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mRNA oxidation and its relation to p53 amyloid formation and disease

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mRNA oxidation and its relation to p53 amyloid formation and disease

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Content MRNA OXIDATION AND ITS RELATION TO P53 AMYLOID FORMATION AND
DISEASE
by
Kayla Love
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMISTRY)
DECEMBER 2024
Copyright 2024 Kayla Love

ii
Acknowledgements
I would like to acknowledge God, my family, friends and mentors. I deeply appreciate
your encouragement, support and guidance along this journey. To the next young Black Queen
or King who finds herself in new spaces, you can be authentic and un-apologetically black, be a
parent, and put your health first. It’s always worth it. To anyone pursuing great goals, never give
up on your aspirations or dim your light, as challenging as it may be. I am happy to pay it
forward, because “The revolution will always be in the hands of the young.” - Dr. Huey P.
Newton.

TABLE OF CONTENTS
Acknowledgements......................................................................................................................... ii
List of Figures................................................................................................................................. 4
Abstract ........................................................................................................................................... v
Introduction..................................................................................................................................... 1
Broader Impact.......................................................................................................................... 13
Approach................................................................................................................................... 14
Circle Sequencing & PLAAC....................................................................................................... 15
Recombinant engineering, expression and purification of the S149F mutant .............................. 17
p53 S149F purification.............................................................................................................. 18
Initial S149F Seeding Congo Red Staining & TEM Analysis.................................................. 18
Characterization of P53/S149f Aggregates: TEM, MALS, and AFM...................................... 19
CHAPTER 1: S149F and R175H SEEDING P53 ........................................................................ 21
Generating the S149F mutant ................................................................................................... 21
P53 and S149F overexpression and purification....................................................................... 24
TEM of amyloids generated by S149F seeding p53 ................................................................. 27
AFM of amyloids/fibers generated by S149F seeding p53....................................................... 30
Amyloid Fiber Hanging Drop Assay S149F Seeding p53.................................................... 30
S149F seeding p53 on Grid for TEM Analysis..................................................................... 33
S149F Seed Size Determination ........................................................................................... 35
R175H Seeding of p53 Induces Fiber Formation ................................................................. 36
S149F and R175H Seeding p53 Discussion ......................................................................... 43
CHAPTER 2: V30M SEEDING MTTR VIA AMYLOID FIBER HANGING DROP ASSAY. 45
Background ............................................................................................................................... 45
RESULTS ..................................................................................................................................... 47
pH Dependent V30M Seeding of MTTR.................................................................................. 47
V30M Seeding of MTTR at pH 5.4 .......................................................................................... 49
pH Dependent V30M Seeding of MTTR at pH 7.4.................................................................. 50
MTTR fibers seeded with V30M on the grid for TEM analysis............................................... 51
Discussion V30M seeding MTTR ............................................................................................ 53
CHAPTER 3: SOD1 SEEDING BY G142E USING AMYLOID HANGING DROP ASSAY.. 56
G142E Seeding of SOD1 using Amyloid Fiber Hanging Drop Assay ..................................... 57

G142E Seeding SOD1 Discussion............................................................................................ 59
CHAPTER 4 ......................................................................................................................... 61
Closing Discussion................................................................................................................ 61
R175H & S149F Fiber Extraction & X-ray Diffraction ........................................................... 64
Separation of Fibers for X-ray Diffraction ........................................................................... 64
Schematic Summary ............................................................................................................. 66
METHODS ................................................................................................................................... 66
Protein Expression of p53, S149F, R175H , MTTR, SOD1, G142A ....................................... 67
Purification................................................................................................................................ 68
Amyloid Fiber Hanging Drop Assay ........................................................................................ 69
MTTR V30M seed preparation............................................................................................. 69
SOD1 G142E Seed Preparation ............................................................................................ 70
R175H/ S149F Seeding of P53 ............................................................................................. 70
S149F Seeding of P53 Setup................................................................................................. 71
R175H Seeding of P53 Setup................................................................................................ 72
G142E Seeding of SOD1 Setup ............................................................................................ 73
Microscopy of Protein Fibers.................................................................................................... 73
SEC-MALS of amyloid seeds................................................................................................... 74
REFERENCES.......................................................................................................................... 75

List of Figures
Figure 1 mRNA Oxidation and its Relation to p53 Amyloid Formation and Disease Logic ......... 6
Figure 2 Detecting p53 mRNA sequence errors by circle sequencing ........................................... 9
Figure 3 Identification of prion-promoting RNA errors using PLAAC ....................................... 10
Figure 4 Schematic of Amyloid Fiber Hanging Drop Assay........................................................ 13
Figure 5 S149F location on p53 structure..................................................................................... 17
Figure 6 Snap Gene design of S149F............................................................................................ 22
Figure 7 Translational analysis of S149F ..................................................................................... 23
Figure 8 p53 and S149F overexpression....................................................................................... 25
Figure 9 p53 and S149F purification ............................................................................................ 26
Figure 10 S149F purification ........................................................................................................ 27
Figure 11 Seeding of p53 with 1% S149F .................................................................................... 29
Figure 12 Seeding of p53 with S149F .......................................................................................... 30
Figure 13 p53 seeded with S149F induces fiber formation .......................................................... 32
Figure 14 S149F & R175H seeding on TEM grid ........................................................................ 34
Figure 15 TEM of P53 seeded with S149F grown on grid Negative staining of fiber samples. .. 35
Figure 16 S149F seed size determination by MALS. ................................................................... 36
Figure 17 R175H location on p53 structure.................................................................................. 37
Figure 18 R175H purification....................................................................................................... 39
Figure 19 Structure comparison between p53 seeded with R175H aggregates vs S149F............ 40
Figure 20 Increase in fibril growth and branching of 0.5% R175H seeding of p53..................... 41
Figure 21 AFM of R175H and S149F seeding wt p53 ................................................................. 42
Figure 22 R175H Seed Size Determination by MALS................................................................. 43
Figure 23 pH 4.3 dependent V30M seeding of MTTR alters morphology................................... 49
Figure 24 Increase in fibril growth and branching of 1% V30M seeding of MTTR at pH 5.4 .... 50
Figure 25 pH 7.4 dependent V30M seeding of MTTR alters morphology................................... 51
Figure 26 MTTR seeded with V30M Fibers at pH 5.4................................................................. 52
Figure 27 V30M Seeding of MTTR on TEM Grid....................................................................... 53
Figure 28 SOD1 structure and location of G142E........................................................................ 57
Figure 29 SOD1 & G142E purification ........................................................................................ 58
Figure 30 1% G142E seeding of SOD1 at isoelectric point leads to fiber formation................... 59
Figure 31 Separation of S149F seeding P53 samples for X-Ray Fiber Diffraction ..................... 65
Figure 32 Schematic Summary ..................................................................................................... 66

v
Abstract
mRNA Oxidation and its Relation to p53 Amyloid Formation and Disease
The tumor suppressor protein p53, often referred to as the "guardian of the genome," plays a
critical role in maintaining genomic stability by regulating cell division and apoptosis. Recent
studies have shown that mutations in the p53 gene can lead to protein misfolding and prion-like
amyloid aggregation, contributing to the pathology of various diseases, including cancer,
Alzheimer's, cardiovascular, and respiratory diseases. Interestingly, approximately 50% of
cancer patients exhibit p53 loss of function without detectable mutations in the p53 gene. This
suggests the involvement of alternative mechanisms. We hypothesize that A small number of
mRNA mutations may cause p53 protein misfolding, leading to p53 amyloid fibrils and prion-like
activity. Current amyloid fiber detection methods, primarily reliant on increases in optical
density and X-ray diffraction, are limited in their ability to distinguish between true amyloid
structures and non-specific background precipitation. There are currently no high throughput
methods for screening amyloid fiber formation that can distinguish from background amorphous
precipitation. To address this limitation, I have developed a novel Amyloid Hanging Drop Assay,
adapted from traditional protein crystallization techniques. This method allows for the direct
observation of amyloid fiber formation under a light microscope, with the use of polarized light
to confirm fiber characteristics through birefringence. The assay significantly enhances the
ability to study the seeding behavior of mutant proteins, providing a robust platform for the
identification of small molecules that inhibit amyloid formation. This assay holds potential as a

vi
valuable tool for drug discovery, targeting amyloid-related diseases such as Alzheimer’s, cancer,
and cardiovascular disorders.

1
Introduction
p53, is one of the most extensively studied tumor suppressor proteins, recognized for its
critical role in regulation of cell cycle and apoptosis 1,2. It is frequently examined in relation to its
expression, mutation profile, and functional dynamics across a wide range of tumor types 1,2
.
Through its regulatory control over cell division and programmed cell death, p53 serves as a key
defense mechanism against tumorigenesis 1,2
.
Mutations in the p53 gene are known to result in loss of function, amyloidosis, or prionlike aggregation, which can act in a gain-of-function manner in conditions such as breast cancer
³, neuroblastoma ⁴, and cardiomyopathy ⁵. The phenomenon of p53 aggregation has been
observed in a wide variety of cells 3,4,5 . Researchers have reported evidence of p53 aggregation
in the pathology of Alzheimer's Disease (AD), forming oligomers and fibrils in the human AD
frontal cortex of the brain 4 . Recent studies propose that p53 aggregation-induced loss of
function may be a contributing factor to these diseases, potentially classifying them as p53
amyloid diseases6
. The formation of amyloid aggregates involving p53 is often associated with
mutations in the p53 gene ⁷. More than 50% of cancer patients are reported to have p53 gene
mutations⁸; however, in some cases, p53 loses function without alterations in its DNA sequence⁸
This observation suggests that factors beyond DNA mutations may contribute to p53
dysfunction and amyloid plaque formation. The mechanisms underlying p53 dysfunction without
gene mutations remain poorly understood. RNA, a major target of oxidative damage in AD
patients⁹, is less studied for oxidative damage compared to DNA because RNA modifications are

2
not typically inheritable and are less likely to be mutagenic 9,10. However, the small amount of
oxidatively damaged RNA may lead to misincorporation of incorrect amino acids during
translation9,10. This may potentially lead to the production of proteins with amyloid or prion-like
activities that can affect wild-type proteins ¹¹.
We postulated that A small number of mRNA mutations may cause p53 mutations, leading to
p53 amyloid fibrils and prion-like activity.
The overarching goal of this study is to determine how mutations in the p53 mRNA
may lead to mutant p53 proteins that can act as a dominant-negative inhibitor toward wild-type
p53, leading to amyloid fibers. Insights from this research could help in developing more
targeted treatments for patients who are facing or are at risk for amyloid-associated diseases,
such as cancer and Alzheimer's disease.
To do so, my thesis research addressed the following key gaps in our knowledge:
1. It is unclear if rare mRNA errors may lead to p53 protein aggregation.
When cancer, Alzheimer’s, and other amyloid-associated diseases arise in a patient,
medical doctors typically use genetic testing to identify sequence variations within the patient's
DNA that may have led to, or increased the risk of, genetic disorders¹². Consequently, in many
cases, it is unclear whether the DNA mutations found in patients were present before the onset of
the disease. This approach also falls short when patients have p53 amyloid plaques but no known
DNA mutations in the p53 gene¹². This creates a challenge in developing targeted treatment
options involving genomic mutations associated with protein aggregation.

3
This led us to ask: how can p53 protein lose its function without mutations in the p53
gene? Considering that damaged mRNAs may persist in human cells for up to 10 hours on
average, this may cause rare mRNA errors23 to produce p53 proteins that can form
amyloids with prion-like activity. Although studies have implicated mRNA oxidation damage
as a potential source of mRNA mutations, attempts to pinpoint which mRNA sequences that lead
to amyloids are often hindered, due to artifacts that may arise during sequencing and library
construction 9,13. Therefore, it leaves a gap in understanding how mRNA damage can lead to
protein aggregation in diseases.
To fill this gap in our knowledge, I investigated the mRNA errors that may translate into
mutant p53 proteins with a high propensity to aggregate and display prion-like activity, using an
optimized version of circle sequencing technology¹³. This work was conducted through an
established collaboration with the Marc Vermulst Lab in the Department of Gerontology at the
University of Southern California. The resulting mRNA mutations were analyzed by Dr. Yi Kou,
a postdoctoral fellow in our lab, using a Prion-Like Amino Acid Composition (PLAAC)
algorithm¹⁴ to assess the propensity of these mutations to result in p53 proteins with prion
activity. Among the mutations identified for prion-like propensity, S149F was selected,
considering its location on a loop of the p53 beta-sandwich core. Mutations in this loop region
may avoid significant disruption of the beta-sandwich folding structure, unlike mutations in the
core beta-sheet regions that are critical for the protein's stable folding¹⁵. Selecting mutations in
the loop region may help ensure that the protein mutation does not lead to amorphous
aggregation due to structural instability, but rather retains a beta-rich structure that can form the
extended cross-beta structures characteristic of amyloid fibrils¹⁵. Hence, S149F was chosen as a
starting example for investigating the ability of p53 mutants to infect wild-type (wt) p53.

4
2. It is unclear if mutant p53 can infect wild type p53 in a prion-like manner.
A key aspect of our hypothesis is that a small amount of amyloid/prion seeds generated
by erroneous p53 mRNA transcripts may induce a conformational change in the wild-type p53
protein, leading to a cascade of amyloid formation in cells that largely contain wild-type p53.
The formed amyloid fibers can then spread from cell to cell via prion-like mechanisms. From a
clinical perspective, amyloid fibers can be targeted by drugs, such as tafamidis, that block
amyloid fiber growth¹⁶. However, this approach does not address the source and formation
mechanisms of amyloid/prion seeds. Among the amyloid fibers formed, it is unclear whether the
seeding process is initiated by dysfunctional wild-type or mutant proteins, posing a challenge in
drug development regarding targeting prion/amyloid seeds¹⁷. Previous studies have shown that
dysfunctional p53 prions may cause functional p53 proteins to form aggregates in a prion-like
manner¹⁷ (Fig. 1). Mutant p53 proteins have also been observed to infect other mutant p53
proteins¹⁷(Fig. 1) Mutant p53 proteins have also been observed to infect other mutant p53
proteins17 (Fig. 1). Although mutant p53 proteins have shown in some cases to co-aggregate with
wt-p53, it has yet to be determined if mutant p53 prion-like seeding of wild type p53 can occur
(Fig. 1).
To fill this gap in our knowledge, S149F p53 mutant seeds were incubated with wildtype p53 to measure the changes in aggregation and amyloid fiber growth that may occur in the
presence or absence of S149F seeds, and to determine the minimum amount of seed needed to
infect p53. Aggregates and/or fibrils were initially investigated for amyloid fiber formation by
Congo Red staining, followed by Transmission Electron Microscopy (TEM). However, due to
technological limitations with Congo Red staining and TEM¹⁸,¹⁹, we recently developed a novel

5
Amyloid Fiber Hanging Drop Assay to visually confirm fiber formation upon seeding. This
method serves as an intermediate and less costly step before TEM analysis, which is discussed in
the next section (innovations).
To further test the general applicability of the Amyloid Fiber Hanging Drop Assay,
another p53 mutant, R175H, was selected and included in our studies. R175H is a common p53
cancer mutation that is prone to amyloid formation²⁰. While R175H has been shown in some
cases to co-aggregate with wt-p53, it has not been shown to infect wt-p53 in a prion-like manner
to form fibers²⁰. Therefore, this approach may help determine if mutant p53 proteins are indeed
capable of infecting wt p53 in a prion-like manner. These studies may provide further insights
into the mechanism of fiber formation and aid in the screening of drug targets against the seeding
process of amyloid fibers.

6
Figure 1
Innovations FIGURE 1
Innovation 1.

7
Detecting mRNA errors that may be associated with p53 protein aggregation and prionlike activity is challenging due to artifacts that can arise during library construction, as seen with
widely used technologies such as RNA-Seq¹³. This is because random errors introduced during
PCR reactions in library preparation can mask the rare errors present in the original mRNA
transcript.
This study is novel in detecting rare mRNA errors by using multiple rounds of mRNA
circle sequencing¹³, which will be cross-analyzed against genomic DNA sequences¹³ and DNA
errors¹³ to reveal mutations that may occur in mRNA without corresponding DNA mutations
(Fig. 2). This innovative approach, optimized by our collaborators in the Dr. Marc Vermulst lab,
improves the detection of mRNA errors linked to p53 mutations with high fidelity and
accuracy¹³. The resulting sequences were further analyzed using Prion-Like Amino Acid
Composition (PLAAC)¹⁴ to identify potential aggregating prion targets (Fig. 3). This approach
not only allows for a more accurate characterization of the relationship between mRNA damage
and p53 amyloid formation, but it may also reveal new mutational targets beyond the scope of
current knowledge about p53 DNA mutations that could lead to disease. Random errors
introduced during PCR reactions in library preparation can obscure the rare errors present in the
original mRNA transcript. This study is novel in its ability to detect rare mRNA errors using
multiple rounds of mRNA circle sequencing¹³, which will be cross-analyzed with genomic DNA
sequences¹³ and DNA errors¹³ to reveal mutations that may occur in mRNA without
corresponding DNA mutations (Fig. 2). This innovative approach, optimized by our
collaborators in the Dr. Marc Vermulst lab, enables the accurate detection of mRNA errors

8
linked to p53 mutations with high fidelity¹³. The resulting sequences were further analyzed using
Prion-Like Amino Acid Composition (PLAAC)¹⁴ to identify potential prion-aggregating targets
(Fig. 3). This method not only allows for a more precise characterization of the relationship
between mRNA damage and p53 amyloid formation but also may reveal new mutational targets
beyond the current understanding of p53 DNA mutations that may lead to disease.

9
Figure
2

10
Figure 3
Innovation 2.
Following the identification of candidate mutations with increased prion propensity,
characterizing the formation of protein aggregates and amyloid seeding is often limited by the
uncertainty of where and when seeding first begins¹⁷. For example, imaging aggregates is
typically done using Transmission Electron Microscopy¹⁹. These techniques require transferring
samples from the solution in which they were formed to a sample holder, which may result in

11
sample fragmentation¹⁹. Since the sample preparation for imaging tends to cause fiber
fragmentation, it is difficult to accurately decipher structural patterns and identify the origin of
seeding. The inevitable fragmentation of fibers or aggregates using these techniques limits
structural characterization, particularly the seeding pattern. Additionally, these techniques are
quite expensive¹⁹,²¹, making it important to have cost-effective preliminary assays to determine
seeding and aggregation potential.
Currently, less expensive preliminary techniques include using Thioflavin T (THT),
which relies on changes in optical density (OD), and Congo Red staining, which measures
protein aggregation and amyloid beta fibril formation²²,²³. However, many of these methods
cannot effectively differentiate between amorphous aggregates and amyloid fibrils. Congo Red
staining is used to detect amyloid structures by binding and staining the cross-β sheet structure,
which consists of short peptide chains that are parallel to each other but aligned perpendicular to
the fibril axis²³. Congo Red, an anionic dye, deposits itself in amyloid fibrils by forming
hydrogen bonds within the β-pleated sheets²³. Although the formation of cross-β sheet
supersecondary structures has become a diagnostic tool for identifying amyloid proteins, not all
amyloids bind and stain with THT or Congo Red. Additionally, THT and Congo Red also bind
to some non-amyloid structures²⁴, raising the risk of false positive or negative results.
To address these uncertainties, our lab developed the novel Amyloid Fiber Hanging Drop
Assay to better observe p53 seeding initiation and aggregation patterns (Fig. 4). In this
technique, droplets (~1 µl) containing mutant protein at various concentrations (~0.6-6 µM),

12
representing less than ~1% of the total wild-type p53 protein, are added to one corner of a larger
droplet (~10 µl) containing fixed amounts of wild-type p53 (~60 µM) on a coverslip (Fig. 4).
These droplets are suspended in a closed chamber equilibrated with a seeding buffer inside the
well (Fig. 4). The seeding buffer, derived from previous studies of amyloid formation¹⁷, contains
increasing amounts of NaCl to shrink the drop size and bring the proteins closer together to
initiate seeding and fiber formation¹⁷ (Fig. 4). Glycerol is also added to the seeding buffer to
reduce background precipitation and enhance the visualization of true fibers²⁵ (Fig. 4).
Using this method, seeding initiation and fiber growth patterns are stable and observable
under polarized light at the micron scale. p53 fiber formation typically takes ~1-2 weeks, with
the rate of fiber formation dependent on the kinetic properties of the protein of interest, as shown
later in our results. Our lab's Amyloid Fiber Hanging Drop Assay may help researchers obtain
preliminary data on protein aggregation and fiber formation and establish the lower limits of
seeding prior to employing more costly methods such as TEM, XRD, and Cryo-EM.
Furthermore, this assay may serve as a screening tool for drugs that inhibit the seeding and
growth of protein amyloid fibers.

13
Figure 4
1. Broader Impact
Numerous studies have shown that p53 amyloid plaques are present in a variety of human
diseases3,4,5,6. Several reports describe abnormal p53 aggregation and amyloid formation in
cancer cells and tissues3,11,15. p53 oligomers and fibrils have also been linked to neurotoxicity
and neuronal death in Alzheimer’s disease patients⁴. In terms of treatment, p53-related cancers

14
are typically managed with chemotherapy agents, but there are currently no effective drugs for
inhibiting and clearing amyloid fibers in patients with Alzheimer’s or cancer¹⁶.
Furthermore, drugs designed to remove amyloid fibrils may not entirely prevent the continued
production of amyloid seeds, which can initiate new rounds of amyloid formation¹⁶. The longterm impact of this proposed study, which investigates mRNA-related mutant p53 proteins and
their propensity to seed with p53 to form amyloid fibers, may provide researchers with new
insights for developing more targeted treatments for amyloid-related diseases. Additionally, our
Amyloid Fiber Hanging Drop Assay may offer a means to observe seeding initiation and serve as
a tool for screening drug therapies aimed at slowing the progression of Alzheimer’s, cancer, and
other diseases associated with protein aggregation and prion activity.
2. Approach
To provide new insight into p53 protein aggregation and prion-like activity and their
impact on human diseases, the following aims were pursued:
Aim 1: Identify Potential mRNA mutations lead to p53 aggregation.
Aim 2: Determine if S149F is capable of seeding wt p53 in a prion like manner.
Rationale:

15
Circle Sequencing & PLAAC
As previously mentioned, artifacts are known to arise during library construction,
amplification, and sequencing in RNA-seq4,13. Thus, p53 mRNA mutations were identified using
an optimized version of circle sequencing, to distinguish artifacts from true mutations. In this
technique, p53 mRNA under-went fragmentation and circularization to enable multiple rounds of
sequencing and reverse transcribed into cDNA’s with multiple copies of tandem repeats 13
.
Multiple rounds of sequencing were key in distinguishing artifacts that were not consistently
present in the tandem repeats when analyzing each sequencing result. (Fig 2). In brief, p53
mRNA was fragmented and circularized using a ligation enzyme 13(Fig. 2). The circularized p53
mRNA is reverse transcribed by a strand displacement polymerase which can make a copy of
only one strand by displacing the other strand 13(Fig. 2). This was followed by library
construction of the resulting p53 cDNAs sequences (Fig. 2). The library of sequences is then
compared to the original Tp53 cDNA sequences used to promptly identify and remove any false
positive mutations that may have arisen during library construction 4,13 (Fig. 2). This approach
helps to reduce the chance of selecting artifacts as potential targets over true RNA mutations 13
.
To ensure that these mutations are specific to p53 mRNA and not present on the TP53 cDNA,
true mRNA mutations identified by cir-seq but not present in the genomic DNA of p53 were
selected. p53 mRNA mutation rates can be enhanced using alkylating agent
1-methyl-3-nitro-1-nitrosoguanidine (MMNG), which has shown to cause transcriptional
mutagenesis due to formation of O6‐methyl guanine (O6MeG) inducing thymine mispair during
DNA replication13,27. The resulting p53 mRNA damaged sequences were cross analyzed against
damaged TP53 sequences to obtain mutations that were specific to p53 mRNA13,27
.

16
To analyze the prion-promoting potential of the detected p53 mRNA mutations, Dr. Yi
Kou in our lab used PLAAC for sequence analysis 14 (Fig. 3). In this technique, mutations were
prioritized by a number of factors including, location within the core domain, changes in amino
acid chemical properties and potential to gain prion-like activity
14. Deciding the location of the
mutation was important in ensuring that the mutation does not only induce structural changes
leading to protein aggregation, but gains prion-like activity as well 14,8. S149F was among those
detected within p53 mRNA (Fig. 5). S149F mutation occurs in the loop region of the p53 beta
sandwich core domain and makes the protein more hydrophobic without drastically changing the
overall structure 28,29,30 (Fig. 5). Thus, we hypothesize that S149F may be more characteristically
prone to aggregation and prion-like activity. Therefore, S149F was selected for initial studies
involving p53 mRNA mutations that may lead to expression of proteins with
prion-like activity.

17
Figure 5
Recombinant engineering, expression and purification of the S149F mutant
S149F was found as a somatic mutation in numerous cancers, but its role in
tumorigenesis is unclear. Prior to this study, S149F had not been tested as a potential mutation
leading to p53 amyloid fibrils. The p53 sequence was used as a template to make the mutant
S149F using the software SnapGene for primer design 30
. P53 mutant primers were used to
amplify the S149F mutation using Quikchange Site-Directed Mutagenesis. The PCR products
were then treated with DpnI, to digest the methylated parental DNA template to select for p53
S149F mutants 31. Both the wt p53 and the S149F mutants were under the control of the T7

18
promoter. The expression plasmids were transformed into BL21(DE3) E.coli cells since these
cells contain T7 RNA polymerase and lack proteases to prevent degradation of the foreign p53
S149F 31. However, due to leaky expression of BL21(DE3), Rosetta (DE3)pLysS E.coli cells
were used instead since they contain a T7 lysozyme gene (LysS) which inhibits residual T7 RNA
polymerase before the induced expression before the application of IPTG to induce protein
expression.31. The original wt p53 plasmids used in this study were generated by our former
postdoc member Dr. Jiang Xu based on which I engineered the S149F mutant.
p53 S149F purification
After successfully transforming wt P53 and S149F into Rosetta (DE3)pLysS, p53 and
S149F proteins were overexpressed using Isopropyl-ß-D-thiogalactopyranoside (IPTG) in a
nutrient rich 2YT media. Protein expression levels and its 25kd molecular weight were
confirmed by running an SDS-PAGE on p53 and S149F cell culture samples. Once confirmed,
p53, S149F and R175H were purified using a Ni-NTA column given the his-tag label on p53 and
mutants. This was followed by gel filtration of p53 and S149F using a Superdex 75 column for
separation on the basis of molecular size.
Initial S149F Seeding Congo Red Staining & TEM Analysis
To date, there are no published studies indicating S149F as a potential prion protein. To
test the seeding capability of S149F, mutant seeds were first produced by mixing 1 mg/ml of
S149F mutant protein, with a high salt working buffer (see detailed methods below) and
incubated for 30 min at room temp to allow seeds to form as seen in previous studies
investigating other mutations. 0.5%-1% of the resulting seeds were incubated with up to 60µM

19
wt p53 protein for four days in 50 µl volume working buffer, as previously shown to be
sufficient for p53 wt seeding to occur 7,11. Preliminary amyloid screening was then tested using
Congo red staining, followed by TEM for structural analysis.
Amyloid Fiber Hanging Drop Assay
To test whether seeding could be observed using our Amyloid Fiber Hanging Drop
Assay. S149F seeding potential of p53, was tested to confirm and visualize aggregation/fibril
growth in solution (Fig. 4). A droplet (1µl) containing 0.06-0.66µM S149F, which was 0.5-1.5%
of wt p53 seeded with 6µM-60µM p53 droplet (10µl) on a coverslip. The negative control
contained only p53 in Working Buffer, in the absence of mutant seed in this assay. This assay
was inspired by the hanging drop method in which protein crystals are grown 34. However,
instead of growing crystals we use the hanging drop vapor diffusion principle to seed and grow
protein fibers. In a hanging drop manner, samples on the coverslip were sealed onto the top of
the wells containing increasing NaCl (0.3-1.2M) amounts in the mother liquor (see detailed
methods) to help shrink the drop size 34 bringing proteins closer together to increase
amyloid/fiber formation potential.
Aggregates/fibrils were then visualized under polarized light given their anisotropic properties 35
.
Characterization of P53/S149f Aggregates: TEM, MALS, and AFM
Potential P53, S149F, R175H and TTR aggregates/fibrils were detected using
Transmission Electron Microscopy (TEM) to characterize aggregates in the sub-micrometer
range. Since the seed is typically below the detectable limits of the light microscope, Multiangle
Light Scattering (MALS) wasused for its ability to measure the light scattered by the seed at

20
various angles to determine the seed size and molecular weight13. This provides insight into the
minimum amount of mutant seed that is needed to initiate prion-like seeding of wildtype. AFM
was also used to provide topological information on protein aggregates and fibril structures4
.

21
CHAPTER 1: S149F and R175H SEEDING P53
RESULTS
Generating the S149F mutant
To generate the S149F p53 mutant, a site-directed mutagenesis approach was employed
using specifically designed primers and a plasmid template. S149F primers were designed using
the software SnapGene which creates primers based on the target sequence of p53
36 (Fig. 6).
Upon uploading the S149F plasmid sequence into the software, S149 in the wt p53 DNA
sequence, which contained the nucleotide coding sequence GGA, was changed to GAA that
codes for phenylalanine in both the forward and reverse directions (Fig. 6) Primers extended
15nt in both directions for specificity in primer binding and
DNA amplification (Fig. 6) The primer DNA was purchased from Integrated DNA
Technologies.
After obtaining the PCR DNA primers, the conversion of serine 149 to phenylalanine was
done using the Quick Change protocol for site directed mutagenesis37,38. In a round of PCR cycle
these primers anneal to the template DNA, replicating the plasmid DNA with the mutation37,38
.
After amplification PCR products were analyzed using an agarose gel to confirm amplification
and S149F was verified by sanger sequencing39,40 (Fig. 7).

22
Figure 6

23
Figure 7

24
P53 and S149F overexpression and purification
Although our data suggest that S149F is likely to lead to prion-like activity, it has not yet
been biochemically characterized as a mutation observed in p53 amyloid fibers. P53 & S149F
were overexpressed in E. coli. Wt p53 and S149F plasmids were initially transformed into
Rosetta (DE3)pLysS E.coli cells. The expression of the wt p53 and the S149F mutant proteins
were induced by the addition of 1 mM IPTG at the mid log phase (OD600~0.5). The
overexpression of wt p53 and the S149F were then analyzed by SDS-PAGE (Fig. 8) SDS-PAGE
confirmed the overexpression of a 25 kDa protein matching the molecular weight of p53 and
S149F (Fig. 8).This was followed by purification of the his-tagged p53, and S149F using a NiNTA column. Next, gel filtration of p53 and S149F using a Superdex 75 column was conducted
for separation on the basis of molecular size (Fig. 9-10).

25
Figure 8

26
Figure 9

27
Figure 10
TEM of amyloids generated by S149F seeding p53
Since S149F has been theoretically suggested to be a prion by PLACC but not yet been
established through experimentation, Congo red staining, was first used as a preliminary test to
confirm S149F amyloid structure by our postdoctoral fellow Dr. Yi. Kou. Although Congo red

28
staining works by hydrogen bonding to amyloid's beta pleated sheets, there was no significant
birefringence upon CR staining, suggesting that S149F amyloid may not bind CR well or may
not have formed typical amyloid structure with high CR binding affinity24. This does not exclude
S149F from being an amyloid or having prion activity. Amyloids in some cases form without
becoming CR stain positive24. Thus, S149F’s ability to seed p53, was further tested. 1 mg/ml
S149F was incubated with a high salt working buffer and incubated for 30 min at room temp to
allow seeds to form. S149F seeds were sonicated, which is to increase the number of
small amyloid seeds for seeding experiments. Heat generated during sonication has been shown
to lead to partial protein unfolding, and tend to result in its prion-like gain of function41
.
Complete fractionation of seeds was confirmed under polarized light. Wt p53 was then seeded
with the resulting S149F seeds by incubating 1%-3% of the S149F seeds with up to 60uM p53,
for four days, in a 50ul working buffer. Seeding morphology was then analyzed by TEM (Fig.
11).
Negative control consisting of p53 samples in the absence and S149F, was also analyzed by
TEM. TEM revealed that wt p53 samples were mostly soluble in solution and a few aggregates,
~20 nm in length, formed (Fig. 11). In contrast, S149F seeds in the absence of wt p53 showed a
size range between 100-150 nm in length, which is consistent with MALS analyses (see below) d
(Fig. 11). Seeding 1% S149F with 60µM wt p53, resulted in aggregates that covered almost the
entire TEM image space of ~700nm (Fig. 11). This suggests that S149F may act in a prion-like
manner inducing p53 aggregation, despite the lack of Congo red staining.

29
Figure 11

30
AFM of amyloids/fibers generated by S149F seeding p53
We used AFM to characterize the cross-seeding behavior between wt p53 and S149F
mutant . Using sonication and centrifugation we prepared a seeding solution of mutant S149F
aggregates. We then mixed these particles into a wt p53 solution so that the S149F mutant
made up to 2% of all protein
material and observed a
remarkable seed-dependent
growth of wt p53 fibers by
AFM (Fig. 12). Some fibers
appeared to be up to 20 µm in
length (Fig. 12). Interestingly,
fibers appear to grow from a
central point and continue in a
wave-like motion that can
eventually form conglomerates
with some degree of symmetry
(Fig. 12).
Amyloid Fiber Hanging Drop Assay S149F Seeding p53
It still remains obscure, whether wt p53 aggregates were initiated by S149F seeding or if
p53 aggregation may have occurred spontaneously. To ensure that the phenomena is not caused
by the sample preparation of AFM, which involves drying the sample on a mica surface or
Figure 12

31
fragmentation involving pipetting and washing samples on a TEM grid for imaging, we created
Amyloid Hanging Drop Assay to characterize the seeding process in solution. As previously
mentioned, our Amyloid Hanging Drop Assay also allows for fiber growth by seeding to be
visually monitored in real time given the limitations of preliminary assays such as ThT and
Congo Red staining 22,23.In efforts to help close these gaps in uncertainty, p53 seeding by S149F
was monitored in solution to minimize risk for fragmentation and observe fiber growth in realtime and help establish seeding potential of mutants. Negative controls contained p53 in Working
Buffer in the absence of mutant seeds. 1 mg/ml S149F seeds were generated to initiate the
seeding. S149F mutant seeds were added to 60µM of p53 on a glass coverslip at increasing seed
concentrations ranging from 0.5-1.5% (Fig. 13). The mutant seed drop, which only makes small
contact to the main drop, will initial fiber growth at the drop-drop interface when the WT protein
reach a certain concentration (Fig. 13).This range was used to help characterize the lower limit of
seed needed to induce amyloid or prion-like activity for biological relevance and future assays.
Samples were sealed onto wells containing increasing NaCl (0.3-1.2M) amounts in the mother
liquor. (Fig.13). Aggregates/fibrils were then visualized under polarized light. Samples
containing as little as 0.5% S149F seeds incubated with p53, led to an increase in aggregation
and fiber formation (Fig. 13). When p53 was seeded with S149F, p53 aggregates formed
elongated branched fibers that increased in size and branching up to 15 µm in length, within
~one week of seeding. The observed branching is a common characteristic of amyloid fibers
even in diseased tissues. We observed consistently seed-dependent growth of the WT p53 fiber
in solution under light microscope (compare (Fig. 13B, Fig. 13C, Fig. 13D) with the control of
(Fig.13A)) as compared with the control. When zooming in using polarized light, strong
birefringence can be observed on the rod-shaped materials, suggesting fiber-like structure (Fig.

32
13B, Fig. 13C, Fig. 13D). These are the largest p53 fibers observed to date as p53 is typically
~200nm in length as shown in literature studies measuring p53 amyloid fibrils by TEM, 11,35
.
Importantly, fibers could be visibly distinguished from background precipitation, which
minimizes false positive fiber formation. These studies not only validated our previous
observations of seed-dependent growth of wt TP53 fiber, but also establish a robust, scalable,
and quantitative seeding assay in solution that will great facilitate our studies of a wide range of
protein mutants from transcription errors.
Figure 13

33
S149F seeding p53 on Grid for TEM Analysis
As previously mentioned, fiber fragmentation during TEM analysis is a limiting factor in
analyzing fiber formation and the seeding initiation process. To better determine the morphology
of fibers formation and monitor the fiber growth trajectory, wt proteins were seeded with mutant
samples on the TEM grid (Fig 14). To begin, P53 samples were seeded with S149F using our
Amyloid Hanging Drop Assay with the alteration of adding the grid inside of the drop. Fibers
formed on the grid for p53 samples seeded with S149F (Fig. 14). TEM was then used to resolve
fibers grown within the drop that formed in the grid area (Fig. 14). Since smaller fibers that
formed may be in the nanometer range or may have grown outside of the grid area, a second
round fiber collection consisted of scooping a new grid within the drop to potentially collect
more fibers. S149F seeded samples contained large fiber-like structures when observed by TEM
(Fig. 15). However, they were not conclusively similar to p53 amyloid structures seen in
literature 11,35. Further optimization and validation of on-grid seeding assay will be needed to
develop this assay.

34
Figure 14

35
Figure 15
S149F Seed Size Determination
The minimum amount of seed present to initiate seeding of wt proteins was determined
using MALS. For seeding of p53, MALS data revealed that the S149F seed size was ~130nm
(Fig.16). Given that the p53 core domain is ~125 Å x ~50Å x 50Å, the number of seeds that
could theoretically fit in this space on average, is ~3-8000 S149F molecules. The amount of
proteins translated by a single mRNA molecule varies between 10 protein molecules to 105
protein molecules, per mRNA molecule in each cell generation. These values are dependent on
varying degradation rates of mRNA.
Thus, ~3K-8K S149F mutant proteins arising from mRNA error, may be sufficient to
initiate seeding, as shown in this study. Moreover, this suggests that as few as one damaged
mRNA transcript may produce enough mutant p53 proteins capable of seeding and propagating
p53 aggregation.

36
Figure 16
R175H Seeding of p53 Induces Fiber Formation
Since S149F is a mutation predicted to form amyloid fibrils, it is important to compare
fiber formation of S149F to a well-established amyloid fiber forming p53 mutation using our
hanging drop method. Thus p53-R175H, a top cancer-related mutation known to form infectious
amyloid fiber 20,35
, was selected for further studies of p53 fiber formation. R175H is considered a
hotspot mutation, having the highest frequency of occurrence in diverse cancers20,25. R175H is
important to DNA binding, it is located in the zinc-binding site, relatively close to the DNA
binding interface 20,35 (Fig. 17). Its DNA binding activity and stability has shown to be dependent
upon its structure and buffer pH 20,35. The R175H mutation affects its stability such that arginine
and histidine pKa values differ significantly even though they are both charged molecules 20,35
.
Arginine has a pKa of 14, while histidine’s pKa is 6 20,35. This drastic reduction in pKa caused by

37
R175H reduces p53 protein stability and loss of DNA binding20,35. Moreover, this mutation leads
to a much larger hydrophobic surface area and an increase in loop flexibility, in comparison to
wild type (wt) p53, making it more prone to aggregation20,35. R175H has shown to coaggregate
with wt-p53 predominantly by trapping 35. Although R175H p53 has been shown to display
prion-like activity to infect wt p53 in yeast 35, its capacity to seed wt-p53has not been
characterized at biochemical level. By investigating the R175H seeding potential in parallel with
S149F, this may serve as a reference for S149F fiber formation and further establish our Amyloid
Fiber Hanging Drop Assay, as a preliminary method to observe protein fibers.
Figure 17

38
R175H was tested for seeding of p53 using our hanging drop technique. To begin, R175H
was purified based on affinity and molecular size using Ni-NTA and Superdex gel filtration
columns as described for S149F (Fig. 17). Characteristically R175H is known to result in a loss
of p53 DNA binding. This was confirmed, using a heparin column which is composed of
linearized polysaccharides to allow DNA binding proteins to bind specifically to the column 35
.
There was an apparent reduction in binding of R175H to the column during purification as
compared with wt p53. This futher confirmed the mutation related effect of R175H on DNA
binding. Next, 4mg/ml of R175H, was added to the Working Buffer and incubated at room
temperature for 30 min to allow seeds to form. The R175H seeds were diluted to 1 mg/ml.
Negative controls contained p53 in Working Buffer in the absence of mutant seeds. Fibrils that
formed were fractionated by sonication and served as seeds.

39
Figure 18
R175H seeds were added to 60µM of p53 on a glass coverslip at increasing seed
concentrations ranging from 0.5-1.5% (Fig. 19). Samples were sealed onto wells containing
0.1M NaCl in the mother liquor (Fig. 19). Aggregates/fibrils were then visualized under
polarized light. R175H in the presence of wt p53 proteins resulted in an increase in p53
aggregation with as little as 0.5% R175H seeding. However, unlike S149F seeding, when
visualizing p53 samples seeded with R175H in the hanging drop, there was a clear increase in
background precipitation, making it difficult to distinguish fibers from the background (Fig. 19).

40
To reduce background precipitation, 0.5% glycerol was added to slightly increase the protein
solubility, reducing background precipitation. As a result, the fibers were more visually
distinguishable forming branches ~12um in length (Fig. 20). In the absence of the S149F and
R175H seeds, p53 control samples were much more soluble in solution and less prone to
aggregation (Fig. 20). When monitoring the formation fibers over the course of ~ 30 days there
was an apparent increase in branching and fiber formation in R175H seeded samples (Fig. 20).
P53 in the absence of R175H seed formed few aggregates & fibers like structures in comparison
to observed in seeded samples (Fig. 20). In addition, R175H seeded samples structurally
resembled S149F seeded samples in terms of forming elongated fibers and branching
capabilities (Fig. 20).
Figure 19

41
Figure 20
R175H seeding P53 samples were also visualized by AFM for topological analysis (Fig.
21). Some fibers appeared to be up to 20 µm in length. Interestingly, the apparent structural
characteristics of R175H can be seen using our lab's hanging drop method, overlapping with
S149F aggregation patterns obtained by AFM (Fig. 12, 19).R175H seed was ~179nm as
determined by MALS (Fig. 22).
R175H seeding as a positive control for our Amyloid hanging drop assay, further suggest
that S149F seeds may be capable of seeding p53 in a prion-like manner to form amyloid fibers.
This suggests that R175H may do more than co-aggregate with wt p53, as shown in previous
studies 20, R175H may be capable of infecting p53 in a prion-like manner, resulting in p53
dysfunction and fiber formation. Moreover, these results suggest that our Amyloid Fiber

42
Hanging Drop Assay may be a sufficient preliminary technique to monitor p53 amyloid fiber
formation.
Figure 21

43
Figure 22
S149F and R175H Seeding p53 Discussion
Overall, our current research study results provide preliminary evidence that a relatively
small amount of mutant p53 seed (0.5-1%) may can indeed seed wt p53 with prion-like
properties using both S149F and R175H as seeds using our Amyloid Fiber Hanging Drop Assay.
R175H served as an excellent positive control for comparison as similar fiber-like structures
formed upon seeding p53. R175H and S149F fibers grow remarkably larger, growing in the µm
range compared to the nm range in previous research studies 3,5,11,20.,35. This increase in size may
allow for complex structures to form to give more insight into fiber formation within the human
body. They may also serve as an indicator that fibers have formed in the nm range prior to more
costly methods such as TEM. Moreover, fiber growth including the formation of branches, may
be observed in real time using our Amyloid Fiber Hanging Drop Assay giving more confidence

44
that fibers are forming in comparison to Congo red staining or the ThT assay that relies on O.D.
and beta sheet formation.
Nevertheless, there is still work to be done to best confirm that the fibers observed are
true amyloid fibers and not artifacts. The resulting TEM structures were not conclusively similar
to amyloid fibers seen in previous studies. This may be due to an increase in fiber size and
background precipitation, limiting their resolving potential.
Immunoelectron microscopy may be used to better determine the fiber composition
using colloidal gold-conjugated secondary antibodies against p53 primary antibodies 3
. If the
fibers are indeed composed of p53 the colloidal gold-conjugated secondary antibodies will
decorate the fibers as a clear indicator3
. In parallel X Ray fiber diffraction may be used to
determine the molecular structure of fibers 3
. Overall, these results indicate that S149F and
R175H may cause p53 dysfunction resulting in fiber formation using our novel approach.

45
CHAPTER 2: V30M SEEDING MTTR VIA AMYLOID FIBER HANGING DROP
ASSAY
Background
Amyloid formation has been associated with a wide range of pathologies. P53 amyloid
activity in cancer is a relatively new study. So to further test the general applicability of the
Amyloid Fiber Hanging Drop Assay, this study included testing seeding potential and fiber
formation of the well-known amyloidosis associated protein, transthyretin (TTR) 45, 46,47. TTR is a
55 kDa homotetrameric transport protein found in the plasma or serum and cerebrospinal fluid
(CSF). TTR is mainly synthesized by the liver and the choroid plexus (CP) in the hippocampus
45,46 . Upon destabilization it is reduced to a monomer, causing the misfolding45,46. The
monomeric form of TTR is prone to aggregation. TTR is a transporter of the thyroid hormone
thyroxine and vitamin A throughout the body 45, 46. Loss of function off TTR often results in
aggregation known as Aggregating Transthyretin (ATTR) and characteristically leads to amyloid
deposits within organs such as peripheral nerves, heart, intestine, and the kidney 45, 46. Particularly
transthyretin amyloid cardiomyopathy (ATTR-CM), a rare but severe cause of restrictive
cardiomyopathy, and potentially fatal disease, demonstrates the need for drugs to target the seeds
to treat amyloid conditions 45, 46. ATTR-CM is characterized by deposits of amyloid protein
fibrils in the walls of the left ventricle, the main pumping chamber of the heart. Diagnosis of
ATTR-CM is often missed or delayed since its early stages, and may mimic the symptoms of
other heart conditions, such as heart failure related to high blood pressure, or hypertension,
enlargement and thickening of the heart, or hypertrophic cardiomyopathy 45, 46. Since TTR is
produced in the liver, the typical treatment for ATTR-CM is a liver transplant46. Although liver
transplant has shown to yield improvements for both wildtype (wt) and V30M patients' health,

46
after some time ATTR-CM begins to progress again46. TTR aggregates begin to re-deposit into
the heart for reasons unknown and may result in ATTR-CM redeveloping in patients46. A liver
transplant does not remove any residual mutant amyloid seeds already present in the heart or
prevent these mutant prion-like seeds in the heart from inducing amyloid formation of wt TTR
generated by the newly transplanted liver. 46. Currently, the acceptable post-transplant treatment
for this disease typically results in temporary reduction in amyloid deposits and fiber formation,
despite reports of continuous deposition, in cardiac tissue46 . Our hypothesis is that mutant TTR
may inherit a gain of function with prion-like activity and serve as seeds resulting in the reinitiation of TTR amyloid deposits in cardiac tissue.
What therapeutic treatment is available for ATTR-CM? Typically, amyloid deposits in
ATTR-CM are treated with the drug tafamidis which binds with high affinity and selectivity to
TTR and kinetically stabilizes the tetramer, slowing monomer formation, misfolding, and
amyloidogenesis 16, 46. Just like the liver transplant, after treatment, there may be TTR prion-like
seeds that still remain 46. Thus, tafamidis may slow down the progression of fiber formation
temporarily in patients but does not completely prevent any remaining TTR seeds from reinitiating fiber seeding process 16,46
TTR amyloidosis resulting in abnormal deposits in various tissues, is well established as a
protein disorder 45, 46,47. TTR amyloid activity was also tested in our study to demonstrate that our
Amyloid Fiber Hanging Drop Assay may be used to screen various proteins for amyloid and
prion-like activity, not just p53 and its mutants. TTR Val30Met mutation (V30M) was selected
as a seed for this study; it is the most common form of hereditary amyloidosis 45, 46,47,48. V30M

47
was used to seed TTR monomers (MTTR) 46,47,48 to monitor their potential to form amyloid fiber
by seeding. There is an overall 5‐10 year survival rate for cardiac patients with the more common
hereditary Val30Met (V30M) mutation47,48. Therefore, being able to visualize MTTR fiber
formation in real time using our Amyloid Fiber Hanging Drop Assay, may help researchers in
developing drugs targeting fiber formation.
RESULTS
pH Dependent V30M Seeding of MTTR
As the name suggests, Amyloid Fiber Hanging Drop Assay was developed based on the
principles of growing protein crystals 49,50 . In the hanging drop the water vapor equilibrates with
the reservoir solution 49,50. This slowly increases the concentrations of the protein and reagent in
the drop, until supersaturation is reached, and crystals form 49,50. On the other hand, growing
protein crystals close to the protein's isoelectric point contributes to protein aggregation and
precipitation, reducing the likeliness of crystal formation 49,50. Although protein aggregation is
unwanted when growing crystals, its isoelectric point dependence is of benefit to the study of
amyloidosis 49,50. Recent studies have shown that changes in pH affects the rate of TTR seeding
and fiber formation 51. The isoelectric point of TTR is 5.4. During fiber formation the TTR
homotetramer begins to destabilize into monomeric form 48,50. This is the early step of potential
TTR aggregation, and its rate of destabilization may be dependent upon pH 48,50. However,
MTTR amyloids form amorphous structures that appear somewhat indistinguishable from
crashed out precipitation by TEM imaging50. Thus, our Amyloid Fiber Hanging Drop Assay

48
could potentially help characterize these as amyloids by monitoring amyloid formation upon
seeding and adjusting parameters to decrease background signal.
To test the pH dependence of MTTR fiber formation, the monomeric TTR (MTTR) was
seeded with 1% V30M. MTTR was used in this study as it is an initial step in destabilizing the
homotetrameric TTR to gain initial insight into fiber formation 48,50. MTTR fibers, V30M seeding
of MTTR was tested between pH 4.3-7.4. This range was tested since pH. 4.3 & 7.4 had both
shown in previous studies to lead to MTTR aggregation 48,50. 1% V30M seeds were used to seed
40µM MTTR. NaCl concentration was maintained at 0.5M as shown in our previous
experiments to be sufficient for efficient seed-protein interactions 34. Both wells and samples
contained 20% glycerol to prevent high background precipitation that was seen in cases using
less than 20% glycerol.
pH Dependent V30M Seeding of MTTR at pH 4.3
pH 4.3 V30M Seeding of MTTR Research studies have shown that MTTR at pH 4.3, is
significantly amorphous when imaged using TEM 50. Previous studies have shown that proteins
in acidic conditions can lead to protonation of residues resulting in structural change and
increased exposure of hydrophobic patches on the protein's surface. Low pH has shown to lead
to decreased protein solubility, structural instability, and consequently, as seen with MTTR,
induce amyloid formation 50. Amorphous structures similar to literature findings, were observed
with 1% V30M seeding MTTR using the hanging drop method at pH 4.3 after ~5 days (Fig.
23). However, aggregates grew much larger, up to ~10 µm on average, providing a clear
indication of amyloid fibers in samples prior to TEM, AFM XRD etc. (Fig. 23). Although no
distinguishable fibers grew at pH 4.3, the structure of the aggregates that appeared to be

49
intricately more connected over time (Fig. 23), suggesting that potential fibers may be
intertwined leading to the resulting precipitant.
Figure 23
V30M Seeding of MTTR at pH 5.4
At pH 5.4, upon seeding of 1% V30M with MTTR, the resulting structures appeared
more fiber-like (Fig. 24). This was a significant reduction in amorphous aggregates as seen at pH
4.3 (Fig. 24). The fibers were distinguishable from the background precipitation and solutions.
Branching of fibers occurred over time (Fig. 24). These MTTR fibers were approximately µm in
diameter which is ~3x-4x smaller in diameter than p53 fibers. NaCl concentration was
maintained at 0.5M, both wells and samples contained 20% glycerol. Visible fibers appeared
between 3-4 weeks. This suggests the relatively slow seeding/ low precipitation controlled by
glycerol, and pH 5.4 may be optimal to visualize fiber formation of seeded MTTR in the μm
range using our Amyloid Fiber Hanging Drop Assay.

50
Figure 24
pH Dependent V30M Seeding of MTTR at pH 7.4
Next, we tested whether we could observe fibers at physiological pH. At pH 7.4 1%
V30M seeding MTTR resulted in fiber formation after ~two weeks (Fig 25). A clear fiber ~8µm
in length could be seen growing from the location where the V30M seed droplet was initially
placed (Fig 25). This further suggests that V30M may initiate seeding of MTTR.

51
Figure 25
MTTR fibers seeded with V30M on the grid for TEM analysis
To grow MTTR fibers seeded with V30M on the grid for TEM analysis, the grid was
placed within the sample directly after seeding (Fig. 26). Seeding was done at pH. 7.4 for 15
days (Fig. 26). Samples were also collected by using the grid to scoop potential fibers out of the
drop that may not have been visible (Fig. 26). Distinct fibers could be first seen growing within
and outside of the grid range ~8µm in length. TEM images of MTTR samples seeded with
V30M produced remarkable fiber-like structures at physiological pH, when compared to
previous studies (Fig 27).

52
Figure 26

53
Figure 27
Discussion V30M seeding MTTR
Overall pH plays a major role in fiber formation and structure in V30M seeding MTTR.
Low pH conditions such as pH 4.3 tend to result in more amorphous structures as compared to
pH 5.4 and 7.4, which appeared more fiber-like. MTTR fibers formed between 5-10 µm’s in
length appeared ~40 % thinner in size compared to p53 R175H and S149F fibers formed and had
less branching. pH 5.4 and 7.4, showed distinguished fiber growth including branching patterns
which is consistent with prion-like properties. Therefore, adjusting parameters around pH 5.4-7.4
may also be helpful in determining the optimal condition for fiber formation. Some areas include
focusing on seed preparation such as using a temperature control room rather than an incubator

54
to prevent temperature fluctuation, using adjusting seed growth time, and modifying sonication
settings to optimize the seed. This may increase and optimize the rate, size, and branching
capacity of seeding MTTR with V30M.
Our preliminary studies show that a relatively high concentration of glycerol of 20% was
vital in helping to reduce background precipitation during V30M seeding of MTTR for better
visibility of fiber formation. Although high concentrations of glycerol results in relatively slower
fiber formation rates compared to studies in which MTTR fibers form within a few days, this
does not indicate fibers are not forming sooner, as fibers may have formed on the nanoscale that
require advanced imaging such as TEM, AFM etc. Thus, the large fibers may serve as an
indicator of the smaller nm sized fibers. We also found that MTTR fiber formation rate
decreased from 4 weeks to two weeks by centrifugation of the initial seed solution to collect the
clear fraction for seeding, compared to using the entire seed solution that includes any
precipitation within the seed sample that may have occurred during the formation of the seeds.
V30M seed size will also need to be confirmed by MALS. This will be necessary to accurately
determine the amount of V30M molecules that may be initiating fiber formation of MTTR. TEM
images produced fiber-like structures when compared to previous studies. However, as
previously mentioned in the cases of S149F and R175H seeding p53, primary antibodies
targeting MTTR may be necessary to confirm the formation of MTTR fibers.
Overall, our Amyloid Fiber Hanging Drop Assay appears to not be only limited to p53
but may be used to screen for other proteins that may form amyloid fibers as well. In the future
this may be beneficial for screening drugs like Tafamidis to test its efficacy in inhibiting fiber

55
formation and targeting the seed to inhibit reformation of fibers in diseases such as hereditary
transthyretin amyloidosis.

56
CHAPTER 3: SOD1 SEEDING BY G142E USING AMYLOID HANGING DROP ASSAY
We further tested the application of Amyloid Fiber Hanging Drop Assay with another
well-established amyloid protein, superoxide dismutase 1 (SOD1). Abnormal protein
aggregation is becoming increasingly associated with many human diseases. Another protein
prone to form amyloid fibers screened by our Amyloid Fiber Hanging Drop Assay was
Superoxide dismutase 1 (SOD1), which protects cells from oxidative damage caused by reactive
oxygen species 52. SOD1 is an antioxidant enzyme which binds to zinc and copper molecules to
break down superoxide radicals, protecting cells from reactive oxygen species toxicity 52. SOD1
is a 32 kDa homodimer which forms a beta barrel (β-barrel) and contains a Cu/Zn site
responsible for catalyzing the dismutation of superoxide radicals 52. Mutations in the superoxide
dismutase 1 (SOD1) gene have shown to cause amyotrophic lateral sclerosis (ALS) by causing
SOD1 to misfold and form amyloid fibrils in motor neurons and astrocytes 52. This can damage
the nervous system and lead to ALS which weakens muscles and impacts physical function by
progressive degeneration of nerve cells in the spinal cord and brain 52. Researchers have
previously identified G142E in familial cases of ALS patients that tend to carry a single point
mutation 52,53(Fig. 28). G142E is located in the alpha helical segment of the protein, which is
located in a loop element that is important for binding zinc 52,53(Fig. 28). This change from a
nonpolar glycine to a negatively charged glutamic acid may lead to non-covalent interactions
resulting in SOD1 aggregation 52,53
.

57
Figure 28
RESULTS
G142E Seeding of SOD1 using Amyloid Fiber Hanging Drop Assay
Wt SOD1 and the G142E mutants were overexpressed and purified using similar
experimental protocols described for p53 S149F (Fig. 29). Both were purified using a Ni-NTA
column given the his-tag label on p53 and mutants. This was followed by gel filtration of p53
and S149F using a Superdex 75 column for separation on the basis of molecular size.

58
Figure 29
G142E was used to seed wt SOD1 at physiological pH using our Amyloid Fiber Hanging Drop
Assay (Fig. 30). However, when attempting to grow seeds at physiological pH, it was observed
that typical precipitation that forms did not occur as seen in p53 and MTTR samples. These
seeds also did not lead to fiber formation (Fig. 30). Upon lowering the pH to its isoelectric point
as was initially done with MTTR seeded samples, fibers started to form. Fibers of ~3µm in
length appeared ~75% thinner in width than p53 and MTTR samples (Fig. 30). The lowering of
the pH slightly increased precipitation such that the SOD1 did not completely crash out, but
instead formed stable amyloid fibers. SOD1 fibers formed branching patterns from aggregated
clumps/precipitants (Fig. 30).

59
Figure 30
G142E Seeding SOD1 Discussion
SOD1 formed fibers using our Amyloid Fiber Hanging Drop Assay at its isoelectric
point. Although SOD1 forms fibers, it will be important to adjust parameters to optimize fiber
formation at its isoelectric point and at physiological pH. For example, 0.5M NaCl was initially
used to shrink the drop size and allow for the SOD1 proteins to move closer together to help
initiate fiber formation. This may be adjusted by increasing the salt concentration overtime to
allow the drop to shrink faster and initiate SOD1 fibers formation that may be delayed by
kinetics. The observed solubility of SOD1 further shows that proteins may have varying

60
properties that lead to prion-like amyloid fibers as seen in the case of using glycerol to reduce
background precipitation in R175H seeding p53 samples. Temperature may also be adjusted to
test the effects it may have on fiber formation rate and structure. Also, higher concentrations of
above the standard 1mg/ml of SOD1 may be needed to better observe fibers at 1% seeding.
Structural assays will need to be performed as well to determine G142E seed SOD1 fiber
composition to gain insight into the structure of these fibers.

61
CHAPTER 4
Closing Discussion
This study initially aimed to determine if mRNA mutations lead to p53 aggregation and if
S149F could do so in a prion-like manner. We hypothesized that a small number of mRNA
mutations may cause p53 protein mutations, leading to p53 amyloid fibrils and prion-like
activity. In order to achieve this aim, we addressed key gaps in technological limitations by
creating our novel Amyloid Fiber Hanging Drop Assay to observe fiber formation in real time
without fiber fragmentation. This technique may help to confirm the formation of fibers prior to
more costly methods that are typically used such as TEM, Cryo-EM, and X-ray fiber diffraction.
Fiber growth exceeded typical size characterization limits from 1-100 nm to 20 µm as seen by
AFM. Through this study it was demonstrated that p53 mutant S149F, which we predicted to be
a result of mRNA mutation may indeed lead to protein aggregation in a prion-like manner. Prior
to this study, researchers have yet to deem S149F as a potential mutation to lead to amyloid fiber
formation or characterized its prion-like properties. S149F may now serve as a model starting
point in understanding how a small number of mRNA mutations may cause p53 mutations,
leading to p53 amyloid fibrils and prion-like activity. Moreover, prior research studies have
linked p53 to DNA mutations but not changes in the mRNA. Therefore, this study may provide
researchers with more insight in determining how ~50% of p53 amyloid fibrils may occur
without a mutation in the p53 gene. Although mRNA mutations may not solely be responsible
given its rarity (~1 in 1,000,000), our MALS calculated seed size of ~3K-8K mutant protein
molecules demonstrates that as few as one damaged mRNA leading to translational error, may be
sufficient to initiate seeding of p53 and propagate fiber formation in a prion-like manner.

62
Testing S149F for prion-like activity also lead to testing more well-established proteins
prone to aggregation in leading diseases such as: p53 R175H which can alter DNA binding and
most commonly found in cancers 7,11, MTTR V30M in ATTR-CM associated with heart failure
7,11 6,52,53
and SOD1 G142E found in the fatal neurodegenerative disease, ALS . In each of these
cases our Amyloid Fiber Hanging Drop Assay was used to show that the mutants may indeed
infect their corresponding wt proteins in a prion like manner, which was previously
undetermined.
Looking forward, to reach the stage of conducting drug screenings, seed optimization will
be necessary as it appears to be the initiator of fiber formation. So, adjusting parameters such as
sonication settings, temperature, NaCl concentrations to determine the best conditions to produce
quality seeds that maintain optimal prion-like capabilities, may help to best ensure fiber
formation and the rate in which fibers are formed. We also observed that overtime seed samples
appeared to crash out of solution, requiring us to make fresh seeds after several weeks. When
the seed begins to crash out of solution, it is known to reduce their seeding capacity. This may
also cause seeds to aggregate overtime thus increasing in size when analyzed by MALS. Going
forward seed size in solution will be analyzed by MALS before and after seeding to better
determine seed size and monitor its activity. Although our lab plans to test more mRNA
predicted mutations associated with prion-like activity based on PLAAC data, this technique is
not limited to mutations linked to mRNA, as we tested mutants associated with DNA error.
Thus, these areas of optimization will be useful for screening a variety of drugs targeting
proteins prone to form amyloid fibers.

63
Given that this is a novel technique, more statistical analysis will need to be conducted.
Fiber growth was repeated 3-4x and yielded fibers between 40-60% of the time. Other
established methods of confirming amyloid formation are currently being tested on TTR & p53
amyloid fiber samples, in our lab as well. For example, our lab is currently using SemiDenaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE) for the detection of amyloid
fibers based on size and detergent insolubility. As previously mentioned Immunoelectron
microscopy may be used to better determine the fiber composition using colloidal goldconjugated secondary antibodies against wt primary antibodies that may bind along the fibers.
This will help us better determine whether mutant protein can truly infect wt protein leading to
amyloid fiber formation in a prion like manner.
TEM as mentioned provided information regarding fiber structure and morphology. However,
there is no depth sensitivity in a single 2D TEM image. Moreover, the relatively large size of
fibers within the µm range often went beyond the resolution limits of TEM making it difficult to
distinguish fibers. Atomic Force Microscopy (AFM) was beneficial in providing true surface
topography of nanoscale fiber samples. To better distinguish fibers from the background, +3
washes of fibers may be useful since they are much larger in comparison to typical nm size
fibers. X-ray Fiber Diffraction may also be a powerful method to help confirm that fiber-like
structures are indeed forming, as seen in previous amyloid fiber studies 17. Subsequently, CryoEM may also be used for higher resolution structure analysis of fibers 21
.
Before this study took a turn leading to the development of the Amyloid Fiber Hanging
Drop Assay, this study aimed to determine oxidative stress effects on rare mRNA errors in vitro
and in vivo. Oxidative stress has been shown to play a role in mutational errors. In future studies,

64
oxidative stress induced treatments with hydrogen peroxide 53 may be used to create damaged
copies of mRNA and used to test the capacity for mutant p53 protein to be produced from
damaged sequences and lead to amyloid fiber formation 53. In vivo future studies may include the
co-expression of mt & wt proteins to measure the protein-protein interactions that may lead to
the formation of amyloid fibers within the same cell 53. Overall, these approaches to optimize
Amyloid Fiber Hanging Drop Assay and further analyze mutant seeding patterns, may help
researchers better target amyloid associated diseases.
Additional Study
R175H & S149F Fiber Extraction & X-ray Diffraction
To confirm that the structures that were present in the hanging drop wells after R175H &
S149F seeding of p53, fibers samples will be analyzed by Fiber X-Ray diffraction. Fiber X-Ray
diffraction of most distinguished fibers will be conducted by our collaborators at the Brookhaven
national lab, long island, New York.
Separation of Fibers for X-ray Diffraction
Fibers were separated in solution using a scalpel tool to collect individual fibers for X
Ray diffraction (see methods) (Fig. 24) . Fibers form tightly bonded aggregates that tend to have
“rubber-like” properties. When attempting to separate single fibers for X Ray Diffraction fibers
would stretch and spring back into place. Background precipitation was separated as much as

65
possible using scalpel tools. Samples were then caught onto a loop and stored in liquid nitrogen
to preserve samples for future X-Ray Fiber Diffraction analysis.
Figure 31

66
Schematic Summary
Figure 32
METHODS
P53/S149F transformation and expression into BL21(DEC) cells.
To express p53 to study its function, p53 was cloned into a pET28a expression vector
which is known to provide high expression levels and contains a Kanamycin resistance selective
marker. To ensure that both the wildtype and mutant p53 plasmids could be transformed into the
same cell type for purification, stellar E. coli cells were selected since they are highly competent
and lack the gene cluster for cutting foreign methylated DNA. To ensure the fidelity of the
sequence, the p53 plasmid was purified using a Qiagen purification Kit, and sanger sequenced.
The obtained sequence was aligned using the Basic Local Alignment Search Tool (BLAST) to

67
confirm p53 sequence. (10, 11,12, 14). Upon validation, the p53 sequence was used as a
template to make the mutant S149F using the software SnapGene for primer design (15). The
primer sequence was sent to Integrated DNA Technologies to produce custom p53
S149F primers. These p53 S149F primers were then used to amplify the mutation using
Quikchange Site-Directed Mutagenesis, in which the S149F primer and p53 template underwent
rounds of PCR cycles to replicate the plasmid DNA using a Q5 high fidelity polymerase (16,17).
The PCR products were then treated with DpnI, to digest the methylated parental DNA template
to select for p53 S149F mutants (17). p53 S149F plasmids were cloned into BL21(DE3) cells
since these cells lack proteases to prevent degradation of the foreign p53 S149F protein (8).
Thus, S149F was successfully designed and cloned into BL21(DE3) Competent E.coli cells to
accurately investigate how this mRNA predicted mutation could induce amyloid formation of
wildtype p53 proteins. SOD1 wildtype and mutant G142A plasmids were cloned into a Pet30b+
vector with kanamycin resistance and transformed into BL21 DE3 Competent E.coli cells.
MTTR wildtype and mutants cloned into a Pet24a+ expression vector provided courtesy of
Professor Lorena Saelices Gomez, UT Southwestern Medical.
Protein Expression of p53, S149F, R175H , MTTR, SOD1, G142A
Starter cultures for protein expression was first prepare as follows: 1 colony or glycerol
stock samples from each transformed cell line (p53,S149F, R175H , MTTR, SOD1, G142A) was
placed into individual glass tubes) containing 10 ml of 2YT and 10μl (1000x) kanamycin.
Cell cultures were grown overnight at 37°C for up to 18 hours at 230 rpm or at 18°C 24
hours. This step helps to ensure that the optical density in each culture increases synonymously
over time. The following day, 1ml from each culture was removed and added to 1L of 2YT

68
media. The cell culture was placed into an incubator, grown at 37°C & shaken at 230 rpm for
approximately 3 hours until the OD reached 0.5-0.8. A control sample was collected before
induction; no IPTG is added to control. Control: 100μl of each tube was taken out and placed
into a 1.5 ml Eppendorf tube and stored at 4°C. Control cell culture samples were spun down,
and majority of supernatant removed before resuspending in a loading buffer: 4μl of SDS to 8μl
of sample. 1μl/ml of 1mM of IPTG was added to the original remaining sample culture and
grown for 24 hours at 18°C and 230 rpm. (Longer induction time seemed to yield better
expression, low temp for protein folding). The next day induced cells were spun down and
majority of supernatant removed before resuspending in SDS loading buffer (4μl SDS to 8 μl of
sample). Samples were heated at 95° C on a PCR machine for 10 min to denature proteins in the
sample. Positive controls included samples of purified p53 and Mef2. Glycerol stocks of purified
R175H, MTTR, SOD1 were provided courtesy of Professor Lorena Saelices Gomez, UT
Southwestern Medical and our collaborator Marc Vermulst Lab, University of Southern
California. For selection, each protein in this study contained a histidine tag and kanamycin
resistance.
Purification
After successfully transforming p53, p53 S149F, p53 R175H , MTTR, MTTR mutants,
SOD1, SOD1 G142A plasmids into Bl21(DE3) E.coli cells, each were overexpressed using
1mM IPTG to induce protein expression (8). SDS-PAGE gel was used to confirm each protein's
overexpression by molecular weight. All protein samples contained histags for purification using
a Ni-NTA column according to Qiagen Quick Start NI NTA Purification of 6xHis -tagged

69
Proteins from E.coli Under Native Conditions protocol. This was followed by separation by size
using a Superdex 70 gel filtration column using the following buffer components: 10mM
HEPES pH 7.5 100mM NACl 0.5mM BME 0.5mM EDTA.
Amyloid Fiber Hanging Drop Assay
P53 S149F/R175H seed preparation
60 μM S149F & R175H seeds were incubated at room temperature, in a working buffer
composed of 100mM NaCl, 0.5mM 2-mercaptoethanol, 10mM HEPES pH 7, and 0.5mM
EDTA, for 30 min at room temperature. Next fibrils were collected by centrifugation at 12,000
rpm for 15 min. The clear supernatant containing fibrils not visible by the eye, were removed,
and used as seeds. The removal of visible precipitation in the supernatant was confirmed by light
microscopy. Initial mutant fibers were sonicated using a Sonic 550 Dimembrator for 3 min (3 sec
on 1 sec off). Complete fractionation of seeds was confirmed under polarized light (LEICA
Microscope MZ12) whereas there were no visible aggregates.
MTTR V30M seed preparation
40µm V30M MTTR seeds were formed using a working buffer composed of 10mM
NaoAc pH 4.3, 100mM KCL, 10mM EDTA and incubated at 37°C for four days. G142E 30μM
SOD1 seeds were prepared using 20mM TRIS pH 7.4, 5mM TCEP and incubated at 37°C for
40-48 hours.

70
V30M mutants and MTTR monomers were generously obtained from our collaborator
Professor Lorena Saelices Gomez, UT Southwestern Medical Center and purified by our post
doc Dr. Yi. Kou. To form V30M seeds, 50μl of 40μM V30M purified protein samples were
incubated in a working buffer composed of 10mM C2H3NaO2 pH 5.4, 100mM NaCl, 1 mM
EDTA, 37°C for 7 days. Samples were diluted with the working buffer to 100μl solution for
sonication. Seed samples were solubilized using a Fisher Scientific 550 Sonic Dismembrator
with the following sonication settings: 5 sec on, 5 sec off, amplitude: 2 for 10 min. Complete
fractionation of seeds was confirmed under polarized light (LEICA Microscope MZ12) whereas
there were no visible aggregates.
SOD1 G142E Seed Preparation
G142E mutants were provided by the Marc Vermulst lab, and cloned into bacterial
expression plasmid by Dr. Jiang Xu in our lab. To form V30M seeds, 30μM G142E SOD1
purified protein samples were incubated in a working buffer composed of 20mM Tris pH 5.3,
5mM TCEP 37°C for 40-48 hrs. Sonication settings were the same as previously mentioned
above for MTTR V30M samples. Complete fractionation of seeds was confirmed under
polarized light. Sonication settings were the same as previously mentioned above for MTTR
V30M samples. Complete fractionation of seeds was confirmed under polarized light.
R175H/ S149F Seeding of P53
Initially, to confirm fiber formation of seeded wt p53 proteins, seeding was initially done by
incubation of wildtype p53 proteins with 1% mutants in an Eppendorf tube for two days as

71
shown in previous studies (). This was followed by TEM. However, since seeding cannot be
observed prior to TEM the preceding Amyloid Fiber Hanging Drop Assay was used.
Amyloid Hanging Drop Assay Setup
To test our Amyloid Fiber Hanging Drop Assay, A hanging drop crystallization plate was
used to observe seeding. Inside each well the mother liquor contained 500μl of the appropriate
working buffer for each protein with increasing concentrations of NaCl 0.1-1mM to maintain the
drop concentration and optimize seeding kinetics. 10μl droplets containing fixed concentrations
6μM-60μM wt samples were mixed with 1ul of 1% mutant seeds, in a hanging drop manner (see
next section on seeding for detailed explanation of each unique protein). For TEM analysis,
carbon formvar grids were placed inside of the drop to allow fibers to grow on the grid.
Concentrations of wt to be used, was determined by varying the minimum amount of wt needed
to observe fiber formation with 1% seeding. Negative control contained wt protein in Working
Buffer in the absence of mutant seeds, while the V30M MTTR was used as a positive control.
After approximately 4-30 days aggregates/fibrils were observed under polarized light.
S149F Seeding of P53 Setup
S149F seeds were diluted in an Eppendorf tube to 4μM for 1% seeding of 40μM p53 with
its appropriate mother liquor (previously mentioned in seed preparation section) . Hanging drop
crystallization plates were used as a seeding apparatus. In brief, 40μM p53 samples were
incubated with 1% S149F seeds onto a glass coverslip and sealed onto the top of wells

72
containing 0.5-1M NaCl concentrations in the mother liquor for optimization. 1M NaCl was
sufficient to observe fibers in 5-7 days.
R175H Seeding of P53 Setup
R175H seeds were diluted Eppendorf tube to 4μM for 1% seeding of 40μM p53 with its
appropriate mother liquor (previously mentioned in seed preparation section). 5% glycerol was
added to seeds to minimize the amount of protein crashing out of solution. Hanging drop
crystallization plates were used as a seeding apparatus. In brief, 40μM p53 samples were
incubated with 1% R175H seeds onto a glass coverslip and sealed onto the top of wells
containing 0.5-1M NaCl concentrations in the mother liquor for optimization. 1M NaCl was
sufficient to observe fibers in 5-7 days.
V30M Seeding of MTTR Setup
V30M seeds were diluted in an Eppendorf tube to 4μM for 1% seeding of 40μM MTTR
with its appropriate mother liquor (previously mentioned in seed preparation section) . 20%
glycerol was added to the V30M seed and MTTR to minimize the amount of protein crashing out
of solution. Hanging drop crystallization plates were used as a seeding apparatus. In brief, 40μM
MTTR samples were incubated with 1% V30M seeds onto a glass coverslip and sealed onto the
top of wells containing 0.5-1M NaCl concentrations in the mother liquor for optimization. 1M
NaCl was sufficient to observe fibers in (14-30 days). To increase the rate of fiber formation,
alternatively 4mg/ml MTTR was used and seeding with 1% V30M.

73
G142E Seeding of SOD1 Setup
G142E seeds were diluted in an Eppendorf tube to 0.1 mg/ml for 1% seeding of 1mg/ml
SOD1 with its appropriate mother liquor (previously mentioned in seed preparation section).
Hanging drop crystallization plates were used as a seeding apparatus. In brief, 1 mg/ml SOD1
samples were incubated with 1% V30M seeds onto a glass coverslip and sealed onto the top of
wells containing 0.5-1M NaCl concentrations in the mother liquor for optimization. 1M NaCl
was sufficient to observe fibers in (14-30 days).
Microscopy of Protein Fibers
For preliminary visualization of fibers, after ~1-4 weeks aggregates/ fibrils were visible
and observed under polarized light (LEICA Microscope MZ12).
Transmission Electron Microscopy (TEM)
3D characterization of protein aggregation was visualized using FEI Talos F200C G2
Biological Transmission Electron Microscope. Negative-stained TEM specimens were prepared
by removing the formvar and carbon-coated 200 mesh copper grid (Electron Microscopy
Sciences) that held the samples, from the drop. As previously mentioned, to collect smaller fibers
that formed in the nanometer range or may have grown outside of the grid area, a second round
fiber collection consisted of scooping a new grid within the drop to collect fibers. After
incubation of the sample for 1 min at room temperature, the excess solvent was removed with
filter paper. The grid was washed three times with water and samples were deeply negatively

74
stained three times with 2% uranyl acetate solution. Grids were examined using the FEI Talos
operated at 80KeV. The quantification of the protein aggregates was performed using the
program TEM Imaging & Analysis (TIA).
Atomic Force Microscopy (AFM)
Topological analysis of protein aggregates was done using a Bruker’s Dimension XR
scanning probe microscope with an SCANASYST-AIR probe. To determine the optimal
condition for scanning, samples were prepared by adding 15μl of samples to MICA sheets with
serial 10x dilutions of 10, 100,100,10k,100k (Typically 1k yielded optimal image visualization).
Samples were dried at room temperature for 10 min then washed ten times with 40μl of
deionized water. Samples were loaded onto the AFM and probed in ScanAsyst mode. Negative
controls included the working buffer and water to distinguish salt formation from aggregates.
SEC-MALS of amyloid seeds
The sizes of mutant seeds used to initiate wt aggregating, were characterized using size
exclusion chromatography coupled to multi-angle light scattering (SEC-MALS). Samples were
resuspended in 10 mM HEPES, 100mM Na2SO4 except for S149F seed samples in which salt
was increased to 500mM Na2SO4 and acidity adjusted to pH to 4 by acetic acid for optimal
resolution. Bovine serum albumin was used as a positive control. After separation of the seed
was achieved on the SEC column, seed size and molecular weight was calculated by MALS
detectors. These detectors include multiple photodiodes positioned at various angles relative to
the illuminating beam, to measure the amount of light scattered by the samples into detectors.

75
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Abstract (if available)

Abstract The tumor suppressor protein p53, known as the "guardian of the genome," is essential for genomic stability, regulating cell division and apoptosis. Mutations in the p53 gene can cause protein misfolding and prion-like amyloid aggregation, contributing to diseases such as cancer, Alzheimer's, cardiovascular, and respiratory disorders. Notably, about 50% of cancer patients show p53 loss of function without detectable mutations in the p53 gene, suggesting alternative mechanisms. We propose that a small number of mRNA mutations could lead to p53 protein misfolding, promoting amyloid fibril formation and prion-like activity. Current amyloid detection methods, which rely on optical density and X-ray diffraction, are limited in distinguishing true amyloid structures from non-specific precipitation. There is a lack of high-throughput screening methods capable of effectively differentiating amyloid fibers from background noise. To address this gap, we have developed a novel Amyloid Hanging Drop Assay, adapted from protein crystallization techniques. This assay allows for the direct observation of amyloid fiber formation under a light microscope, with polarized light confirming fiber characteristics through birefringence. The method enhances the study of mutant protein seeding behavior and provides a robust platform for identifying small molecules that inhibit amyloid formation. This assay could be a valuable tool in drug discovery for amyloid-related diseases, including Alzheimer's, cancer, and cardiovascular disorders.

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Creator Love, Kayla K. (author)

Core Title mRNA oxidation and its relation to p53 amyloid formation and disease

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

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Degree Conferral Date 2024-08

Publication Date 10/08/2024

Defense Date 08/19/2024

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Tag aggregation,amyloid hanging drop,cancer,mutation,OAI-PMH Harvest,p53,s149f,Tumor

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Language English

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