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Huntington’s disease vaccine development using fungal prion HET-s

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Huntington’s disease vaccine development using fungal prion HET-s

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Huntington’s Disease Vaccine Development Using Fungal Prion HET -s
by
Fengxing Chen

A Thesis Presented to the
FACULTY OF USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Medicine)

August 2021

Copyright 2021 Fengxing Chen
ii

Acknowledgements
I would like to express my most profound gratitude to my research mentor and thesis committee chair,
Dr. Ansgar Siemer, for his patient mentoring, helpful comments, and constant correction of this
research. It has been a great opportunity and honor for me to have a mentor with such rigorous
demeanor and professionalism. He has conveyed his passion for science and will have a profound impact
in my career.
In addition, I would also like to express my sincere gratitude to the other members of my dissertation
committee, Dr. Ralf Langen and Dr. Young-Kwon Hong, for their generous help in my PhD application
and their invaluable guidance in this project.
I would also like to thank Ms. Silvia Cervantes for guiding me through my master’s program and being a
strong support throughout the project. She gives me warm encouragements and professional help
whenever I need them. I would like to give sincere thanks to other members in Siemer Lab as well for
the supportive lab environment and great experience.

iii

Table of Contents
Acknowledgements ....................................................................................................................................... ii
List of Figures ................................................................................................................................................ v
Abstract ........................................................................................................................................................ vi
Chapter I: Introduction ................................................................................................................................. 1
1.1 Huntington’s Disease .......................................................................................................................... 1
1.2 Developing Huntingtin Vaccine ........................................................................................................... 1
1.3 HET-s ................................................................................................................................................... 3
1.4 Site-Directed Mutagenesis .................................................................................................................. 6
1.5 Glutamines on Different Surfaces of HET-s Fibril Core ....................................................................... 7
Chapter II: Results ....................................................................................................................................... 10
2.1 Expression and Purification of HET-s (218-289) ................................................................................ 10
2.2 Betaine Reduces the Non-Specific Binding of HET-s Mutagenesis .................................................. 12
2.3 Expression and Purification of HTT HET-s Fusion Protein ................................................................ 13
2.4 Expression and Purification of HET-s Mutants ................................................................................. 17
2.5 Thioflavin T Fluorescence Assay ....................................................................................................... 19
Chapter III: Discussion ................................................................................................................................. 20
3.1 The PRD Region of HTT Exon 1 Solubilize HET-s ............................................................................... 20
3.2 The Point Mutations Disrupt the Fibrilization Ability of HET-s ........................................................ 20
Chapter IV: Conclusion ................................................................................................................................ 20
Chapter V: Materials and Methods ............................................................................................................. 22
iv

4.1 Transformation and Protein Expression ........................................................................................... 22
4.2 Inclusion Body Purification Protocol ................................................................................................. 22
4.3 Soluble Protein Purification Protocol ................................................................................................ 23
4.4 Immunoblot ...................................................................................................................................... 23
4.5 Thioflavin T Staining .......................................................................................................................... 24
References .................................................................................................................................................. 25

v

List of Figures
Figure. 1 Illustration of the bottlebrush model proposed by Isas et al. ...................................................... 2
Figure. 2 3 β-sheet surfaces construct HET-s solenoid fibril core. ............................................................... 4
Figure. 3 Site-directed mutagenesis. ........................................................................................................... 6
Figure. 4 HET-s fibril core with 2 continuous glutamines mutation on the most exposed β1,3b surface. .. 7
Figure. 5 HET-s fibril core with 2 continuous glutamines mutation on the most hidden β2, 4a surface. ... 8
Figure. 6 HET-s fibril core with 2 continuous glutamines mutation on the kink of the β1,3a and β1,3b
surfaces. ....................................................................................................................................................... 9
Figure. 7 HET-s purification gel. ................................................................................................................. 10
Figure. 8 HET-s fibrils centrifugation. ......................................................................................................... 11
Figure. 9 Betaine reduces the non-specific binding of HET-s mutagenesis. .............................................. 12
Figure. 10 HTT HET-s fusion protein purification gel. ................................................................................ 13
Figure. 11 HTT HET-s fusion protein immunoblot. ..................................................................................... 14
Figure. 12 HTT HET-s fusion protein expression immunoblots. ................................................................. 15
Figure. 13 HTT HET-s fusion protein purified with soluble purification protocol. ..................................... 16
Figure. 14 HET-s mutant A, B, and C purification gel. ................................................................................ 17
Figure. 15 HET-s mutant A, B, and C fibrils centrifugation. ........................................................................ 18
Figure. 16 HET-s mutant A is not ThT positive. .......................................................................................... 19

vi

Abstract
Huntington’s Disease is a progressive neurodegenerative disease caused by abnormal protein folding
and aggregation in the neurons. Similar to other neurodegenerative disorders like Alzheimer’s Disease
and Parkinson’s Disease. The long poly glutamine repeats in huntingtin exon 1 leads to toxic fibril
formation in the neurons and therefore leads to neuron death. Previous studies have shown that
lowering prion protein expression therapies are a promising path forward against prion disease. Another
approach of decreasing toxic fibrils is developing a vaccine against huntingtin fibrils. In this work, we are
designing chimeric amyloid fibrils based on fungal prion HET-s that can serve as such vaccine. We
developed 2 approaches towards huntingtin imitations and by studying the structural changes of the
mutant HET-s, we found valuable insights about HET-s solubility and fibrilization.

1

Introduction
1.1 Huntington’s Disease
Huntington’s Disease (HD) is a rare disorder with a prevalence of 5-10 individuals per 100,000 in the
Caucasian population (Ross et al., 2011). The neuropathology of HD is characterized by the dysfunction
and death of specific neurons within the brain (Vonsattel et al., 2011). The CAG repeats in huntingtin
(HTT) codes for a polymorphic polyglutamine (polyQ) stretch. In the non-HD population, the CAG
sequence is repeated 9 to 35 times, with an average median of between 17 and 20 repeats (Kremer et
al., 1994). Previous studies also show that HTT exon 1 with an expanded CAG repeat is sufficient to
cause a progressive neurological phenotype in transgenic mice (Mangiarini et al., 1996). Suggesting that
mutant HTT exon 1 is causing the protein misfolding and aggregation.
1.2 Developing Huntingtin Vaccine
Due to the growing HD population and the incurable nature of HD. A promising therapeutic approach
towards this disease is protein vaccine development against HTT fibrils. HET-s is a well-studied model
protein that is known to form fibrils like HTT and other amyloids (Wasmer et al., 2008). Our aim is to
develop a HTT fibril mimicry that can act as a HTT vaccine against Huntington’s Disease.
Huntingtin is a multiple conformation protein; the well-studied N-terminal region has the expandable
polyQ. It is preceded by 17 amino acids and a proline-rich domain (PRD) (Sadou & Humbert, 2016). The
structure of HTT fibrils is unknown. Isas et al. have proposed a bottlebrush model for HTT exon 1 fibrils.
The studies suggest that polyQ domain of HTT exon 1 forms the static amyloid core. The rest of HTT
exon 1, especially the proline-rich C-terminus is relatively dynamic, which was found to be in a
polyproline II helical and random coil conformation (Isas et al, 2015) (Figure 1).

2

Figure. 1 Illustration of the bottlebrush model proposed by Isas et al. N17 (orange) and polyQ (blue)
domains form the center of the fibril. The proline-rich C-termini (green) are flexible, partially disordered,
and pointing away from the center forming the bristles in this bottlebrush model.
Polyglutamine core Prolin richdomain
saset al., 2 1
3

In comparison to the 17 amino acids and the PRD in huntingtin. Previous studies suggest that the polyQ
plays a vital role in the fibrilization process by playing two important roles in seeding peptide
aggregation in HD similar to polyQ and glutamine-rich peptides in a plethora of pathological aggregation
events. In the first phase, glutamines mediate seeding by pairing monomeric peptides, which serve as
primers for higher-order nucleation. According to the glutamine content, these low molecular-weight
oligomers may then proceed to create larger aggregates. Once within the aggregates, buried glutamines
continue to play a role in their maturation by optimizing solvent-protected hydrogen bonds networks
(Barrera et al., 2020). However, other studies suggest that the C-terminal proline-rich domain (PRD) of
huntingtin are effective at seeding polyQ aggregation and makes up a large fraction of the fibril surface
(Falk et al., 2020; Lin et al., 2017).
To imitate and investigate the HTT fibril core, we hypothesized 2 methods of HTT mimicry. First is to
make the HET-s fibril core to have exposed glutamines sticking outside of the HET-s fibril core. These
glutamines on the HET-s β -solenoid fibril core will mimic the structure of polyQ fibril core. Another
approach of HTT mimicry is by attaching the C terminal PRD region of HTT exon 1 directly to the end of
HET-s. This fusion protein will have a fibril core of HET-s with an attached HTT exon 1 PRD surface.
1.3 HET-s
HET-s is a prion protein involved in a genetically controlled cell death in the fungus Podospora anserina
(Coustou et al., 1997). Previous studies show that HET-s is a self-perpetuating amyloid aggregate. Wild-
type HET-s is 289 amino acids in length. However, the region of the HET-s protein that is responsible for
amyloid formation and propagation is residues 218-289 (Balguerie et al., 2003). Therefore, in this study,
HET-s 218-289 is used as the template protein for HTT mimicking modification.
Wasmer et al. present a structural model based on solid-state nuclear magnetic resonance restraints for
amyloid fibrils from the prion-forming domain (residues 218-289) of the HET-s protein. They find that
4

HET-s(218–289) forms a left-handed β solenoid, with each molecule forming two helical windings, a
compact hydrophobic core, at least 23 hydrogen bonds, three salt bridges, and two asparagine ladders
(Wasmer et al., 2008). The HET-s solenoid fibril core has 3 β-sheet surfaces which is ideal for glutamine
mutations (Figure. 2).

Figure. 2 3 β-sheet surfaces construct HET-s solenoid fibril core. β1, 3b surface is the most exposed
surface of HET-s fibril core. β2, 4b surface is the most hidden surface that is hidden by β2, 4a surface and
the C terminus.
2,4a
2,4b
1,3b
1,3a
5

Mutagenesis of glutamines point mutations allow a specific surface of the solenoid to be a “pseudo-
glutamine surface”. Which will potentially be a good HTT fibril core imitator. This mutant thus can be a
good candidate for HD vaccine.
6

1.4 Site-Directed Mutagenesis
To mimic the HTT fibril core, either 3 or 4 mutation sites need to be mutated into glutamines. Site-
directed mutagenesis (SDM) is a good technique to achieve this goal. This technique uses synthesized
oligonucleotides (primers) which is complementary to part of a ssDNA template but with one or few
mismatch points (Carter, 1986)(Figure. 3). In this experiment, in order to mimic different structural
features of HTT fibril core, 3 plans of mutation sites are chosen for SDM.

Figure. 3 Site-directed mutagenesis. Schematic representation of the site-directed mutagenesis (SDM)

Polymerase and
igase
7

1.5 Glutamines on Different Surfaces of HET-s Fibril Core
For the mutation plan A, glutamines are put on the most exposed β1,3b surface. Defining the first HET-s
(218-289) residue as residue 1, the mutation sites are D14Q, R16Q on N terminus and T50Q, V52Q on
the C terminus of HET-s. These sites are specifically chosen so that there will be 2 continuous glutamines
on this surface (Figure. 4).

Figure. 4 HET-s fibril core with 2 continuous glutamines mutation on the most exposed β1,3b surface .
A) and B) are different point of view of the HET-s fibril core. Blue arrows on both images are β-sheets on
the fibril core. Highlighted yellow molecules are the target molecules for glutamines mutation.
1,3b surface
1,3b surface

lutamines
8

Mutation plan B is to put glutamines on the most hidden β2, 4a surface. Defining the first HET-s (218-
289) residue as residue 1, mutation sites for the second plan are R22Q on N terminus and R58Q, L60Q
on the C terminus. Because site 24 is already a glutamine, this plan will be a 3 mutations mutagenesis
(Figure. 5).

Figure. 5 HET-s fibril core with 2 continuous glutamines mutation on the most hidden β2, 4a surface.
A) and B) are different point of view of the HET-s fibril core. Blue arrows on both images are β-sheets on
the fibril core. Highlighted yellow molecules are the target molecules for glutamines mutation.

2, 4a surface
2, 4a surface lutamines

9

Mutation plan C does not have any mutation on any surface. Instead, the glutamines are put on the kink
of the β1,3a and β1,3b surfaces. Defining the first HET-s (218-289) residue as residue 1, mutation sites
are K13Q, D14Q on the N terminus and E49Q, T50Q on the C terminus (Figure. 6).

Figure. 6 HET-s fibril core with 2 continuous glutamines mutation on the kink of the β1,3a and β1,3b
surfaces. A) and B) are different point of view of the HET-s fibril core. Blue arrows on both images are β-
sheets on the fibril core. Highlighted yellow molecules are the target molecules for glutamines mutation.

ink ink
lutamines

10

Results
2.1 Expression and Purification of HET-s (218-289)
The plasmid was transformed into lab-made calcium chloride chemically competent BL21 E.coli cells.
The cell culture reached OD of 0.5 in approximately 4 hours. After 4 hours of expression after Isopropyl
β-D-1-thiogalactopyranoside (IPTG) induction. The protein was dissolved in the denaturing buffer then
apply to nickel column. The protein was eluted with 200mM imidazole buffer. The SDS-PAGE image
indicated that the protein was successfully expressed in BL21 cells (Figure. 7).

Figure. 7 HET-s purification gel. The molecular weight of HET-s is 9. 4 kDa. The “Denature” and “Flow
through” columns were affected by the 6M guanidine denaturing buffer, which affects protein running
down the SDS-PAGE results.
kDa

2
11

HET s
11

The elution was dialyzed in 4 ℃ for fibrilization. μ fibrilized samples are collected and centrifuged
down, and the SDS-PAGE indicated that most of the soluble protein were fibrilized (Figure. 8).

Figure. 8 HET-s fibrils centrifugation. The band width of centrifuged fibrils pellet is a lot larger than the
supernatant, suggesting that most of the HET-s fibrils fibrilized in one week.

kDa

2
11

12

2.2 Betaine Reduces the Non-Specific Binding of HET-s Mutagenesis
We started with primers for the SDM reactions with 2 mismatches on each primer and primer lengths
are between 45-62 nucleotides. The SDM reaction did not yield significant amount of mutant plasmid
(Figure 9A). This is likely due to the high GC content in the sequence that leads to relatively high Tm
(78.50℃-79.88℃).
In order to minimize the energy cost for the mismatches. We redesigned the primers in a stepwise
manner with only 1 mismatch on each primer. However, the SDM reactions still did not yield a
significant amount of mutant plasmid but smears (Figure. 9B).
Based on the agarose gel electrophoresis, the PCR reactions seem to have faint smears which indicated
unspecific binding of the polymerase. Therefore, betaine was added to both reactions for reducing
unspecific binding of the polymerase. It seems like a high concentration of betaine (1M) does help with
unspecific binding in the HET-s mutagenesis remarkably (Figure. 9C). However, does not help with the
yield of the reaction. The mutant plasmids were synthesized from GenScript for expression and
purification.

Figure. 9 Betaine reduces the non-specific binding of HET-s mutagenesis. Smears in B) are eliminated
by 1M betaine.

13

2.3 Expression and Purification of HTT HET-s Fusion Protein
The plasmid was transformed into the same cell line as HET-s. Same expression and denaturing
purification protocol was applied. The SDS-PAGE indicated that there is not a specific protein that binds
to the column (Figure. 10).

Figure. 10 HTT HET-s fusion protein purification gel. The molecular weight of HTT HET-s fusion protein is
13. 1 kDa. The “Denature” and “Flow through” columns were affected by the 6M guanidine denaturing
buffer, which affects protein running down the SDS-PAGE results.

kDa

2
11

14

The immunoblotting analysis result suggests that there is low level of the fusion protein is in the soluble
fraction of the cell instead of inside the inclusion body (Figure. 11).

Figure. 11 HTT HET-s fusion protein immunoblot. The molecular weight of HTT HET-s fusion protein is
13.01 kDa. The primary antibodies used in this immunoblotting are 6X-his tag antibodies. The target
protein exists in whole cell lysate, supernatant, and the wash indicating that the target protein is in the
soluble portion of the cell.

kDa

2
11

15

The expression and purification protocols were then altered. According to the immunoblot, the protein
expression volume in 37℃ is extremely low comparing to it in 18℃ or 25℃ (Figure. 12).

Figure. 12 HTT HET-s fusion protein expression immunoblots. The molecular weight of HTT HET-s fusion
protein is 13.01 kDa. The primary antibodies used in this immunoblotting are 6X-his tag antibodies. The
fusion protein does not exist in pre-induction. The fusion protein had low expression level at 37℃. 25℃
overnight expression is the optimum condition for fusion protein expression.

kDa

2
11

16

The purification protocol was then altered. A soluble protein purification protocol which uses the
supernatant of the whole cell lysate instead of the denatured pellet to apply to the nickel column.
According to the SDS-PAGE analysis, the protein was successfully purified (Figure. 13).

Figure. 13 HTT HET-s fusion protein purified with soluble purification protocol. The molecular weight of
HTT HET-s fusion protein is 13.01 kDa. HTT HET-s fusion protein was successfully purified.

HTT HET s
fusion protein
kDa

2
11

17

2.4 Expression and Purification of HET-s Mutants
All HET-s mutagenesis mutants are expressed and purified using protocols from HET-s. According to the
SDS-PAGE image, mutant A and B were expressed and purified successfully (Figure. 14). However, HET-s
mutant C has low yield compare to A and B.

Figure. 14 HET-s mutant A, B, and C purification gel. A) HET-s mutant A was successfully expressed and
purified. B) HET-s mutant B was successfully expressed and purified. C) HET-s mutant C had very low
yield compared to mutant A and B. The “Denature” and “Flow through” columns were affected by the
6M guanidine denaturing buffer, which affects protein running down the SDS-PAGE results.

HET s A HET s HET s
18

Elution from all 3 plans were dialyzed in 4℃ and aggregation formed for all 3 mutants. 50 μL of each
aggregated sample is collected and centrifuged down, and the SDS-PAGE indicated that most of the
soluble protein are in the insoluble aggregates (Figure. 15).

Figure. 15 HET-s mutant A, B, and C fibrils centrifugation. Most of the proteins aggregate in HET-s
mutant A, B, and C in one week.

kDa

2
11
19

2.5 Thioflavin T Fluorescence Assay
To investigate the mutants’ ability to form fibrils , thioflavin T fluorescence assay (ThT) was used for
quantifying the β-sheet formation in the aggregates. Samples from most aggregated mutant A is ThT
negative. (Figure. 16)

Figure. 16 HET-s mutant A is not ThT positive. A positive ThT value should be more than 10 folds greater
than the values in the negative control. In our ThT assay analysis, the value is too low to be conclusive.
3 .3
.
62.3
9.
2 .3
1 .
93.
91.

2
4
6

1
12
ThT nega ve control u er nega ve
control
u er ThT nega ve
control
H ThT posi ve
control
Empty well Sonicated sample
with ThT
Sample ThT Sample extra ThT
Absorbance 4 4nm
HET s Mutant A ThT esualt
Fluorescence
20

Discussion
3.1 The PRD Region of HTT Exon 1 Solubilize HET-s
Surprisingly, the PRD region of the HTT exon 1 solubilize the fungal prion HET-s in vitro. Even though this
result makes it difficult for structural study since the protein is soluble and unable to fibrilize under
standard condition, the results give interesting insights about HET-s fibrilization. The C terminus tail of
the HET-s molecule seems to play an unexpected role in HET-s fibrilization. Moreover, our results further
confirms that the PRD region of the HTT is soluble and not aggregation prone.
3.2 The Point Mutations Disrupt the Fibrilization Ability of HET-s
The ThT fluorescence data indicated that the aggregation in the mutant solution seems to be non-
specific aggregation instead of fibrilization. Sonication of the aggregation on 20% amplification for 3
seconds seems to help with the ThT value slightly. However, the values are not conclusive to be β-sheet
positive. Unfortunately, electron microscopy is in maintenance and we cannot further confirm the
structure of the aggregates.
Based on the ThT data, point mutations of glutamines which cause fibrilization in HTT exon 1 seems to
disrupt the fibrilization ability of HET-s. The HET-s protein is, however, still aggregation prone. This result
gives unique insights on prion aggregation and fibrilization. Further experiments for structural study of
this protein are needed.
Conclusion
In this study, we proposed 2 approaches towards HTT mimicry and several versions of the imitations.
The physical characteristics of some mutants change dramatically due to the mutation. The fusion
protein with the HTT exon 1 PRD tail becomes soluble unlike the original prion. The change in solubility
21

of the HET-s suggest that the C terminus of HET-s molecule might be a key fragment for HET-s solubility.
In the future, experiments on HET-s C terminus modification will gives insights on prion fibrilization.
Furthermore, the point mutations on the HET-s disrupt the secondary structure of HET-s. It would be
interesting to investigate the structure of the mutant aggregation. It seems like even though the mutant
protein remain aggregation prone, it does not form fibrils anymore.

22

Materials and Methods
4.1 Transformation and Protein Expression
BL21 DE3 cells are used for protein expression. 200μL of cells were taken out from -80 ℃ freezer and
thawed on ice. The cells were then transferred to a pre-cooled 5mL culture tube and at least 50 ng of
DNA were added to the culture tube under sterile condition. Cell DNA mixture were incubated on ice for
10 minutes then heat in 42 ℃ water bath for exactly 30 seconds without shaking. Cell DNA mixture were
put back on ice for another 2 mins before 800μL of SOC medium were added under sterile conditions.
The cell culture was then incubated for about 1 hour at 37 ℃ and 225rpm. 100μL of incubated cell
culture was plated on LB agarose plates containing the corresponding antibiotics. The plates were then
incubated overnight at 37 ℃.
A single transformed colony of the was incubated with 25mL of LB Miller broth with 25μ of antibiotics.
Cell culture were incubated overnight at 30 ℃ 200rpm. 1mL of overnight culture was then transferred to
a flask of 1L LB broth with 1mL of antibiotics. The culture was incubated at 37 ℃ and 225rpm for about 4
hours until the optical density reached . . Protein expression was then induced with μ of 1M
sopropyl β - d-1-thiogalactopyranoside (IPTG). The culture was incubated either at 37℃ and 225rpm for
4 hours or 25℃ and 225rpm overnight depending on the mutants. To isolate cell pellet, cells were
centrifuged at 4,000G for 20 minutes. Cell pellets were stored in -80℃ freezer.
4.2 Inclusion Body Purification Protocol
Cell pellet was thawed on ice and lysed in 35-40mL of 150 mM NaCl and 100 mM Tris-HCl, pH 8. Pellet
was vortexed till completely dissolved in the buffer. The mixture was then placed on ice and sonicated
four times at 75 amplification for 30 seconds. The mixture was then sonicated the fifth time at 80
amplification for 30 seconds. The whole cell lysate was centrifuged at 20,000G for 20 minutes.
Supernatant was discarded and the pellet was resuspended and washed in the same buffer and
23

centrifuged down again. The supernatant was again discarded, and pellet was suspended in 40mL of
denaturing buffer with 6 M guanidinium HCl, 150 mM NaCl, and 100 mM Tris-HCl, pH 8. The solution
was incubated with pre-equilibrated Ni-NTA column for 1 hour at room temperature, and the resin was
washed with 8 M urea, 150 mM NaCl, and 100 mM Tris-HCl, pH 8. 5). The protein was eluted from the
resin in the same buffer containing 200 mM imidazole.
4.3 Soluble Protein Purification Protocol
The cell pellet was thawed on ice and dissolved in 25mL of denaturing buffer with 100mM NAH2PO4,
10mM Tris-HCl, and 8M Urea, pH 8. The mixture was sonicated at 75 amplification for 6 minutes. Lysate
was centrifuged at 20,000G for 20 minutes. Supernatant was then incubated with pre-equilibrated Ni-
NTA column for 1 hour at room temperature. The column was washed with 1) 25mL denaturing buffer
with 0.5% Triton X-100, 2) 25mL of denaturing buffer with 500mM NaCl, 3) 25mL of denaturing buffer of
pH 6.75, 4) 25mL of renaturing buffer with 50mM NAH2PO4, 200mM NaCl, and 10% glycerol, pH 8, 5)
25mL renaturing buffer with 20mM imidazole. The protein was eluted from the resin using renaturing
buffer with 200mM imidazole.
4.4 Immunoblot
Normal protocol of SDS-PAGE with a reference ladder was performed. 1liter of 10X transfer buffer was
prepared by mixing 30.3 g Tris, 144.1 g Glycine. 1X transfer buffer was prepared with 100ml of 10X
transfer buffer, 200ml methanol and 700 ml water. Membrane transfer was done with 1X transfer buffer
at 100V for 1 hour. The membrane was soaked in blocking solution (5% milk powder) followed by 3
washing steps with TBST buffer (8g NaCl, 2.4g Tris, 10µl Tween 20 in 1liter water, pH = 7.6). The
membrane was then incubated with primary antibody solution (Anti- 6XHis epitope tag, rabbit IgG
antibody) by either incubating for 1 hour at room temperature of incubating overnight at 4°C with
constant agitation. The membrane was again washed with TBST buffer and the same step was repeated
24

for secondary antibody (Anti Rabbit -IgG, goat peroxidase conjugated antibody). After poring off the
secondary antibody and washing the membrane with TBST, the membrane was soaked in 2ml of GE
detection solution 1 and GE detection solution 2 for 30 seconds before imaging.
4.5 Thioflavin T Staining
Amyloid fibrils formation was confirmed using ThT fluorescence at 482nm. 100μL of the sample
containing the protein as well as the baseline solution (150 mM NaCl and 100 mM Tris-HCl, pH 8) were
incubated with 50μL of Thioflavin T stock solution. Excitation at 450nm, slit width 1nm and emission
460-500nm. Slit width 10nm were used as measuring parameters.

25

References
Balguerie, A., Dos Reis, S., Ritter, C., Chaignepain, S., Coulary-Salin, B., Forge, V., Bathany, K., Lascu,
I., Schmitter, J.-M., Riek, R., & Saupe, S. J. (2003). Domain organization and structure–function
relationship of the HET-s prion protein of Podospora anserina. The EMBO Journal, 22(9), 2071–
2081. https://doi.org/10.1093/emboj/cdg213
Barrera, E. (n.d.). Dissecting the role of glutamine in seeding peptide aggregation. 15.
Carter, P. (1986). Site-directed mutagenesis. Biochemical Journal, 237(1), 1–7.
Coustou, V., Deleu, C., Saupe, S., & Begueret, J. (1997). The protein product of the het-s heterokaryon
incompatibility gene of the fungus Podospora anserina behaves as a prion analog. Proceedings of
the National Academy of Sciences, 94(18), 9773–9778. https://doi.org/10.1073/pnas.94.18.9773
Hoop, C. L., Lin, H.-K., Kar, K., Magyarfalvi, G., Lamley, J. M., Boatz, J. C., Mandal, A., Lewandowski,
J. R., Wetzel, R., & van der Wel, P. C. A. (2016). Huntingtin exon 1 fibrils feature an
interdigitated β-hairpin–based polyglutamine core. Proceedings of the National Academy of
Sciences of the United States of America, 113(6), 1546–1551.
Isas, J. M., Langen, R., & Siemer, A. B. (2015). Solid-State Nuclear Magnetic Resonance on the
Static and Dynamic Domains of Huntingtin Exon-1 Fibrils. Biochemistry, 54(25), 3942–
3949. https://doi.org/10.1021/acs.biochem.5b00281
Kim, M. W., Chelliah, Y., Kim, S. W., Otwinowski, Z., & Bezprozvanny, I. (2009). Secondary Structure
of Huntingtin Amino-Terminal Region. Structure, 17(9), 1205–1212.
https://doi.org/10.1016/j.str.2009.08.002
Kremer, B., Goldberg, P., Andrew, S. E., Theilmann, J., Telenius, H., Zeisler, J., Squitieri, F., Lin, B.,
Bassett, A., Almqvist, E., Bird, T. D., & Hayden, M. R. (1994). A Worldwide Study of the
Huntington’s Disease Mutation: The Sensitivity and Specificity of Measuring CAG Repeats. New
England Journal of Medicine, 330(20), 1401–1406.
https://doi.org/10.1056/NEJM199405193302001
26

Makin, O. S., & Serpell, L. C. (2005). Structures for amyloid fibrils. The FEBS Journal, 272(23), 5950–
5961. https://doi.org/10.1111/j.1742-4658.2005.05025.x
Mangiarini, L., Sathasivam, K., Seller, M., Cozens, B., Harper, A., Hetherington, C., Lawton, M.,
Trottier, Y., Lehrach, H., Davies, S. W., & 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. https://doi.org/10.1016/S0092-8674(00)81369-0
Minikel, E. V., Zhao, H. T., Le, J., O’Moore, J., Pitstick, R., Graffam, S., Carlson, G. A., Kavanaugh, M.
P., Kriz, J., Kim, J. B., Ma, J., Wille, H., Aiken, J., McKenzie, D., Doh-Ura, K., Beck, M.,
O’Keefe, R., Stathopoulos, J., Caron, T., … Vallabh, S. M. (2020). Prion protein lowering is a
disease-modifying therapy across prion disease stages, strains and endpoints. Nucleic Acids
Research, 48(19), 10615–10631. https://doi.org/10.1093/nar/gkaa616
Muller, S., Zhao, Y., Brown, T. L., Morgan, A. C., & Kohler, H. (2005). TransMabs: Cell-penetrating
antibodies, the next generation. Expert Opinion on Biological Therapy, 5(2), 237–241.
https://doi.org/10.1517/14712598.5.2.237
Reis, S. D., Coulary-Salin, B., Forge, V., Lascu, I., Bégueret, J., & Saupe, S. J. (2002). The HET-s Prion
Protein of the Filamentous Fungus Podospora anserina Aggregates in Vitro into Amyloid-like
Fibrils *. Journal of Biological Chemistry, 277(8), 5703–5706.
https://doi.org/10.1074/jbc.M110183200
Review: History of the Amyloid Fibril | Elsevier Enhanced Reader. (n.d.).
https://doi.org/10.1006/jsbi.2000.4221
Riek, R., & Saupe, S. J. (2016). The HET-S/s Prion Motif in the Control of Programmed Cell Death. Cold
Spring Harbor Perspectives in Biology, 8(9), a023515.
https://doi.org/10.1101/cshperspect.a023515
Ritter, C., Maddelein, M.-L., Siemer, A. B., Lührs, T., Ernst, M., Meier, B. H., Saupe, S., & Riek, R.
(2005). Correlation of structure and infectivity of the HET-s prion. Nature, 435(7043), 844–848.
https://doi.org/10.1038/nature03793
27

Ross, C. A., & Tabrizi, S. J. (2011). Huntington’s disease: From molecular pathogenesis to clinical
treatment. The Lancet Neurology, 10(1), 83–98. https://doi.org/10.1016/S1474-4422(10)70245-3
Scherzinger, E., Sittler, A., Schweiger, K., Heiser, V., Lurz, R., Hasenbank, R., Bates, G. P., Lehrach, H.,
& Wanker, E. E. (1999). Self-assembly of polyglutamine-containing huntingtin fragments into
amyloid-like fibrils: Implications for Huntington’s disease pathology. Proceedings of the
National Academy of Sciences of the United States of America, 96(8), 4604–4609.
https://doi.org/10.1073/pnas.96.8.4604
Saudou, F., & Humbert, S. (2016). The Biology of Huntingtin | Elsevier Enhanced Reader. (n.d.).
https://doi.org/10.1016/j.neuron.2016.02.003
Vonsattel, J. P. G., Keller, C., & Cortes Ramirez, E. P. (2011). Chapter 4—Huntington’s disease –
neuropathology. In W. J. Weiner & E. Tolosa (Eds.), Handbook of Clinical Neurology (Vol. 100,
pp. 83–100). Elsevier. https://doi.org/10.1016/B978-0-444-52014-2.00004-5
Wasmer, C., Lange, A., Melckebeke, H. V., Siemer, A. B., Riek, R., & Meier, B. H. (2008). Amyloid
Fibrils of the HET-s(218–289) Prion Form a β Solenoid with a Triangular Hydrophobic Core.
Science, 319(5869), 1523–1526. https://doi.org/10.1126/science.1151839

Abstract (if available)

Abstract Huntington’s Disease is a progressive neurodegenerative disease caused by abnormal protein folding and aggregation in the neurons. Similar to other neurodegenerative disorders like Alzheimer’s Disease and Parkinson’s Disease. The long polyglutamine repeats in huntingtin exon 1 leads to toxic fibril formation in the neurons and therefore leads to neuron death. Previous studies have shown that lowering prion protein expression therapies are a promising path forward against prion disease. Another approach of decreasing toxic fibrils is developing a vaccine against huntingtin fibrils. In this work, we are designing chimeric amyloid fibrils based on fungal prion HET-s that can serve as such vaccine. We developed 2 approaches towards huntingtin imitations and by studying the structural changes of the mutant HET-s, we found valuable insights about HET-s solubility and fibrilization.

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Asset Metadata

Creator Chen, Fengxing (author)

Core Title Huntington’s disease vaccine development using fungal prion HET-s

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Medicine

Degree Conferral Date 2021-08

Publication Date 07/29/2021

Defense Date 06/04/2021

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

Tag HET-s,huntingtin,Huntington's disease,OAI-PMH Harvest,vaccine development

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Siemer, Ansgar (committee chair), Hong, Young-Kwon (committee member), Langen, Ralf (committee member)

Creator Email 497478695@qq.com,fengxing@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC15659707

Unique identifier UC15659707

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