract 5
Chapter 1: Introduction 6
Chapter 2: Research Design and Methods 11
Chapter 3: Results 13
Chapter 4: Discussion 24
Works Cited 28
2
Acknowledgements
I would like to thank Dr. Ralf Langen and Natalie Kegulian for their guidance and
patience with the entire process.
3
List of Figures
Figure 1. Polyglutamine length dependent conformational change of the native monomer
precedes aggregation and fibrilization
Figure 2. Top) Difference spectra between CD spectra at -10 and 37
o
C for Trx-HDx1 tested at
each polyglutamine length. Bottom) Mean residue ellipticity (MRE) values at 222 nm at
each temperature tested for Trx-HDx1 of the indicated Q-lengths, with weighted Trx
MRE values subtracted. Constructs with longer Q-lengths show greater helicity at all
temperatures, including 37
o
C. (In collaboration with Natalie Kegulian)
Figure 3. Temperature-dependent fold enhancement in helicity of Q7, Q16, Q25, and Q46 Trx-
HDx1 wildtype and PTM-mimicry containing constructs, as observed via circular
dichroism. (In collaboration with Natalie Kegulian)
Figure 4. A) MRE values at 222 nm of Trx-HDx1 with phosphomimetic mutations. The
weighted thioredoxin contribution to each MRE value has been subtracted. B and C) Fold
enhancement of helicity (measured as MRE at 222 nm) upon lowering the temperature
from 37 to -10 °C for wild-type and PTM-mimicry containing mutants of Trx-HDx1. (In
collaboration with Natalie Kegulian)
Figure 5. The effect of phosphomimicry on temperature-dependent conformational change in Q7,
Q16, Q25, and Q46 Trx-HDx1 wildtype constructs as viewed by circular dichroism (CD)
Figure 6. EPR spectra measured at the indicated temperatures for Trx-HDx1 of various
polyglutamine lengths spin labeled at residue 5 (Adapted from thesis work by Natalie
Kegulian)
Figure 7. Temperature-dependent scaled mobility analyses of Q25 and Q46 Trx-HDx1 fusion
4
proteins spin labeled at a single position, via continuous wave EPR. (Adapted from thesis
work by Natalie Kegulian)
Figure 8. Differential scaled mobility plot of spin labeled Q46 Trx-HDx1 constructs at 37
o
C and
0
o
C. (Adapted from thesis work by Natalie Kegulian)
Figure 9. X-band EPR spectra of Q46 Trx-HDx1 constructs labeled at position 11 with the native
S16 (black) or the S16D mutant (red). Both spectra contain two components arising from
different mobility as shown below. The red spectrum contains more of the mobile
component while the black spectrum contains more of the immobile component. Spectra
were obtained at 0
o
C. Similar differences were obtained at other temperatures. The
spectra on the bottom are the difference spectra between the respective spectra on the top.
Scan width is 100 G.
5
Abstract
Expansion of the polyglutamine (polyQ) region in the first exon of huntingtin (htt) is the root of
Huntington’s disease (HD). Misfolding and aggregation have long been observed to a greater
extent in polyQ-expanded htt, but studies have also uncovered many implications of polyQ
expansion in alterations to the dynamics and intermolecular interactions of monomeric htt. We
find that htt exon 1 (HDx1), which is composed of the polyQ tract and flanking regions (namely,
the 17-aa N-terminus [N17] and the C-terminal proline-rich domain [PRD]) and in expanded
form is sufficient to cause HD-like symptoms in transgenic mice, has a greater tendency for
adopting α-helical structure in the presence of a polyQ expansion. Our preliminary circular
dichroism (CD) studies showed that cooler temperature causes a gain in α-helical structure in
HDx1 of a variety of polyQ lengths but a greater gain at disease-level polyQ length. We had
performed continuous wave electron paramagnetic resonance (EPR) with site-directed spin
labeling (SDSL) and had found the gain in structure to occur in the HDx1 N-terminus and the N-
terminal region of the polyQ tract. Our initial findings supported the previously proposed ‘rusty
hinge’ hypothesis, which suggests that the normal, but not expanded, polyQ region serves as a
flexible hinge that enables intra- and intermolecular interactions of htt. Stiffening of the hinge by
polyQ expansion may lead to gains and losses of physiological functions for the protein. In this
project, we investigate the effect of N-terminal post-translational modifications by repeating
temperature-dependent CD and EPR analyses on HDx1 constructs containing mutations that
mimic phosphorylation and acetylation. Here we report that phosphomimetic mutation at
position 16 modulates the conformational dynamics of htt exon 1 most consistently across a
range of polyQ lengths and that the observed change in secondary structure is due to reduction in
local structuring found in the N-terminus. In conclusion, the restoration of conformation
flexibility due to S16D implicates a structure based mechanism of huntingtin aggregation and
toxicity which could serve as a target for therapeutics.
6
Chapter 1: Introduction
Proteins are biomolecules that form a network to mediate and regulate many functions in
the cell and in the living system as a whole. The proper functionality of a protein is dependent
on its three dimensional structure, which is determined by its native amino acid sequence. A
specific group of proteins, known as chaperones, associate closely with newly synthesized
proteins to oversee their correct conformational change (or folding), and to remove any
incorrectly synthesized proteins (Carrell and Lomas, 1997). In many cases, mutant or
malfunctioning proteins can misfold, associate with each other, and form aggregates. Such
accumulation of misfolded proteins has emerged as a unifying, hallmark characteristic of human
neurodegenerative pathologies (Ross and Poirier, 2004).
Neurodegenerative diseases are categorically characterized by a neurological physiology
that progressively deteriorates over time. Due to the fact that the mechanisms of their
pathophysiology have not been yet elucidated, there are no effective treatments available. Initial
biochemical studies have
Figure 1. Polyglutamine length dependent conformational change of the native monomer
precedes aggregation and fibrilization (Nagai et al., 2008)
7
brought attention to protein aggregates and inclusions found localized inside and outside of the
neuron, but the understanding of whether they are causative or product of pathology has been
controversial at best. Subsequent genetic studies have clarified that mutations that cause the
neurodegenerative diseases to be inheritable consistently result in the misfolding and aggregation
of disease-associated proteins. It has been so far understood that mutant protein causative of
neurodegeneration assumes an abnormal structure during the formation of aggregates,
characterized by beta sheet and fibrillar structuring (Davies et al., 1997).
In the effort to study the relationship between protein misfolding and neurodegeneration,
it is important to identify the type of protein aggregate that mediate toxicity. So far, studies have
suggested that misfolded and aggregating protein species that is most responsible for functional
pathology is oligomers that precede amyloid fibrils. The oligomeric species of disease proteins
have largely evaded structural characterization due to the fact that they are heterogeneous and
transient by nature (Campioni and Silvia et al., 2010).
Huntington’s disease (HD) is a neurodegenerative disorder that manifests as a
progressive attenuation of muscle coordination and mental cognition in patients (Walker, 2007).
HD is caused by an abnormal expansion of the polyglutamine (polyQ) region in the gene that
encodes the huntingtin protein (htt). Htt with polyQ expansions longer than 36Q repeats is
associated with toxicity and can form amyloid deposits in striatal and cortical neurons of patients
(DiFiglia et al., 1997). The severity of the disease is directly correlated with polyQ length while
age of onset is inversely correlated with polyQ length. Whether the predominant role of such
amyloid deposits is mediation of toxicity or neuroprotection has been the center of much debate
in the understanding of HD.
8
The mechanism with which polyQ expansion leads to neuronal cell death in HD is
difficult to articulate in the context of multiple function of htt (Harjes and Wanker et al., 2003).
A growing body of research suggests that such polyQ expansions modulate monomeric htt
structure, potentially influencing normal interactions with binding partners well before
aggregation and amyloid formation take place (Modregger et al., 2002). Although it is not fully
understood how the polyglutamine expansion causes disease, it is now generally understood that
the increase in polyglutamine length alters the conformational properties of htt. For example,
studies have shown that increase in polyQ length induced a higher propensity to misfold into
toxic species. There are also studies that have demonstrated that polyQ expansion can induce to
different secondary and tertiary structures in htt monomer. This has led to the formation of
conformational hypothesis where the precursor protein monomer undergoes a conformational
change before triggering a nucleation process to oligomerize into larger aggregates (Soto, 2001)
(Figure 1, Nagai et al., 2008). Meanwhile, most structural studies of amyloid proteins have
focused on short polyQ based peptides including htt exon 1 (HDx1), or the N-terminal fragment
of htt. HDx1 retains the structural features that make htt toxic as HDx1 with expanded polyQ is
sufficient to cause disease in mice (Mangiarini et al., 1996). Using HDx1 as a model peptide,
understanding the correlation between polyQ length and toxic gain of function mediated by
changes in htt structure is vital to the development of structure-based therapies against HD.
Htt and other polyglutamine proteins have previously evaded structural characterization
by traditional methods of X-ray crystallography and nuclear magnetic resonance (NMR) due to
the lack of availability of crystals and the homopolymeric nature of the protein, respectively (Jao
et al., 2008). An alternative method known as circular dichroism (CD) analysis allows
circumvention of these problems and monitoring of conformational changes in proteins and
9
DNA (van Stokkum et al., 1990). CD is defined as the differential absorption of left and right
circularly polarized light and the differential absorption can be expressed as molar ellipticity via
Beer-Lambert’s law. In addition, electron paramagnetic resonance (EPR) spectroscopy and side-
directed spin labeling (SDSL) provide another way to characterize the structure of amyloid
proteins, in a manner that is residue specific. EPR in the context of studying protein structure
has been an effective method for elucidating localized structure where a spin label has been
introduced (McHaourab et al., 1996). Studies have shown that spectra obtained from EPR can be
utilized to indicate whether a labeling site is buried, exposed, or in tertiary contact.
Post-translational modifications that can potentially restore the conformational flexibility
of mutant htt can be promising targets for developing structure based therapy for HD. There has
been some evidence in literature that suggest that phosphorylation specifically can play a
protective role in pathogenesis of neurodegenerative diseases, including HD. It was initially
identified that serine 13 and 16 in the N-terminal region of the htt can be phosphorylated in
cultured mammalian cells (Thompson et al., 2009). In a mammalian HD model expressing full
length mutant htt, phosphomimetic mutations (aspartic acid) were introduced at sites serine 13
and 16 and were found to be able to modulate aggregation in vitro and HD pathogenesis in vivo
(Gu et al., 2009). However, the neuroprotective potential of N-terminal phosphorylation in htt
has not been demonstrated in solution dynamics to explain how attenuation of toxicity is
mediated by htt structure. In this project, we investigated the impact of phosphorylation on the
conformational dynamics of htt by generating HDx1 protein constructs that harbor Asp
mutations at N-terminal phosphorylation target sites Thr3, Ser13, and Ser16 to mimic the
negative charge of phosphate. We then performed CD studies of these phosphomimetic
constructs at previously tested temperatures to provide direct information on the impact of
10
phosphorylation on the overall secondary structure of HDx1. It was our goal to identify and
highlight any increase in conformational flexibility of HDx1 constructs of different Q lengths
due to phosphorylation at the N-terminal region. Having a better understanding of the impact of
PTMs on HDx1 structure allows us to continue our long-term work in not only determining the
complete three-dimensional structure of HDx1 but also elucidating structural differences that
confer modulation of htt conformational behavior.
11
Chapter 2: Research Design and Methods
Overview
Previous work on the huntingtin protein structure has failed to yield a cohesive and
fundamental picture of the disease. In attempts to characterize structurally mediated mechanisms
underlying the disease, we relied on observing temperature and polyglutamine length dependent
conformational changes in our model peptide monomers. With the hypothesis that increasing the
temperature promotes flexibility of huntingtin and that increasing the polyglutamine length
reduces disorder, we identified changes in global, secondary structure using circular dichroism
(CD). We also attempted to locate the region of the peptide that is most affected by changes in
temperature, polyglutamine length, and post-translational modifications, using electron
paramagnetic resonance (EPR).
Circular dichroism was performed on these Trx-HDx1 constructs using a Jasco 815
spectropolarimeter and temperature was regulated by a Jasco PFD-425S Peltier type FDCD
attachment connected to a PolyScience recirculator. Temperature dependent measurements were
taken in 1 nm intervals from 200 nm to 260 nm with a scan speed of 50 nm/min. Ten scans were
averaged for each spectra corresponding to the construct and the background spectra was
subtracted before smoothing by Savitsky-Golay algorithm. Single wavelength measurements
were performed in one second intervals for 5 minutes at 222 nm to characterize ellipticity of the
construct.
Thioredoxin huntingtin exon 1 fusion protein (Tx-Htt) constructs were expressed in
bacteria and the cell pellets were lysed according to the protocol that has been previously
published (Bugg et al., 2012; JBC). Following lysis, proteins were centrifuged at 16,500 rpm for
30 min, incubated with nickel-nitrilotriacetic acid-agarose beads at 4
o
C for 1 hour, washed with
12
buffer (20 mM Tris-HCl, pH 7.4, 300 mM NaCl, 50 mM imidazole), and eluted in high salt
buffer (20 mM Tris-HCl, pH 7.4, 300 mM NaCl, 250 mM imidazole). The proteins were then
incubated at room temperature with 5-15 fold excess of MTSL spin label. Spin labeled proteins
were purified in FPLC on a mono-Q column using salt-containing phosphate buffer gradient (pH
7.4). Glycerol was added to the samples up to 25% by volume (1:1 dilution with 50% glycerol
phosphate salt buffer). As a control, monomeric Tx-Htt fusion protein samples (25% glycerol,
20 mM Phosphate, pH 7.4, 300 mM NaCl) were filtered through 100kD Amicon Ultra-4
centrifugal filters at 6000 rpm for 4 minutes.
Spin labeled Tx-Htt fusion proteins were loaded into quartz capillaries and continuous
wave EPR spectra were recorded on an X-band EMX spectrometer at 0
o
C. The scan width was
100 gausses at an incident microwave power of 12 dB.
13
Chapter 3: Results
Figure 2. Top) Difference spectra between CD spectra at -10 and 37
o
C for Trx-HDx1 tested at
each polyglutamine length. Bottom) Mean residue ellipticity (MRE) values at 222 nm at each
temperature tested for Trx-HDx1 of the indicated Q-lengths, with weighted Trx MRE values
subtracted. Constructs with longer Q-lengths show greater helicity at all temperatures, including
37
o
C. (In collaboration with Natalie Kegulian)
Temperature-dependent alpha helix formation in Trx-HDx1 is dependent on polyglutamine
length
From our previous studies (Fodale et al., 2014), we had initially characterized
temperature-dependent secondary structures of HDx1 fusion protein composed of thioredoxin
attached to its N-terminal end (Trx-HDx1), using circular dichroism. At the time, we compared
Q25 and Q46 Trx-HDx1 constructs for any difference(s) in conformational dynamics with
respect to temperature. Across a wide range of temperatures, Q46 Trx-HDx1 constructs not only
exhibited greater alpha helicity but also greater temperature-dependent secondary structural
change than Q25 Trx-HDx1 constructs (Fodale et al., 2014). The initial observations suggested a
potential trend for other physiologically relevant polyglutamine lengths where alpha helicity and
conformational sensitivity to temperature are directly associated with polyglutamine length. In
14
order to confirm such a relationship, we proceeded to extend our initial temperature dependent
circular dichroism studies to Trx-HDx1 constructs containing Q7, Q16, and Q55.
We now report that Q55 Trx-HDx1 construct exhibited the greatest alpha helicity and
that Q7 Trx-HDx1 construct exhibited the least alpha helicity (Figure 2). Q16 and Q25 Trx-
HDx1 constructs were observed to have intermediate helicity between Q7 and Q46 Trx-HDx1
constructs. As such, we can generally conclude that previously suggested trend is valid; alpha
helicity of wildtype Trx-HDx1 monomers increases with increasing polyglutamine length at each
of the seven temperatures tested.
Figure 3. Temperature-dependent fold enhancement in helicity of Q7, Q16, Q25, and Q46 Trx-
HDx1 wildtype and PTM-mimicry containing constructs, as observed via circular dichroism. (In
collaboration with Natalie Kegulian)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Fold Enhancement from 37
o
C to -10
o
C
15
Figure 4. A) MRE values at 222 nm of Trx-HDx1 with phosphomimetic mutations. The
weighted thioredoxin contribution to each MRE value has been subtracted. B and C) Fold
enhancement of helicity (measured as MRE at 222 nm) upon lowering the temperature from 37
to -10 °C for wild-type and PTM-mimicry containing mutants of Trx-HDx1. (In collaboration
with Natalie Kegulian)
Phosphomimetic mutation at position 16 (S16D) impacts temperature-dependent secondary
structuring of Trx-HDx1 monomer
In order to investigate the effect of post-translational modifications (PTMs) on the
secondary structure of Trx-HDx1 construct monomers, we introduced two types of PTM-
mimicking mutations via site directed mutagenesis into the N-terminal region of the Q25 and
Q46 Trx-HDx1 wildtype constructs. Aspartic acid and glutamine mutations would mimic
phosphorylation and acetylation, respectively. Subsequently, circular dichroism was utilized to
characterize the temperature-dependent conformational dynamics of these Trx-HDx1-PTM
constructs.
We had previously noted the enhancement of alpha helicity with increasing
polyglutamine length in the wildtype constructs, especially at low temperatures. Additionally,
16
the difference CD spectrum between -10
o
C spectrum and 37
o
C spectrum of wildtype constructs
had shown that temperature-induced enhancement itself is dependent on polyglutamine length
(Figure 2). Increase in helicity as well as in its change over a wide range of temperatures and
polyglutamine lengths was a consistent conformational trend in Trx-HDx1 monomers that was
again confirmed by mean residue ellipticity (MRE) analysis at 222 nm.
Figure 5. The effect of phosphomimicry on temperature-dependent conformational change in
Q7, Q16, Q25, and Q46 Trx-HDx1 wildtype constructs as viewed by circular dichroism (CD)
The completion of tempreature-dependent circular dichroism (CD) analysis of Q7, Q16,
Q25, and Q46 Trx-HDx1-PTM fusion proteins showed that the phosphomimietic mutation S16D
modulates the MRE distribution of 222 nm of Trx-HDx1 to shift to that of a lower polyglutamine
length fusion protein (Figure 4). Specifically, the S16D mutation reduced temperature-
dependent helicity for each polyglutamine length to exhibit that of the wildtype form of the next-
lower polyglutamine length fusion protein tested (Figure 5). The observed loss of helicity and
structure due to the S16D mutation was consistent in Q16 and Q7 Trx-HDx1 derivatives as well,
implicating a potential role of phosphorylation in altering huntingtin’s monomeric structure.
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
Trx-HDx1 Fold Enhancement (37
o
C to -10
o
C)
17
Only the S16D mutation, not S13D or T3D mutations, had a consistent influence on the
temperature-dependent conformational dynamics, as observed by circular dichroism. The other
serine to aspartic acid mutation at position 13 shifted the MRE distribution to exhibit a higher
amount and enhancement in helicity, modulating the protein to assume a higher polyglutamine
length conformation. The S13D/S16D double phosphomimetic mutation had an intermediate
effect. The results were a part of a more comprehensive analysis of individual T3D, S13D,
S16D mutations, combined triple mutant containing all phosphorylation sites, double mutant
containing S13D and S16D, triple alanine mutant containing alanine at all three phosphorylation
sites, and a K6Q, K9Q double mutant designed to mimic acetylation (Figure 3). Each of these
types of mutations that mimic phosphorylation and acetylation in the N-terminal region of Trx-
HDx1 was tested in the Q7, Q16, Q25, and Q46 Trx-HDx1 wildtype backgrounds.
S16D mutation has a small but detectable influence on the local structuring in N-terminal
region of Q46 Trx-HDx1 monomer
Our collaborative work (with Natalie Kegulian ’15) that preceded the current study of
phosphomimetic huntingtin exon 1 structure employed side-directed spin labeling (SDSL) and
electron paramagnetic resonance (EPR) spectroscopy to initially characterize local structure and
ordering in Q25 and Q46 Trx-HDx1 fusion proteins. Preliminary data had found that htt exon 1
monomer maintains a structural equilibrium between disordered and ordered (alpha-helical)
states (Figure 6). The existence of multiple structural states suggested that certain internal and
external parameters, polyglutamine length and temperature respectively, could alter the native
monomeric structure of huntingtin. It was subsequently established through our temperature-
18
dependent continuous wave EPR studies that the propensity for ordering or helical state is
modulated by polyglutamine length and temperature. The data had suggested that alpha helicity
is predominantly localized in the N-terminal region of the protein and can spread into the N-
terminal part of the polyglutamine domain of the protein (Figure 7). The ordered regions were
found to vary among different temperatures despite the fact that the N-terminal regions were
generally more structured than C-terminal regions (Figure 8).
Figure 6. EPR spectra measured at the indicated temperatures for Trx-HDx1 of various
polyglutamine lengths spin labeled at residue 5 (Adapted from thesis work by Natalie Kegulian)
Figure 7. Temperature-dependent scaled mobility analyses of Q25 and Q46 Trx-HDx1 fusion
proteins spin labeled at a single position, via continuous wave EPR. (Adapted from thesis work
by Natalie Kegulian)
0
1
5 9 11 15 18 21 22 35 50 55 60 90
M
S
Position
Q25 Trx-HDx1
-20 deg
-10 deg
0 deg
4 deg
10 deg
15 deg
20 deg
25 deg
30 deg
37 deg
0
1
1 3 5 7 9 11 15 17 19 21 30 35 48 62 63 66 76
M
S
Position
Q46 Trx-HDx1
-20 deg
-10 deg
0 deg
4 deg
10 deg
15 deg
20 deg
25 deg
30 deg
37 deg
19
Figure 8. Differential scaled mobility plot of spin labeled Q46 Trx-HDx1 constructs at 37
o
C
and 0
o
C. (Adapted from thesis work by Natalie Kegulian)
In order to further characterize the influence of phosphorylation in the N-terminal region
of Trx-HDx1, we utilized continuous wave electron paramagnetic resonance (cw-EPR)
spectroscopy to complete mobility analysis of singly spin labeled Trx-HDx1-S16D fusion
constructs in Q25 and Q46 backgrounds. Trx-HDx1-S16D fusion constructs containing single
cysteine spin label sites at positions 5, 11, and 21 were generated, spin-labeled, and observed
under cw-EPR at 0
o
C. We report that the region around residue 16 exhibited an increase in
mobility upon introduction of the phosphomimetic mutation, aspartic acid (D). The
representative EPR spectra illustrate EPR spectrum of native Trx-HDx1 and phosphomimetic
Trx-HDx1-S16D fusion constructs in the Q46 background spin labeled at position 11 (Figure 9).
We observed that the spectra for position 11 contained two structural components (immobile and
mobile) whether the phosphomimetic mutation was present (Q46 Trx-HDx1-S16D) or not (Q46
Trx-HDx1). Spectral subtraction analysis further highlighted the fact that phosphomimetic
mutation containing Q46 Trx-HDx1 construct contained more of the mobile component while
0
0.2
0.4
0.6
0.8
1
0 10 20 30 40 50 60 70 80
M
S
Position
Q46 37 deg Q46 -10 deg
20
the native Q46 Trx-HDx1 construct contained more of the immobile component (Figure 9). The
influence of S16D mutation on the equilibrium between ordered and disordered structure was
Figure 9. X-band EPR spectra of Q46 Trx-HDx1 constructs labeled at position 11 with the
native S16 (black) or the S16D mutant (red). Both spectra contain two components arising from
different mobility as shown below. The red spectrum contains more of the mobile component
while the black spectrum contains more of the immobile component. Spectra were obtained at
0
o
C. Similar differences were obtained at other temperatures. The spectra on the bottom are the
difference spectra between the respective spectra on the top. Scan width is 100 G.
also detectable in Q46 Trx-HDx1 fusion constructs spin labeled at position 5, where the
phosphomimetic mutation increased the presence of mobile structure (Table 4). However, the
S16D mutation did not affect the structural equilibrium at position 21, which is found in the N-
terminal region of the polyglutamine domain, in the Q46 background. In the Q25 background,
the phosphomimetic mutation at site 16 did induce a small but a detectable increase in the mobile
states at spin label positions 5, and 11 (Table 3). We were able to conclude that the S16D
mutation restores flexibility of Trx-HDx1 monomeric structure, to a level in mobility comparable
to a huntingtin containing a shorter polyglutamine domain.
21
Pre-Filtration Mix Flow Through Overlay
5R1
11R
1
Table 1. EPR spectra of filtered Q25 Trx-HDx1 constructs with SDSL at 5R and 11R positions
Pre-Filtration Mix Flow Through Overlay
5R1
11R1
21R1
Table 2. EPR spectra of filtered Q46 Trx-HDx1 constructs with SDSL at 5R, 11R, and 21R
positions obtained at 0
o
C
22
Spin Label Position Q25 (0
o
C) Q25_S16D Phosphomimicry (0
o
C)
5R1
11R1
21R1
Table 3. EPR spectra of Q25 Trx-HDx1 and phosphomimicry constructs with SDSL at 5R, 11R,
and 21R positions obtained at 0
o
C
23
Spin Label Position Q46 (0
o
C) Q46_S16D Phosphomimicry (0
o
C)
5R1
11R1*
21R1
Table 4. EPR spectra of Q46 Trx-HDx1 and phosphomimicry constructs with SDSL at 5R, 11R,
and 21R positions obtained at 0
o
C
24
Chapter 4: Discussion
In order to study the effect of post-translational modifications, specifically
phosphorylation, we characterized secondary and local structuring of Trx-HDx1 fusion
constructs via circular dichroism (CD) and continuous wave electron paramagnetic resonance
(cw-EPR) spectroscopy, respectively. Generally, prior to this project, we had an initial
understanding that alpha helicity and its temperature-dependent enhancement increase with
higher polyglutamine length (Fodale et al., 2014). The observed, direct relationship in
conformational dynamics was confirmed to be extend to Q7 and Q55 by temperature-dependent
circular dichroism studies (Figure 1). We had begun to build the hypothesis that structuring of
the huntingtin monomer is mediated by alpha helicity and that helical structure increases with
longer polyglutamine length and lower temperature. It was important to further characterize the
location of such helical structure via cw-EPR.
Preliminary cw-EPR analysis of Q25 and Q46 Trx-HDx1 fusion constructs spin labeled
individually at three domains (N-terminus, polyQ, and polyP) showed that majority of helical
structure or ordering is found to be in the N-terminus and the N-terminal portion of the
polyglutamine domain. The mobility analysis was necessary to initially provide us with a
residue-specific assignment of local structure and ordering in the Trx-HDx1 monomer.
Therefore, the polyglutamine length-dependent increase in secondary structuring could be
attributed to the decrease in mobility of the N-terminus and the N-terminal region of the polyQ
domain. The differential mobility was based on normalizing the central linewidths of EPR
spectra from 0 (lowest) to 1 (highest mobility) and represented by using a residue-specific
mobility unit—scaled mobility Ms. In this project, it was our goal to characterize the influence
25
of N-terminal phosphomimetic mutation not only on secondary structure but also on residue-
specific local structuring of Trx-HDx1 monomer.
Before introducing mutations that mimic post-translational modifications
(phosphorylation and acetylation), we performed several control experiments to confirm that any
difference in polyglutamine length-dependent enhancement of helical structure and immobility
was indeed due to modulation of polyglutamine length, not due to a larger presence of
aggregates. As longer polyglutamine domain containing huntingtin protein has been found to
aggregate faster than that of shorter polyglutamine length, it was important that our samples’
identity could be demonstrated biophysically as a monomer. We subjected native Q25 and Q46
Trx-HDx1 fusion constructs spin labeled at position 5, 11, and 21 to 100kD Amicon filtration to
compare the cw-EPR spectra of the pre-filtration mix and the respective post-filtration flow-
through. We demonstrated via our spectral overlays that Trx-HDx1 fusion protein exhibited the
same equilibrium of structural states before and after 100kD filtration (size comparable to
monomer), giving us confidence that the samples we had characterized for preliminary and
current studies were indeed monomeric in solution (Table 1 and 2). In order to further minimize
the propensity of Trx-HDx1 fusion constructs to oligomerize and aggregate, we performed all of
our CD and cw-EPR analysis with samples that were lower than 20 uM in concentration.
Through our temperature-dependent CD analysis of Trx-HDx1 fusion constructs
containing various types phosphorylation and acetylation mimicry mutations, we could
demonstrate that the phosphomimetic mutation at S16 (S16D) impacts the secondary structure of
huntingtin the most. The shift in the MRE distribution of Trx-HDx1 to that of a lower polyQ
construct was induced by the S16D mutation, while the S13D mutation shifted the MRE
distribution to that of a higher polyQ construct. Upon testing the influence of S16D mutation in
26
Q7, Q16, Q25, and Q46 wildtype Trx-HDx1 backgrounds, we found that temperature-dependent
conformational rigidity is lost to an approximate extent in fold enhancement that resembles that
of the next-lower polyglutamine length construct. We also saw a similar but minor attenuation in
fold enhancement in double acetylation mimicking mutants. Among all of the PTM-mimicking
mutations that we have introduced, S16D mutants exhibited the most consistent and significant
change in conformational dynamics due to change in temperature. The results suggest that
phosphorylation in the N-terminal region of the huntingtin exon 1 can play a role in modulating
its monomeric structure in solution.
After confirming that post-translational modifications, specifically phosphorylation, can
influence secondary structure of huntingtin exon 1 monomer, we utilized cw-EPR spectroscopy
to further characterize how and where phosphomimetic mutation at position 16 affects residue-
specific local structure. Phosphomimetic single cysteine Trx-HDx1-S16D mutants were
individually spin labeled at positions 5, 11, and 21 in Q25 and Q46 backgrounds. Differential
spectra analysis of native and phosphomimetic constructs showed that the S16D mutation can
shift the structural equilibrium between ordered and disordered states of Q46 Trx-HDx1 towards
a higher mobile state. The increase in mobile components in Q46 constructs spin labeled at
positions 5 and 11 was a small but a detectable effect of N-terminal S16D mutation. We did also
observe a detectable, but smaller effect of S16D mutation in Q25 Trx-HDx1 fusion proteins
under cw-EPR. The results overall could indicate that the S16D mutation restores flexibility of
huntingtin protein with longer polyglutamine stretch by reducing the local structuring found in
the N-terminus of the huntingtin exon 1. This shift in global and local monomeric structure
towards that of shorter Q native huntingtin protein could implicate restoration of normal
huntingtin function and ultimately prevention of aggregation and toxicity. For future studies, we
27
intend expand our cw-EPR structural analyses of S16D phosphomimetic mutation to Trx-HDx1
fusion constructs containing other polyQ backgrounds such as Q7, Q16, and Q55. Additionally,
huntingtin exon 1 monomers containing real phosphorylation and spin label need to be
characterized under EPR to test whether the phosphomimicry is a valid model for studying the
effect of phosphorylation on local structure.
28
Works Cited
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Davies SW, Turmaine M, Cozens BA, DiFiglia M, Sharp AH, Ross CA, et al. Formation of
neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the
HD mutation. Cell 1997; 90: 537-48.
DiFiglia, M., Sapp, E., Chase, K. O., Davies, S. W., Bates, G. P., Vonsattel, J. P., & Aronin, N.
(1997). Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in
brain. Science, 277(5334), 1990-1993.
Fodale, V., Kegulian, N. C., Verani, M., Cariulo, C., Azzollini, L., Petricca, L., & Caricasole, A.
(2014). Polyglutamine-and temperature-dependent conformational rigidity in mutant huntingtin
revealed by immunoassays and circular dichroism spectroscopy. PloS one, 9(12), e112262.
Gu, X., Greiner, E. R., Mishra, R., Kodali, R., Osmand, A., Finkbeiner, S., ... & Yang, X. W.
(2009). Serines 13 and 16 are critical determinants of full-length human mutant huntingtin
induced disease pathogenesis in HD mice. Neuron,64(6), 828-840.
Harjes, P., & Wanker, E. E. (2003). The hunt for huntingtin function: interaction partners tell
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Jao, C. C., Hegde, B. G., Chen, J., Haworth, I. S., & Langen, R. (2008). Structure of membrane-
bound α-synuclein from site-directed spin labeling and computational refinement. Proceedings of
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Mangiarini, L., Sathasivam, K., Seller, M., Cozens, B., Harper, A., Hetherington, C., & Bates, G.
P. (1996). Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive
neurological phenotype in transgenic mice. Cell, 87(3), 493-506.
McHaourab, H.S., M.A. Lietzow, K. Hideg, and W.L. Hubbell, Motion of spin-labeled side
chains in T4 lysozyme. Correlation with protein structure and dynamics. Biochemistry, 1996.
35(24): p. 7692-704
Modregger, J., DiProspero, N. A., Charles, V., Tagle, D. A., & Plomann, M. (2002). PACSIN 1
interacts with huntingtin and is absent from synaptic varicosities in presymptomatic Huntington's
disease brains. Human molecular genetics, 11(21), 2547-2558.
Nagai, Y., & Popiel, H. A. (2008). Conformational changes and aggregation of expanded
polyglutamine proteins as therapeutic targets of the polyglutamine diseases: exposed β-sheet
hypothesis. Current pharmaceutical design,14(30), 3267-3279.
Ross CA, Poirier MA. Opinion: What is the role of protein aggregation in neurodegeneration?
Nat Rev Mol Cell Biol 2005; 6: 891-8.
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Soto C. Protein misfolding and disease; protein refolding and therapy. FEBS Lett 2001; 498: 204-
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Thompson, L.M., Aiken, C.T., Kaltenbach, L.S., Agrawal, N., Illes, K., Khoshnan, A., Martinez-
Vincente, M., Arrasate, M., O’Rourke, J.G., Khashwji H., et al. (2009). IKK phosphorylates
Huntingtin and targets it for degradation by the proteasome and lysosome. J. Cell Biol., in press.
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Abstract (if available)
Abstract
Expansion of the polyglutamine (polyQ) region in the first exon of huntingtin (htt) is the root of Huntington’s disease (HD). Misfolding and aggregation have long been observed to a greater extent in polyQ-expanded htt, but studies have also uncovered many implications of polyQ expansion in alterations to the dynamics and intermolecular interactions of monomeric htt. We find that htt exon 1 (HDx1), which is composed of the polyQ tract and flanking regions (namely, the 17-aa N-terminus [N17] and the C-terminal proline-rich domain [PRD]) and in expanded form is sufficient to cause HD-like symptoms in transgenic mice, has a greater tendency for adopting α-helical structure in the presence of a polyQ expansion. Our preliminary circular dichroism (CD) studies showed that cooler temperature causes a gain in α-helical structure in HDx1 of a variety of polyQ lengths but a greater gain at disease-level polyQ length. We had performed continuous wave electron paramagnetic resonance (EPR) with site-directed spin labeling (SDSL) and had found the gain in structure to occur in the HDx1 N-terminus and the N-terminal region of the polyQ tract. Our initial findings supported the previously proposed ‘rusty hinge’ hypothesis, which suggests that the normal, but not expanded, polyQ region serves as a flexible hinge that enables intra- and intermolecular interactions of htt. Stiffening of the hinge by polyQ expansion may lead to gains and losses of physiological functions for the protein. In this project, we investigate the effect of N-terminal post-translational modifications by repeating temperature-dependent CD and EPR analyses on HDx1 constructs containing mutations that mimic phosphorylation and acetylation. Here we report that phosphomimetic mutation at position 16 modulates the conformational dynamics of htt exon 1 most consistently across a range of polyQ lengths and that the observed change in secondary structure is due to reduction in local structuring found in the N-terminus. In conclusion, the restoration of conformation flexibility due to S16D implicates a structure based mechanism of huntingtin aggregation and toxicity which could serve as a target for therapeutics.
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Chung, Sean Seungjoon
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Uncovering the influence of N-terminal phosphorylation on conformational dynamics of huntingtin exon 1 monomer
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Keck School of Medicine
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Master of Science
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Biochemistry and Molecular Biology
Publication Date
08/03/2016
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