AN

INVESTIGATION PROBLEM REPORT ON

A) Cloning of TTR-GFP in a yeast vector to examine protein aggregation
and toxicity.
And
B) Screening of inhibitors of aggregation of TTR-GFP and HTT-GFP
For partial fulfillment of the requirement for the degree of
MASTER OF SCIENCE
IN
(Session: 2012-2014)








2

ABBREVIATION
AA Amyloid A Protein Amyloidosis
AL Amyloid Light Chain Amyloidosis
ATP Adenosine Triphosphate
ATTR Transthyretin-mediated Amyloidosis
CNS Central Nervous System
CSF Cerebrospinal fluid
DNA Deoxyribonucleic acid
dNTP Deoxynucleotide triphosphates
EGCG Epigallocatechin gallate
Etbr Ethidium bromide
FAP Familial amyloid polyneuropathy
GABA Gamma-aminobutyric acid
GAG Glycosaminoglycan
Gal Galactose
GFP Green fluorescent protein
HD Huntingtons disease
Htt Huntington
Ig Immunoglobulin
Kb Kilobase
LB Luria- Bertaini
LiAc Lithium acetate
NF Nuclease-Free water
PCR Polymerase Chain reaction
PEG Polyethylene glycol
PNS Peripheral nervous system
Raf Raffinose
RBP Retinol binding protein
RNA Ribonucleic acid
rSAP Shrimp alkaline phosphatase
RT Room Temperature
SAP Serum Amyloid P
SC Synthetic complete
3

SGD Saccharomyces gene database
SNP Sodium nitroprusside
SSA Senile systemic amyloidosis
TE Tris EDTA
TTR Transthyretin
Ura Uracil
WHO World Health Organization
WT Wild-type
YNB Yeast nitrogen base
YPD Yeast potato dextrose
4

LIST OF FIGURES

Figure Description
2.1 Structure of wild-type Transthyretin
2.2 The TTR amyloid cascade
4.1 Isolated plasmid DNA on 1% agarose gel
4.2
Restriction digestion of WT and variant TTR-GFP and p426 plasmids with
BamH1 on 1% agarose gel.
4.3 Shows p426 vector treated with rSAP enzyme on 1% agarose gel
4.4 Purified fragments on 1% agarose gel
4.5 PCR products on 1% agarose gel
4.6 Restriction digestion with AgeI to check orientation on 1% agarose gel.
4.7 Toxicity assay
7.1 Chemical structure of Tafamidis.
7.2 Chemical structure of luteolin
9.1 Shows isolated plasmids on 1% agarose gel
9.2
Graph showing the percentage of protein aggregation after treatment with
different conc. of Luteolin
9.3
Graph showing the percentage of aggregation after treatment with different
conc. Of Luteolin( II attempt)
9.4
Graph showing the percentage of aggregation after treatment with different
conc. Of Tafamidis (attempt I)
9.5
Graph showing the percentage of aggregation after treatment with different
conc. Of Tafamidis (attempt II)
5

LIST OF TABLES

Table No. Description
3.1 List of equipments used
3.2 List of reagents
3.3 Composition of LB broth
3.4 Composition of YPD broth
3.5 Composition of 10X Amino acid mix
3.6 Composition of SC media
3.7 Composition of Raf-Gal media
3.8 Reaction mixure for restriction digestion
3.9 Reaction mixture for Shrimp Alkaline Phosphatase (rSAP) treatment
3.10 Reaction mixture for ligation
3.11 Reaction mixture for colony-PCR
3.12 Thermal profile of the thermal cycler for colony- PCR
3.13 Reaction mixture for digestion with Age I restriction enzyme
9.1 Represents the percentage of cells with aggregates for one to three transformant
treated with luteolin (Attempt I)
9.2 Represents the percentage of cells with aggregates for one to three transformant
treated with luteolin (Attempt II)
9.3 Represents the percentage of cells with aggregates for one to three transformant
treated with tafamidis (Attempt I)
9.4 Represents the percentage of cells with aggregates for one to three transformant
treated with tafamidis (attempt II)


6

ABSTRACT
Amyloidosis encompasses a wide spectrum of disorders characterized by abnormal
accumulation of misfolded proteins. Mutation in transthyretin (TTR), one of the 20 amyloid
proteins, is known to cause a rare but lethal hereditary amyloidosis. More than 80 mutations
in TTR gene have been identified that are associated with amyloidosis. The aim of this work
was to clone wild-type and mutant TTR-GFP gene in the p426, a high copy number vector to
study the protein aggregation pattern and toxicity in yeast model system. The wild type TTR-
GFP and variant TTR-GFP constructs demonstrated high aggregation. However, no toxicity
in yeast was observed on overexpressing p426-TTR-GFP constructs. Yeast model for
transthyretin and huntingtin aggregation were used to test the efficiency of two shortlisted
molecules:Tafamidis and Luteolin. Tafamidis, a small molecule known to kinetically stabilze
the TTR tetramer did not exhibit any effect on aggregation of TTR in yeast model.
Interestingly, Luteolin, a flavonoid, showed two-fold decrease in aggregation of huntingtin
protein in yeast. However, these results need to be further validated.

Keywords: Amyloidosis, Transthyretin, Tafamidis, Luteolin, Huntington, yeast
Saccharomyces cerevisae

7

TABLE OF CONTENTS


SECTION ONE: CLONING OF TTR-GFP IN A YEAST VECTOR TO EXAMINE
PROTEIN AGGREGATION AND TOXICITY ....................................................................... 8
INTRODUCTION ................................................................................................................. 9
REVIEW OF LITERATURE .............................................................................................. 13
MATERIALS AND METHODS ......................................................................................... 20
SUMMARY AND CONCLUSION .................................................................................... 38
SECTION TWO: SCREENING OF INHIBITORS OF AGGREGATION OF TTR-GFP
AND HTT-GFP ....................................................................................................................... 39
INTRODUCTION ............................................................................................................... 40
REVIEW OF LITERATURE .............................................................................................. 41
MATERIALS AND METHODS ......................................................................................... 45
RESULTS AND DISCUSSION .......................................................................................... 47
SUMMARY AND CONCLUSION .................................................................................... 52






8











SECTION ONE: CLONING OF TTR-GFP IN A YEAST VECTOR TO
EXAMINE PROTEIN AGGREGATION AND TOXICITY



















9

INTRODUCTION

1.1. Amyloidosis
Amyloidosis refers to a group of diseases characterized by extracellular deposition of
normally soluble proteins as pathogenic insoluble fibrils in tissues and organs (Falk et a..,
1997). In the mid 19
th
century, Virchow adopted the term amyloid, from botany, meaning
starch or cellulose, to describe iodine stained abnormal extracellular deposits in liver at
autopsy (Kyle, 2001). After this initial study it was found that other diseased organs showed
same iodine reaction pattern and were name amyloid was used for these diseases. Later on,
chemical analysis showed that nature of these amyloid deposits was proteinaceous (Sipe and
Cohen, 2000).
More than 25 human proteins have been found to be associated with amyloidosis,
sharing no sequence homology but having common features. All amyloid proteins share a
core of cross sheet structure in which the sheets are parallel to the fibril direction and where
the strands run perpendicular to the fibril (Westermark, 2005). These amyloid proteins have
affinity for the dye Congo red and show green birefringence in polarized light after staining
with Congo red. Type II diabetes and neurodegenerative disorders like Alzheimers disease,
Huntington and prion disease are well known examples amyloid diseases (Pepys, 2001). The
modern nomenclature for amyloid diseases as established by WHO is based on the chemical
nature of the fibril protein, denoted by letter A which stands for amyloid, followed by the
abbreviated form of the precursor protein. Thus according to this nomenclature, for e.g.
transthyretin amyloidosis is represented by ATTR and when the amyloid is derived from
immunoglobulin light chain, the amyloidosis is caused by AL amyloidosis (Sipe, 2010).

1.2. Classification of amyloidosis
Systemic or Localized:
Accumulation of amyloid deposits may be localized, remaining confined to a specific
tissue or organ, or it may be systemic, involving many tissues and organs and eventually
leading to progressive organ dysfunction (Westermark, 2005). Systemic amyloidosis is
usually fatal owing to the involvement of heart and kidneys. On the other hand, localized
forms may either be clinically silent or associated with severe diseases like organ failure, as
in senile cardiac amyloidosis, haemorrhage in local respiratory tract (Pepys, 2001).
10


Primary or Secondary:
Amyloidosis may also result because of an underlying disease, such as chronic
inflammation and infective diseases, in which case it is termed secondary amyloidosis, or it
may involve no underlying disease and thus be primary or idiopathic (Loizos, 2013).
Amyloid light chain (AL) amyloidosis, formerly known as primary amyloidosis, is caused
due to fibrils formed by monoclonal immunoglobulin (Ig) light chain and their deposition in
different tissues and organs. AL amyloidosis is the most common and severe form of
systemic amyloidosis (Desport, 2012) and affects both men and women with the same
incidence with usual symptoms onset at ages between 60 and 65 (Dubrey et al., 2011;
Saraiva, 2002). In this disease, the amyloid fibrils deposit in multiple organs including heart,
kidney, liver, peripheral nervous system (PNS) and autonomic nervous system (Dubrey et al.,
2011; Saraiva, 2002) Reactive AA amyloidosis, also known as secondary amyloidosis, is
caused by the overproduction of serum amyloid A protein resulting in a sustained acute phase
response (Peps, 2014) and it involves deposition in liver, spleen, GI tract and predominately
in kidneys (Dember, 2006).

Hereditary or Acquired:
Amyloidosis can either be hereditary, if caused by deposition of genetically variant
proteins as amyloid fibrils, or acquired, if due to a higher expression of a normal protein with
amyloidogenic potential or expression of an abnormal protein as a result of a preexisting
disease (Pepys, 2006). Hereditary amyloidosis are rare diseases and are all inherited in an
autosomal dominant manner with variable penetrance (Hawkins, 2003). The most common
hereditary amyloidosis is caused by variants of transthyretin and usually presents as familial
amyloid polyneuropathy (FAP) (Pepys, 2001). Besides FAP, other hereditary amyloidosis
have been reported such as senile systemic amyloidosis (SSA), also termed as senile cardiac
amyloidosis (SCA) which is due to wild-type TTR deposition (Dubrey et al., 2011). Other
less common hereditary amyloidoses including gelsolin amyloidosis causing lattice corneal
dystrophy and cystatin C amyloidosis presenting as cerebral amyloid angiopathy (Benson,
2001).


11

1.3. Amyloid fibril formation
Amyloid fibrils are insoluble, long, straight and unbranching fibres of 70-120 in
diameter (Serpell, 1999). They specifically bind certain dyes such as Congo red and
thioflavin T, and they demonstrate a characteristic cross- pattern on X-ray diffraction,
reflecting distances between -strands (4.7 ) and distances between -sheets (911 )
(Sipe, 2005; Sunde, 1998).
The amyloid deposits are not merely composed of amyloid fibrils but also contain
heparin and dermatan sulphated glycosaminoglycans (GAGs) and proteoglycans. Though the
role glycosaminoglycans in amyloidogenesis in not yet clear, they are thought to contribute to
fibrillogenesis by either influencing protein folding or enhancing resistance to proteolytic
cleavage (Pepys, 2001). In addition, all amyloid deposits contain serum amyloid P
component (SAP) which highly resistant to proteolysis and its binding to amyloid fibrils
protects them against proteolytic digestion (Hawkins, 2002).
Although the detailed mechanism of amyloid fibril formation is not entirely clear, the
first step includes the protein misfolding (Soto, 2006) in which normal souble protein forms
insoluble amyloid involves the production of a partially unfolded intermediate molecule. This
is a thermodynamically unfavourable state and thus rapidly advances toward amyloidogenic
form (Dobson, 2003; Jahn, 2006). Kinetic studies have suggested that the fibril formation is a
nucleation dependent process, resembling crystallization (Come, 1993; Soto, 2008).
According to this model, the aggregation starts after the protein concentration exceeds
threshold concentration (Soto, 2008). Above this critical concentration, a peptide micelle or
seed (nuclei) forms and fibrils nucleate within these, elongating by adding monomers to their
ends (Westermark, 2005; Rambaran, 2008). Lag phase of fibril formation is significantly
shortened in the presence of seeds (Soto, 2008). The biophysical studies of the intermediates
in the amyloid formation process indicate that diverse species with progressive degrees of
aggregation are present simultaneously and in dynamic equilibrium between each other (Soto,
2008).






12

1.4. Diagnosis
Precise and early diagnosis of amyloidosis is essential for its effective treatment.
Correct diagnosis of amyloidosis can be quite difficult as it can cause variety of syndromes
that have all or some clinical features similar to other diseases. Earlier diagnosis of amyloid
was based on histochemical staining of the amyloid deposit by the dye congo red and
observing the characteristic apple-green birefringence in polarized light. This still remains the
gold standard for histological diagnosis (Pepys, 2001). However, histological tests do not
confirm the type of amyloid fibril or its deposition. Diagnosis made by the biopsy of an
affected tissue, for e.g. kidney, heart, and liver is highly sensitive method with 73%
sensitivity and 90% specificity (Bogov, 2008). But it is not definitive for AL amyloidosis,
therefore the presence of plasma cell dysrasia is to be demonstrated for its confirmation. This
can be accomplished by showing plasma cell dyscrasia by a bone marrow biopsy showing
predominance of - or -producing plasma cells or by the presence of a monoclonal light
chain in the serum or urine by serum and urine electrophoresis (Sanchorawala, 2006).
Hereditary amyloidosis can be indicated by the presence of famility history of amyloidosis
(Hawkins, 2003).

1.5. Treatment
The current therapeutics to treat amyloid disorders in humans target on reducing the
concentration of the amyloidogenic protein (Pepys, 2014). For example, one strategy to treat
Alzheimers disease is to reduce the production of amyloid (A) by inhibiting the - or -
secretases that generate the A from the trans-membrane amyloid precursor protein
(Sambamurti, 2011). Another strategy is organ transplantation to control or stop the
production of toxic amyloidogenic protein (Pepys, 2014), for example, heart transplantation
has been performed in FAP suffering from cardiomyopathy to improve transthyretin (Falk,
2005). In light chain (AL) amyloidosis, chemotherapy has been used to eliminate clonal
plasma cells in the bone marrow to dramatically reduce the concentration of the
amyloidogenic light chain protein in the blood. Antisense (RNA interference) strategies have
also been employed to lower the mRNA levels of the amyloid precursor protein in some
amyloidosis such as TTR (Johnson et al., 2012). Effective anti-inflammatory treatments can
help in improving AA amyloidosis (Lachmann, 2005).

13

REVIEW OF LITERATURE

2.1. Transthyretin amyloidosis
The transthyretin amyloidoses (ATTR) are the most prevalent type of hereditary
systemic amyloidosis (Benson, 1984). They are rare, autosomal dominant diseases caused by
the deposition of the mutated TTR protein (Saraiva, 2001). The principle manifestation of
transthyretin amyloidosis is peripheral neuropathy. The first case of ATTR was discovered in
a portuguese family as familial amyloid polyneuropathy (FAP) in 1952, since then several
similar cases have been seen in kindreds in several countries (Dwulet and Benson, 1983). In
1978, Costa et al. showed that the main constituent of amyloid fibril in FAP was a variant of
transthyretin protein (Terry et al.., 1993).
More than 80 point and double mutations in TTR have been identified which are
associated with different amyloidosis. The first and most common type of ATTR, the FAP1
or Portuguese type, is characterized by the substitution of a methionine residue for a valine at
position 30 of TTR (V30M) (Terry et al., 1993). Other mutations include L55P, which is
most aggressive (Jacobson, 1992) and V122I associated with cardiac amyloidosis (Rapezzi,
2010).

2.1.1. Clinical features of Transthyretin amyloidosis
Transthyretin amyloidosis is a muti-systemic disease with heterogenous clinical
presentation, which is manifested by sensory and motor peripheral neuropathy, autonomic
neuropathy, cardiomyopathy, nephropathy, gastrointestinal impairment, or ocular deposition.
The initial symptoms include sensory polyneuropathy in the lower limbs, with loss of
superficial sensation to pain and temperatures, accompanied by motor impairement, in the
later course of the disease, causing wasting and weakness (Misrahi et al.., 1998). Other early
features include impairment of autonomic nervous system, which is manifested by
dyshydrosis, sexual impotence, disturbance of gastrointestinal motility (alternating diarrhea
and constipation), orthostatic hypotension and urinary incontinence (Benson, 2009). Cardiac
and renal dysfunction may also be also observed (Saraiva, 1992). In some cases, ocular
involvement such as vitreous opacity, dry eye, glaucoma, and papillary disorders, is also seen
(Ando et al., 1997).
14

Furthermore, symptoms of TTR-FAP include coldness, hoarseness, decreased skin
temperature, dyscoria, dysesthesia, muscle weakness and atrophy, weight loss, burning,
edema, and arrthymia. Since it is a muti-systemic disease, the deposition of mutant forms of
TTR can occur in various tissues and organs. Based on studies, axonal degeneration and
neuronal loss have shown to be associated with endoneurial amyloid deposits formed from
TTR (Sousa and Saraiva, 2003). In patients with TTR-FAP associated with V30M TTR
mutation, amyloid deposition is seen in nerve trunks, plexuses and sensory and autonomic
ganglia (Takahashi, 1997). Amyloid deposits have also been seen in the choroid plexus,
cardiovascular system and kidneys (Araki and Yi, 2000).

2.2. Transthyretin
The transthyretin (TTR) is a plasma protein that was originally named as
prealbumin for its ability to run faster than of albumin in the presence of an electric field
(Fung et al., 1988). It was first observed in cerebrospinal fluid (CSF) and later in the serum
(Hou et al., 2007).
In humans, transthyretin is encoded by a single copy gene located on the long arm of
the chromosome 18 at the postion 18q12.1 (Sakaki, 1989; Hou et al., 2007). The TTR gene
spans approximately 7 kb and has 4 exons, each with approximately 200 bases (Tsuzuki, et
al., 1985; Sasaki et al.., 1985). An 18-amino acid signal peptide is synthezed by the first exon
but it is cleaved before secretion of mature TTR (Hou et al.., 2007). The sequence of TTR
gene has remained highly conserved during evolution, having about 87% sequence homology
among mammals (Sunde et al.., 1996). In human plasma, TTR is present at a concentration of
0.2 mg/ml (Miroy et al.., 1996).
TTR is synthesized predominantly in liver and also in the choroid plexus of the brain,
contributing to blood and brain proportion of the TTR, respectively (Zheng, 2000). As its
name implies, TransThyRetin, it is involved in the transport of thyroxine (T4) and retinol
(Vitamin A) (Sousa and Saraiva, 2003). In brain, TTR is the main transporter of thyroxine
(80%) (Hamilton and Benson, 2001) whereas in plasma in contributes to about 15 % - 20% of
thyroxine (Richardson, 2007). In plasma, it serves as a major transporter of retinol by binding
to RBP. About 30% of plasma TTR is bound to RBP (Monaco, 2000).
15


Figure 2.1 Structure of wild-type Transthyretin (Source:Pdb)

Transthyretin is a tetrameric protein of 55 kDa composed of four identical -sheet rich
subunits having molecular mass of 14 kDa and containing 127 amino acids residues each
(Power et al., 2000). Each polypeptide chain forms eight strands named A-H and one
helix (Yokoyama et al., 2013). The eight -strands are arranged in a -sandwich made of two
four stranded -sheets and one -helix formed between -strands E and F (Blake, 1978).
TTR monomers assemble into dimers which are stabilized by extensive hydrogen
bonding, which in turn associated to form tetramers through hydrophobic interaction (Damas
and Saraiva, 2000). X-ray crystallography reveals that each dimer results from the association
of two monomers extending two -sheets composed of four -strands, from each monomer,
into two -sheets of eight -strands (Almeida et al., 2004). The dimerdimer interface of the
tetramer forms a central hydrophobic channel that has two T4 binding sites presenting
negative binding co-operativity, so no TTR molecule can bind to more than one T4 (Blake,
1978). There are four binding sites for RBP which are located on the surface of the TTR
molecule. However, due to steric hinderance, at any given time, only two RBP can bind to a
TTR molecule (Monaco, 1995).
16

Transthyretin molecule, apart from being a carrier molecule, also plays an important
role in PNS, maintaining cognitive function, neuropeptide processing and nerve regeneration
(Fleming et al.., 2009).
In the fully functional TTR molecule, TTR monomers interact via hydrogen bonds
between antiparallel, adjacent -strands H-H and F-F to form dimer. The two dimers (A-B
and C-D) form the tetramer through hydrophobic interaction between the residues of the A-B
and G-H loops (Blake, 1971). However, structural studies on oligomers of aggregated mutant
TTR protein have revealed a disruption of the D strand which affects hydrogen bonding with
the A strand. Thus resulting in alteration of conformation of the protein which might may be
associated with its tendency to aggregate (Sousa et al.., 2001). However, several authors have
also contributed -sheet rich composition of the TTR molecule towards its ability to
aggregate (Sousa and Saraiva, 2003).

2.2.1. Pathogenesis of Transthyretin amyloidosis
Amyloid can either form from intrinsically disordered proteins that have no defined
tertiary structure (e.g. Huntington disease) or it can result from the partial misfolding of
proteins that adopt a well-defined tertiary or quaternary structure. Transthyretin protein is an
example of such proteins that undergo partial misfolding (Johnson et al.., 2012).
Transthyretin protein, in native state, seems to contain unoccupied thyroxine (T40-
binding sites that are formed by the weaker dimer-dimer interface of the TTR tetramer (Foss
et al., 2005). Rate-limiting dissociation of the tetramer at this interface generates of dimers,
which then rapidly dissociated to form monomers (Foss et al., 2005; Johnson et al., 2005).
Partial misfolding of these monomers promotes their misassembly into soluble oligomers and
amyloid fibrils through a thermodynamically favorable process (Johnson et al., 2005). These
misfolded monomers and oligomers have been regarded as neurotoxic species (Reixach,
2004).
17


Fig.2.2. The TTR amyloid cascade. Amyloid formation by TTR requires rate-limiting
tetramer dissociation to a pair of folded dimmers, which then quickly dissociate into folded
monomers. Partial unfolding of the monomers yields the aggregation-prone amyloidogenic
intermediate. The amyloidogenic intermediate of TTR (Lower Right) retains much of its
native structure. The amyloidogenic intermediate can misassemble to form a variety of
aggregate morphologies, including spherical oligomers, amorphous aggregates, and fibrils.
Tafamidis binding to the TTR tetramer (Upper Left) dramatically slows dissociation, thereby
efficiently inhibiting aggregation. (Adapted from Bulava et al., 2012)

2.2.2. Current therapeutics for TTR amyloidosis
Strategies to treat transthyretin and other amyloidosis focus on reducing the
concentration of amloidogenic protein. The currently practiced strategy to treat FAP
associated with TTR amyloidosis is liver transplantion (Herlenius et al., 2004). In this
procedure, patients with heterozygous TTR mutation have their liver replaced with those
people that are homozygous for WT TTR. This procedure has been 90% effectiveness in
patients with TTR-FAP (Holmgren et al., 1991).
Another strategy to ameliorate the TTR amyloidosis is the use of antisense
oligonucleotides and RNA interference that lowers mutant TTR mRNA levels (Benson,
2006). Another effective strategy that is being extensively researched is the use of small
molecules. These small molecules act to kinetically stabilize the TTR tetramer, thus
preventing its dissociation. A wide variety of small molecules that stabilize TTR tetramer
18

have been identified including natural derived flavonoid and xanthone derivatives as well as
synthetic compounds belonging to five families that are bisaryloxime ethers, biphenyls, 1-
aryl-4,6-biscarboxydibenzofurans, 2- phenylbenzoxazoles and biphenylamines. TTR kinetic
stabilizers have also been identified by halogenations of NSAIDs such as salicylic acid, etc
(Connelly, 2010).

2.3. Yeast as a model organism
The budding yeast Saccharomyces cerevisae, also known as bakers yeast, is the most
extensively studied eukaryotic organism. It has emerged as a versatile and robust model to
study complex protein interactions, molecular basis of pathogenesis of various diseases,
cloning genes and so on.
S. cerevisae was the first eukaryotic organism to have its genome fully sequenced
(Bostein, 1997). Several features of budding yeast have made it an ideal tool for research
purposes. First, it is a unicellular organism, easy to propagate and handle in laboratory and
have a short generation time (90 minutes on rich medium) (Miller-Fleming et al., 2008).
Second, it has it entire genome sequenced, which makes it easy to genetically manipulate it
(Gitler, 2008). Also, about 50% of human genes involved in diseases have homologues in
yeast (Suter et al., 2006). There are several databases online that provide genetic information
on yeast. One such database is Saccharomyces Genome Database (SGD) that provides
information available about every yeast gene such as genetic deletion, alteration, protein
functions, among others (Bostein and Fink, 2011).
Yeast, Saccharomyces cerevisae contains nearly 6000 genes located on 16
chromosomes, thus its genome is very compact. As microbes, yeasts are grown in batch
liquid culure and isolated as colonies derived from single cells on solid media. Because their
doubling time is short, large populations of individuals can be rapidly grown and analysed.
This property of yeast is essential for genetic studies (Mell and Burgess, 2002).
Yeast as a model organism for amyloidosis diseases such as Alzheimers diseases,
Huntington disease and Transthyretin amyloidosis has been very useful for understanding
their pathogenesis, as protein misfolding and aggregation is the core reason of these diseases
(Miller-Fleming et al., 2008). Several breakthroughs have been achieved in understanding
diseases such as Alzheimers and Parkinsons Diseases using yeast-based systems (Gitler,
2008). Yeast model has facilitated the study of disease-associated genes and verify the
implications of that gene in the disease itself (Miller-Fleming et al., 2008).
19

Transformation of yeast cells with recombinant DNA (rDNA), first became fissible in
1978 (Hinnen et al., 1978). Since then, recombinant DNA technology in yeast has established
itself, and a multitude of different vector constructs are available. Shuttle vectors that can
propagate in both bacteria and yeast are fundamental tools of study in molecular genetic
analysis (Sikorski, 1989). Yeast shuttle vectors contain components that allow their
replication and selection in both E.coli and S.cerevisae. The components that allow its
propagation in E.coli include origin of replication (ori) (e.g. from pBR322 plasmid) and
selectable marker for antibiotic resistance (e.g. -lactamase). The components for yeast
include autonomously replicating sequence (ARS), a yeast centromere (CEN) and a yeast
selectable marker (e.g. URA3 that codes for enzyme for uracil synthesis)
Yeast shuttle vectors are extensively useful for cloning human genes that are
associated with diseases and study their effect in yeast. Purpose of this study was to clone
TTR-GFP genes (WT and Variant) in p426 (shuttle vector) and study their effect in specific
yeast strain.

20

MATERIALS AND METHODS

3.1. Materials
3.1.1. Equipments
Table 3.1 List of equipments used
Equipments
Supplier
Spectrophotometer
Eppendorf Biophotometer plus
Incubators
ThermoScientific MaxQ600,
IB-05G,
Jeiotech
Inverted fluroscence microscope
Nikon
Laminar air flow
Klenz Flo
Agarose gel electrophoresis unit
BioRad
Gel documentation system
Alpha Innotech
pH meter
Sartorius
Weighing balance
Sartorius
Centrifuges
ThermoScientific Heraeus Biofuge
Stratos centrifuge, ThermoScientific
Heraeus Fresco21 centrifuge
Dry bath
GeNei
Thermo cycler
BioRad C1000
96-well plate reader
Tecan infinite M200
Speed vac
Labconco
Vortex
Tarson
21

Pipette tips
Axygen, Tarson
Centrifuge tubes
Tarson, Axygen
Falcons and petri plates
Tarson
Ice Machine
Zexter


3.1.2. Reagents
All reagents used were of molecular biology grade and belonged to different
manufacturers like SIGMA, HIMEDIA, AMRESCO, MERCK, and etc. Different chemicals
used as listed below:
Table 3.2 List of Reagents
Reagents Company
Ethidium
bromide
Sigma
Agarose Sigma
Lithium acetate Sigma
PEG Sigma
Tris EDTA Amresco
DMSO Sigma
Ethanol Merck

3.1.3. Kits
Plasmid isolation kit - Qiagen
Gel purification kit Qiagen
3.1.4. Micro-organisms
E.coli strain DH5
Saccharomyces cerevisae

22

3.1.5. Media
All Medias were prepared in MilliQ water and autoclaved for 20 min. at 15 psi.
I. Growth medium for Bacteria
LB (Luria Bertani) broth
Composition of LB broth (HiMedia) is given in the table 3.2
Table 3.3 Composition of LB broth




LB broth was prepared as described on the bottle.
LB agar
2% agar was weighed and added to the LB broth before autoclaving.

II. Growth media for yeast
YPD media (broth)
Table 3.4 Composition of YPD broth
Ingredient Gms
Peptic digest of animal
tissue
20.00
Yeast extract 10.00
Dextrose 20.00
pH (at 25C) 6.50.2
YPD broth was prepared as per instructions given on the bottle.
YPD agar
2.5% agar was weighed and added to YPD broth before autoclaving.



Ingredients Gms
Casein enzymic 10.00
Yeast extract 5.00
Sodium Chloride 10.00
pH (at 25C) 7.50.2
23

Amino acid mix
Table 3.5 Composition of 10X amino acid mix
Amino Acid mg
Alanine 80
Arginine 40
Aspartic acid 200
Glutamic acid 200
Histidine 40
Leucine 120
Lysine 60
Methionine 40
Phenylalanine 100
Threonine 400
Tyrosine 60
Tryptophan 80
Valine 300
Serine 750
Uracil 40

Synthetic Media (SC)
Table 3.6 Composition of SC media
Ingredients Amount
Yeast Nitrogen Base (YNB)
0.17%
Ammonium Sulphate
0.5%
Glucose
2%
10X Amino acid mix ( minus
amino acid)
1X
Agar(if added)
2.5%








24

SC Raf-Gal Media minus amino acid
Table 3.7 Composition of Raf-Gal media
Ingredients Amount
Yeast Nitrogen Base (YNB) 0.17%
Ammonium Sulphate 0.5%
Raffinose 2%
Galactose 3%
10X Amino acid mix( minus
amino acid)
1X
Agar(if added) 2.5%

3.1.6. Antibiotics
Ampicillin
Stock solution 100 mg/ml
Working solution 1mg/ml
3.1.7. Enzymes
All Restriction enzymes as well as shrimp alkaline phosphatise and T4- DNA ligase
used, including their appropriate buffers were from THERMOSCIENTIFIC or NEB
(New England Labs).
3.1.8. DNA ladder and loading dye
All DNA markers and loading dye used were from THERMO SCIENTIFIC and
FERMENTAS
6X Loading dye
GeneRuler 1KB DNA ladder







25

3.2 Methods
3.2.1 Plasmid isolation from DH5 E.coli strain using QIAprep Plasmid Miniprep kit
(Qiagen)
Method:
a) 5-10 mL of overnight E.coli culture (LB medium) was centrifuged for 10 min at
3500 rpm to pellet down the cells.
b) The pellet was resuspended in 250 L Buffer P1 and was transferred to 1.5 ml
microcentrifuge tube.
c) 250 L of Buffer P2 was added and mixed thoroughly by inverting the tube 5-6
times.
d) After that, 300 L of Buffer N3 and mixed immediately and thoroughly by
inverting the tube 5-6 times.
e) The samples were centrifuged at maximum speed (13,000 x g) for 10 min in a
table-top microcentrifuge.
f) The supernatant was then transferred to column.
g) The column was then centrifuge at 11,000 x g for 1 min.
h) Flow through was discarded. Then, 750 L of Buffer PE was added to column.
i) The samples were then incubated at RT for 5 min and centrifuge at 11,000 x g for 1
min.
j) Flow through was discarded and the columns were then centrifuged at 11,000 x g
for 1 min.
k) Flow through and the column was discarded. The column was then placed in a 1.5
ml microcentrifuge tube.
l) The plasmid was eluted by adding 60 L of Buffer EB to the centre was the
column.
m) The samples were incubated at RT for 2 min and centrifuged at 11,000 x g for 1
min.
n) Isolated plasmid was then checked by performing agarose gel electrophoresis using
1% agarose gel and stored at -20C.



26

3.2.2 Restriction Digestion
Method:
a) Large-scale restriction digestion reaction was set up as described in the table 3.6
Table 3.8 Reaction mixure for restriction digestion
Components Volume (L)
Plasmid 8
BamH1 6
10X Buffer Tango 5
Nuclease-free water 31
Final Volume 50

b) The mixture was then incubated for 3 hrs at 37C.
c) After that, digested products were checked on 1% agarose gel.

3.2.3 Gel extraction using QIAquick gel extraction kit.
Method:
a) The DNA fragment was excised from the agarose gel with a clean, sharp scalpel.
b) The gel slice was weighed in a colorless tube. 3 volumes of Buffer QG to 1 volume
gel.
c) The microcentrifuge tube was incubated at 50C until the gel slice was completely
dissolved. The tube was vortexed every 2 min to help dissolve gel.
d) After the gel slice was completely dissolved, 1 gel volume of isopropanol was
added to the sample and mixed.
e) A QIAquick spin column was placed in a collection tube (2 ml)
f) The sample was then applied to the QIAquick spin column to bind the DNA and
centrifuged for 1 min. Flow through was discarded and QIAquick spin column
was placed back in the same tube.
g) For sample volumes of >800 L, the remaining samples were loaded and
centrifuged for 1 min.
h) To wash, 0.75 ml Buffer PE was added to QIAquick spin column and incubated at
RT for 5 min and then centrifuged for 1 min. Flow through was discarded and
QIAquick spin column was placed back in the same tube.
27

i) The QIAquick column was centrifuge again in the 2 ml collection tube for 1 min at
13,000 rpm to remove the residual wash buffer.
j) QIAquick column was placed into a clean 1.5 ml microcentrifuge tube.
k) To elute DNA, 50 L Buffer EB (10mM Tris:Cl, pH 8.5) was added to the center
of the QIAquick membrane and the column was centrifuged for 1 min.
l) For second elution, flow through was added to the center of the QIAquick
membrane and centrifuged for 1 min.
m) The samples were then analysed on agarose gel.
3.2.4 rSAP treatment
Method:
1. The reaction mixture was prepared as follows:
Table 3.9 Reaction mixture for Shrimp Alkaline Phosphatase (rSAP) treatment
Ingredients Volume (L)
Digested vector 49
rSAP enzyme 2.5
NF water 23.5
Total 75

2. Then, the reaction mixture was incubated at 37C for 45 mins.
3. Next, the mixture was incubated at 65C for 15 mins.
4. The SAP enzyme treated vector was then checked on 1% agarose gel.

3.2.5 Ligation
Method:
a) The ligation reaction was set up for 2 ratios (1:3 and 1:6 molar ratio vector:insert)
as given in the table 3.10:




28

Table 3.10 Reaction mixture for ligation
Volume (L)
Components Control 1:3 1:6
Vector (digested) 1.5 1.5 1.5
Insert (digested) - 1.5 2.5
10X ligase Buffer 1 1 1
T4 DNA ligase 2 2 2
Nuclease-free water 5.5 4 3
Final Volume 10 10 10


b) The ligation mixture was incubated overnight (16-17 hrs) at 16C in a thermal
cycler.
c) Then ligated product was transformed in bacteria (DH5 strain of E.coli).
d)
3.2.6 Bacterial Transformation
Method:
I. Preparation of Comp Cells
Material and Reagents: All the reagents and chemicals used were chilled before use.
a) Autoclaved MilliQ water
b) 100mM CaCl
2
solution
c) Solution of 100mM CaCl
2
solution and 20% glycerol
Method:
a) 180 mL Fresh LB broth was inoculated with the saturated primary culture (E.coli
DH5) at 0.14 O.D.
b) The inoculated media was then incubated on shaker at 37C until it reaches 0.5-0.6
O.D. (about 1 hr).
c) The media was then incubated at 4C for 30 min.
d) The culture was centrifuged at 4C for 10 min to pellet down the cells.
e) The cell pellet was resuspended with autoclaved MilliQ water (chilled) and
centrifuged at 4C for 10 min.
f) Then, the cell pellet was washed with 100mM solution of CaCl
2
(chilled) and
centrifuged at 4C for 10 min.
29

g) The step 6 was repeated once more.
h) The cell pellet was then resuspended in the chilled solution of 100mM CaCl
2
and
20% glycerol and stored at -80C until further use.

II. Transformation of ligation mixture
Material and Reagents:
a) Competent cells
b) Ligation mixture
c) LB media
d) LB ampicillin plates
Method:
a) 100 L of competent cells were thawed on ice and transferred into 2
microcentrifuge tube (50 L each).
b) Then 3 L of the ligated mixture was added to one tube and other was kept as
control.
c) Then samples were incubated for 20 min on ice.
d) Dry bath was set as 42C and the samples were given a heat-shock at 42C for 90
sec.
e) Then, the tubes were spanned chilled on ice for 2 min.
f) After this, 700 L of fresh LB broth was added to the samples at RT.
g) The samples were then incubated at 37C for 45 min on shaker.
h) Then 100 L of samples were spread plated on LB-amp plates and incubated at
37C overnight.
3.2.7 Screening of transformants
I. Colony-PCR
Method:
a) The reaction setup for colony-PCR
Table 3.11 Reaction mixture for colony-PCR
Components Volume (L)
Positive Control Negative
control
Samples
10X Taq buffer 2.5 2.5
30

25mM MgCl2 1.5 1.5 1.5
10M dNTPs 0.7 0.7 0.7
10M Forward
primer
1 1 1
10M Reverse
primer
1 1 1
Colony/Plasmid 1+9(water) - 10(colony+water)
Taq polymerase 0.2 0.2 0.2
Nuclease-free water 18.1 28.1 18.1
Final volume 25 25 25

b) Thermal profile of the thermal cycler
Table 3.12 Thermal profile of the thermal cycler for colony- PCR
Cycles Stage Temperature Time
1 Initial Denaturation 95.0 C 6 min


35
Denaturation 95.0 C 1 min
Annealing 62.0 C 50 sec
Extension 72.0 C 90 sec
1 Final extension 72.0 C 10 min
1 Hold 10.0C Forever

II. Restriction digestion with AgeI
Method:
a) The reaction setup for double digestion
Table 3.13 Reaction mix for digestion with Age I restriction enzyme
Components Volume (L)
10X buffer NEB1 2
Plasmid 2
AgeI 1
Nuclease free water 15
Final volume 20

31

b) The mixture was then vortexed and was given a short spin to clear the lid.
c) Then the tubes were incubated at 37C for 3 hrs
d) The digested products were analysed on 1% agarose gel.

3.2.8 Yeast transformation using Lithium acetate (LiAc)
Material and Reagents:
a) 100mM Lithium Acetate (LiAc)
b) Polyethylene glycol (PEG) 3500, 50% w/v
PEG solution: 1X TE buffer + 100mM lithium acetate mixed in 50% PEG.
c) Boiled salmon sperm DNA (ssDNA), 10 mg/mL
Method:
Day 1
a) VL2 strain was streaked on YPD plate and incubated at 30C.
Day 2:
a) The patch was scooped and mixed in about 500 L of autoclaved water in 1.5ml
microcentrifuge tube.
b) The microcentrifuge tube was centrifuged at 5,000 rpm for 2 mins and the
supernatant was discarded.
c) The pellet was resuspended in 650 L 1X LiAc solution made by mixing 100mM
LiAc and 10X TE buffer in MQ, and incubated for 30-45 min at 30C.
d) After incubation, the samples were centrifuged and supernatant was discarded.
e) Then the pellet was resuspended in (50 X N) L of 1X lithium acetate solution,
where N is the number of samples.
f) 300l of PEG solution was taken in a clean microcentrifuge tube and 50l of
mixture from the previous step was added to it and mixed thoroughly.
g) Then, 9l of sperm DNA (preheated at 95C) was added and mixed thoroughly.
h) Finally, 4l of plasmid with the insert was added and mixed.
i) Then, the samples were incubated at 30C, 200 rpm for 1.5 hrs.
j) After incubation, samples were given heat shock at 42C for 8 min and centrifuged.
k) The supernatant was discarded and the pellet was resuspended in 100l of
autoclaved water and plated onto SC-ura plate and incubated for 48 hrs at 30C.

32





3.2.9 Analysis of aggregate formation
Method:
a) The transformant colonies were patched onto SC-ura plates and incubated for 24
hrs.
b) Then, 2mL of SC-ura broth was inoculated with the transformants and incubated
overnight at 30C, 200 rpm.
c) Next day, O.D was taken at 600nm and noted down.
d) Then 3 mL of RafGal-ura broth was inoculated at 0.2 O.D. and incubated for 48
hrs at 30C, 200 rpm.
e) After 48 hrs, the aggregation was observed at 100X on the inverted fluorescence
microscope by manually counting 200-300 cells and percentage of aggregation was
calculated and noted.

3.2.10 Toxicity Assay
Method:
a) Fresh 2 mL of SC-ura media was inoculated at 0.1 O.D. using SC-ura cultures of
variants and incubated for 3-4 hrs at 30C, 200 rpm or till O.D. reached 0.2 0.3.
b) Then the O.D. was checked after the 3-4 hrs and then all cultures were normalized
at 0.2 O.D. in 200 L of autoclaved water.
c) Then the following dilutions of the cultures were prepared:
5
-1
, 5
-2
, 5
-3
, 5
-4

d) Then 10 L of every dilution of each variant was spotted on SC-ura and RafGal-
ura square plates.
e) Then, after the plates had dried, they were incubated at 30C for 48-72 hrs or till
the growth appeared.



33





RESULTS AND DISCUSSION

Aggregation and Toxicity of WT and Variant TTR

Cloning of WT- TTR and its variants in a high copy number plasmid, p426
Good quality of plasmids with WT and variant TTR inserts and plasmid with p426
vector into which the TTR inserts were to be cloned was also isolated. Figure 4.1 (A and B)
shows good quality and RNA free preparation of all the plasmid.



Fig 4.1 shows isolated plasmid DNA on 1% agarose gel. (A) Lane 1 shows ladder (1Kb)
and lanes 2-5 shows WT-TTR, TTR variant 1-3. (B) Lane 1 shows ladder (1Kb) and lanes 2-
3 show p426 plasmid.

The isolated vector and TTR plasmids were digested with BamH1. Two DNA
fragments of size 6kb corresponding to the vector backbone and1Kb size corresponding to
the gene of insert were obtained after digesting with BamH1 restriction enzyme.

34





Fig 4.2 Restriction digestion of WT and variant TTR-GFP and p426 plasmids with
BamH1 on 1% agarose gel. (A) Lane 1 shows ladder (1kb), Lane 2-5 shows WT and
Variant TTR vector backbone and insert and (B) Lane 1 shows ladder (1kb) and lane 2
shows p426 vector backbone

Then, the p426 vector was treated with rSAP (Shrimp Alkaline Phospphatase) enzyme
to prevent its self ligation of the vector and analyzed on the gel (Figure 4.3). The vector
backbone and the inserts was purified from the gel using QIAquick gel extraction kit and
further used for ligation.




Fig.4.3. shows p426 vector treated with rSAP enzyme on 1% agarose gel. Lane 1-
ladder(1Kb) , lane 2-3 shows the p426 vector backbone.

Vector backbone
Fragment of
Interest (TTR
gene)
Vector backbone
treated with rSAP
enzyme

V
35


Fig.4.4 Purified fragments on 1% agarose gel. Lane 1- ladder(1Kb) , lane 2 shows plasmid
vector backbone and Lane 3 shows gene of interest.

The vector and the insert were ligated as explained in materials and methods and the
ligated product was transformed in E.coli DH5 strain on LB-amp selection plate. No
colonies were seen in negative control (without plasmid) and a high number of transformant
colonies (10
7
-10
8
) were observed. These transformants were randomly picked and screened
for gene of interest by colony PCR using TTR specific and GFP specific primers. (Figure 4.5)
Transformants showed a fragment of 1.2 kb corresponding to TTR-GFP fusion construct.



Fig 4.5 PCR product on 1% agarose gel. Lane shows the ladder and lane 6, 10, 13 and
16 shows clones with gene of interest

Vector backbone
V
Purified Insert
36

In the above Figure 4.5, 10 clones were found positive. All the positive clones of the
WT and variant TTR p426 plasmid were then confirmed by restricted digestion. The plasmids
were isolated from these transformants and digested with enzyme AgeI to confirm the
orientation of the inserts. Expected sizes for correct orientation are: 7108 kb and 477 kb. The
sizes of incorrect orientation are: 6091 kb and 1494 kb. The clones showing about 400 kb
band upon digestion with AgeI restriction enzyme were positives. Based on the sizes of the
fragments, clones 1, 2, 3, 4, 7, 8, 10, 13 were positive (Figure 4.6)



Fig.4.6 Restriction digestion with AgeI to check orientation on 1% agarose gel. Lane
6- ladder (1Kb) and lane 1-4, 8-9, 11 and 14 depicts clones with gene of interest in the
right orientation

Aggregation and toxicity of WT and variant TTR-GFP in p426 plasmid
These positive plasmids with inserts in right orientation, were transformed in the yeast
using LiAc protocol and selected on SC-ura plates, as explained in Material and methods.
After 48 hrs of incubation at 30C or until the colonies appear, the colonies were picked and
inoculated in 2 ml of SC-ura culture and incubated at 30C for 24 hrs. Next, 2 ml of RafGal-
ura was inoculated at 0.2 O.D. and incubated at 30C for 48 hrs for induction. After
induction, aggregation of WT and variant TTR plasmids was checked on inverted
fluorescence at 100X. The WT and variant TTR gene in these plasmids are GFP tagged so the
cells showed green fluorescence. The percentage of aggregation was calculated by manually
counting 200-300 cells per clone. All plasmids showed very high expression (>40%) and no
significant difference was observed in aggregation between WT and variant TTR-GFP
plasmids.
We also examined the toxicity of these constructs by dilution spotting as explained in
material and methods. 10l of 10 transformants of one clone of each construct was spotted on
37

SC-ura and RafGal-ura (for induction of toxic protein) agar plates. After 72 hrs incubation at
30C, no toxicity was observed in neither WT or variant TTR-GFP construct as can been
seen in the fig.4.7.



Fig. 4.7 Toxicity assay. A(i), B(i)and C(i) shows growth of 10 transformants
of each variant TTR-GFP and WT-TTR construct on SC-ura agar plate. A(ii),
B(ii)and C(ii) shows the growt pattern of the same transformants on
2%Raf3%Gal-ura (induction media) agar plate.
38

SUMMARY AND CONCLUSION
Transthyretin is a tetramer polypeptide associated with transport of thyroxine (T4) in CSF
and retinol in the plasma, respectively. However, due to mutation in the TTR gene, the
propensity of tetramer dissociation increases significantly. More than 80 point mutations have
been identified in the TTR gene such as V30M, L55P, etc. The dissociation of the TTR
tetramer into dimers is a rate-limiting step. Due to mutation in the TTR gene, a misfiled TTR
protein is formed, and this misfolded protein is easily dissociated. Once the TTR tetramer
dissociates into dimmers, it is rapidly converted to monomers, these monomers are the toxic
species and results in the dissociation of other TTR molecule.
Despite several studies going on transthyretin amyloidosis, the exact trigger of TTR
pathogenesis is not yet clearly understood. Thus, several model organisms are being exploited
to study TTR tetramer dissociation and aggregation. One such model organism is yeast,
Saccharomyces cerevisae. In this work, WT and three variant TTR-GFP genes were cloned in
p426, a high copy number plasmid, and aggregation and toxicity of these plasmids was
studied in yeast model organism. The conclusions of this study are as follows:
1. The WT and variant TTR-GFP inserts were successfully cloned in the p426
vector.
2. Increased aggregation pattern of p426 variant TTR-GFP constructs and of p426
WT-TTR-GFP construction was observed in the fluorescence microscope. Also,
there was no difference in the aggregation pattern of WT and variant TTR-GFP
constructs.
3. When these new constructs were assayed for toxicity, no toxicity was seen. The
growth pattern of transformants of each p426 WT and variant TTR-GFP clone
was same on SC-ura (minimal media) plates and RafGal-ura (induction media)
plates. Therefore, no transfomants was toxic.


39















SECTION TWO: SCREENING OF INHIBITORS OF AGGREGATION
OF TTR-GFP AND HTT-GFP
























40

INTRODUCTION

6.1. Screening of inhibitors of TTR amyloidosis
Transthyretin amyloidosis, as explained in chapter 3, is caused to due to the variant TTR
tetramer dissociation, which is the late-limiting step of TTR amyloidogenesis, and further
misfolding into TTR aggregates. Several studies have demonstrated that binding of the small
molecules to the unoccupied thyroxine (T4) sites on the TTR protein stabilizes the protein
and reduces its rate of dissociation. These small molecules can prove to be quite helpful in
treating TTR amyloidosis (Connelly, 2010) Therefore scientists at The Scripps Research
Institute, California, USA have discovered tafamidis (Fx-1006A), a small molecule that acts
as a pharmacological chaperone for TTR and prevents misfolding. Tafamidis is the first drug
to be approved for the treatment of patients with stage I symptoms of transthyretin familial
amyloid polyneuropathy (TTR-FAP).

6.2. Screening of inhibitors of Huntington Disease (HD)
Huntingtons disease (HD) is a late-onset progressive neurodegenerative disorder that is
characterized by impairement of motor, cognitive and psychiatric functions (Bonnila, 2000).
It is caused by the abnormal expansion of CAG repeats in the first exon of HD gene that
encodes huntingtin (Htt) protein. These CAG repeats are translated as polyglutamine (polyQ)
repeats which make the mutant Htt protein was prone to cleavage by proteases and thus
misfolding and their subsequent aggregation (Zoghbi, 2000). The misfolding toxic mutant
Huntington protein causes impairement of axonal transport, interferes with the regulation of
transcription, causes mitochondrial dysfunction (Landles, 2004)
At present, there are no effective treatments for this devastating disease. However, one
therapeutic strategy to ameliorate the Huntingtons disease includes the use of small
molecules. One such example is Luteolin, which is a naturally occurring flavonoid abundant
among the plant kingdom (Harborne, 1992). Luteolin is a potent anti-inflammatory and
antioxidant agent (Asif, 2012). Several in vitro and in vivo studies have demonstrated that
luteolin exhibit neuroprotective effects such as it protects from toxicity due to oxidative
stress, and so on (Nazari, 2013)

41

REVIEW OF LITERATURE
7.1. Transthyretin amyloidosis and small molecules
Transthyretin amyloidosis
Transthyretin amyloidosis, as explained in chapter 2, is caused to due dissociation of
TTR tetramer into monomer, which is an initial rate-limiting step of TTR pathogenesis.
Several studies have been undergoing to synthesize small molecules exhibit structural
complementarity with T4.
Tafamidis
Tafamidis meglumine is a novel, first-in-class drug for the treatment of transthyetin
familial amyloid neuropathy (TTR-FAP) (de Lartigue, 2012). Transthyretin amyloidosis is a
fatal, late-onset neurodegenerative disease caused by accumulation of misfolded mutant TTR
protein, as explained in chapter 2. It is the first drug to be used for treatment of TTR-FAP
patients with early stage I symptpms (Connelly, 2010). Tafamidis mimics the natural
hormone T4 and prevent amyloid fibril formation (Nencetti, 2013). Tafamidis acts to
kinetically stabilize the variant TTR tetramer and thus preventing TTR dissociation which is
a rate-limiting step in TTR amyloidogenesis (Bulawa et al.., 2012).


Figure 7.1 Chemical structure of Tafamidis. (Source: PubChem)

Tafamidis (Vyndaqel), previously named as Fx 1006A, was discovered in the
Jeffrey W. Kelly laboratory using the structure based design strategy (Connelly, 2010). The
42

drug was approved by European commission in November, 2011 for the treatment of
transthyretin amyloidosis (Said, 2012). Tafamidis thermodynamically stabilize TTR tetramer
against both acid-mediated misfolding and urea denaturation by increasing the activation
barrier to tetramer dissociation, which is a crucial rate-limiting step for amyloid formation
(Razavi et al.., 2003). In a recent 12-month study, where oral dose of 20 mg tafamidis was
given to patient, TTR stabilization was observed in 94.1% of patients and 93.3% of placebo.
It was also reported that is safe and well tolerated for long-term use (Coelho, 2013).

7.2. Huntington disease and small molecules
Huntington disease
Huntington disease (HD) is an autosomal dominant neurodegenerative disease with
late onset (Ross, 2002). HD is estimated to affect five to seven people per 100,000 throughout
the word (Landles, 2004). Huntington disease is characterized mainly by chorea, which are an
abnormal involuntary writhing movements, psychiatric impairment and cognitive defects due
to selective neuronal degeneration (Zoghbi, 2000). In 1872, George Huntington gave the first
detailed description of this disorder, and since then this disorder has been named after him as
Huntington disease (Roos, 2010).
Huntington (HD) disease is caused by the expansion of CAG triplet in the first exon
of a single-copy gene located on short arm of the chromosome 4 (4p16.3) (Bonilla, 2000).
The Htt gene encodes the Htt protein with 3140 amino acid and a molecular mass of about
349 kDa. The wild-type Htt gene contains 6-39 CAG repeats, whereas the mutant allele has
more than 36 CAG expansion repeats (Wood and Everett, 2004). The unstable CAG repeats
in the mutant gene are translated as polyglutamine (PolyQ) stretch near the N-terminal of the
Htt protein (Zoghbi, 2000). This abnormal Htt protein is cleaved by caspases and generates
N-terminal fragments (Wellington, 2002) that exhibit toxic gain-of-mutation resulting in
impaired transcriptional regulation, intracellular transport, mitochondrial function (Landles,
2004).
The expression of normal Htt protein is ubiquitous; however, its expression is higher
in brain. It is mostly found in cytoplasm but is also seen in nucleus and vesicle membranes
(Ross, 2011). The normal Htt protein have an important role is vesicular transport,
embryogenesis, gene expression (Bonilla, 2000).
Key feature of pathogenesis of Huntington disease include the misfolding of the
expanded polyQ stretch of mutant Htt protein and formation of a toxic soluble oligomer,
43

which gradually accumulates into intracellular aggregates containing insoluble -sheet rich
amyloid deposits (Kim, 2013). Toxicity of mutant Htt is also enhanced by its accumulation
in neuron, impaired cellular metabolic pathways and translocation of mutant Htt protein into
nucleus where it affects transcription (Ross, 2011).
Clinical features of Huntington diseases are CNS degeneration, metabolic
dysfunction, muscle wasting and weight loss. In brain, there is massive straital neuronal death
with loss of 95% medium size spiny GABAerigic neurons. In addition, there is atrophy of the
cerebral cortex (Ross, 2011).
Since the pathogenesis of Huntington disease is not clearly understood yet, it is very
difficult to develop effective therapies. One therapeutic strategy is to reduce the concentration
of pathogenic protein, either by decreasing its production or increasing its clearance. Partial
recovery of both behavioral and pathological features was seen in an inducible transgenic
mouse, in which expression of mutant HTT was switched off (Yamamoto, 2000). Promising
results have been shown with using antisense oligonucleotides against Htt mRNA in HD
mouse models (Pfister, 2009). Another strategy is to enhance the activity of molecular
chaperones. Overexpression of one or both of the chaperones HS104 and HSP27 can suppress
mutant HTT-mediated neurotoxicity in mouse and rat models of Huntington disease (Perrin,
2007). Recent studies are screening for natural compound to alleviate symptoms of the
disease. For example, epigallocatechin gallate (EGCG), a polyphenol, decreases the toxic
forms of Htt (Ehrnhoefer, 2008).

Luteolin
Luteolin or 3,4,5,7-tetrahydroxyflavone is a flavonoid that is found in many plants.
Flavonoids are plant polyphenols that play an important role in plants as antioxidants, UV
light protectants and defense against phytopathogens (Harborne, 1992). Flavonoids comprise
a large group of plant secondary metabolites and are characterized by diphenylpropane
structure (C6-C3-C6) (Lopez-Lazaro, 2009). In plants, most of the flavonoids present in
plants are attached to sugars (glycosides), although occasionally they are found as aglycones
(Ross and Kasum, 2002). Flavonoids have been reported to posses many beneficial
properties, including anti-inflammatory, antiallergic, antitumor activity, oestrogenic
regulators, antimicrobial agents and antioxidant activity (Asif, 2012).

44


Figure 7.2 Chemical structure of luteolin (Source: Pubchem)

Luteolin, belongs to flavone class of flavonoids and has a typical C6-C3-C8 structure
and possess two benzene rings (A, B), a third, oxygen-containing ring, and a 2-3 carbon
double bond. Luteolin also have hydroxyl groups at carbons 5, 7, 3, and 4 position (Lin et
al.., 2008). The 2-3 double bond and hydroxyl moieties are important structures features of
luteolin that are associated with its biological and biochemical properties (Rice-Evanset al.,
1995). Luteolin is often found in glycosylated form and it hydrolysed to glucuronides when
passing through the intestinal mucosa. Luteolin is heat-stable and its loss due to cooking is
relatively low. Luteolin is found in vegetables and fruits such as celery, parsley, broccoli,
onion leaves, carrots, peppers, cabbages, apple skins, and other plant species (Lin et al..,
2008).
Luteolin exhibit increased vascular permeability, antioxidant and anti-inflammatory activities
(Seelinger, 2008) and is reported to reduce the chances to cancer, cardiovascular and
neurodegenerative disease (Nazari, 2013). Recently, luteolin have been reported to show
neuroprotective effective in invitro and invivo (Dajas, 2003). It has also been reported to be
effective against many neurological disorders such as amnesia, anxiety and depression.
Recently, luteolin has reported to exhibit protective effect against oxidative stress induced by
SNP toxicity in mouse brain (Nazari, 2013). Luteolin has also demonstrated neuroprotection
against oxidative stress via activation of nuclear factor erythroid-2-related factor 2(Nrf2). It
also protects rat neural PC12 and glial C6 cells from n-methyl-phenyl-pyridinium (MPP+)
induced toxicity invitro (Wruck, 2007)
45

MATERIALS AND METHODS
8.1. Materials
All materials like equipments, reagents, media etc were same as mentioned previously in
section 3.1
8.2. Methods
8.2.1. Plasmid isolation
Plasmids were isolated using QIA prep kit from Qiagen. The methodology is same as
explained in section 3.2.1
8.2.2. Yeast transformation
Yeast transformation was done using LiAc solution as mentioned in the section 3.2.8

8.2.3. Luteolin and Tafamidis treatment
Method:
a) 2 mL of Sc-ura was inoculated in 15 mL falcon tube with three transformants and
incubated at 30C, 200 rpm overnight.
b) Then, O.D. of the SC-ura cultures was taken.
c) The same evening, RafGal-ura media was inoculated with these SC-ura cultures for 0.2
O.D.
d) Next morning, O.D. was taken to check if the RafGal-ura cultures have reached 0.2-0.3
O.D.
e) Then, the remaining culture was added to 96-wells with each well containing 200 L of
culture.
f) A blank containing only culture, control(DMSO) and different concentration of luteolin
and/or tafamidis was added to the wells as follows:

Blank
Control
Varying conc. Of
Luteolin/Tafamidis
46

g) After adding luteolin/ Tafamidis, the culture was incubated for 8 hrs at 30C, 200
rpm.
h) After 8 hrs, cultures were washed with MilliQ water.
i) Then the cultures were resuspended in 200 L RafGal-ura media and incubated for 16
hrs at 30C, 200 rpm.
j) After this, aggregation was observed at 100X on inverted fluorescence microscope by
counting 200-300 cells manually.
47

RESULTS AND DISCUSSION

Effect of Luteolin/Tafamidis on protein aggregation of TTR-GFP and HTT-GFP
respectively
The plasmids containing WT and 3 variant TTR-GFP sequences and plasmid
containing expanded poly Q repeats (Htt72Q) under galactose promoter were isolated from
DH5 strain of E.coli.


Figure 9.1 shows isolated plasmids on 1% agarose gel. Lane 1 &6- DNA ladder,
Lane 2 WT-TTR, Lane 3, 4 and 5- TTR Variant 1, 2 and 3 respectively, Lane 7-
Htt72Q plasmid.
The plasmids were then transformed in yeast by LiAc protocol and the transformants
were selected on SC-ura media. The transformants were induced by growing them in media
containing raffinose and galactose. The transformants with Htt72Q plasmid were treated with
luteolin and the transformants with variant TTR plasmids were treated with tafamidis as
described in material and methods and were studied under the fluorescence microscope and
the percentage of aggregation was evaluated by manually counting approximately 300 cells
for each transformant.

48


EFFECT OF LUTEOLIN ON HTT AGGREGATION
IST ATTEMPT


Conc. T1 T2 T3 Average Std. Dev Std. Error
DMSO 13.5 15.8 11.9 13.733333 1.9604421 1.1318618
300 M 7.98 9.1 9.4 8.8266667 0.7484206 0.4321008
500 M 7.78 8.5 7.9 8.06 0.385746 0.2227106





As can be observed from the graph, significant difference was seen between treated
and untreated samples. However, not much difference was observed among samples treated
with different conc. of Luteolin. Thus, the experiment was repeated again.



0
2
4
6
8
10
12
14
16
18
DMSO 300 M 500 M
A
g
g
r
e
g
a
t
i
o
n

P
e
r
c
e
n
t
a
g
e

Varying conc. of Luteolin
Fig 9.2 Graph showing the percentage of protein aggregation after
treatment with different conc. Of Luteolin
Table 9.1 represents the percentage of cells with aggregates for one to three transformant
treated with luteolin (attempt I)

49


IIND ATTEMPT
In the 2
nd
attempt, the experiment was repeated with different concentration of
luteolin. The table below shows the aggregation percentage in each transformant.

Table 9.2 represents the percentage of cells with aggregates for one to three
transformant treated with luteolin (Attempt II)
Conc. T1 T2 Average Std Dev. Std. Error
DMSO 18 22 20 2.8284271 2
50 M 11 17 14 4.2426407 3
300 M 10 6 8 2.8284271 2


Fig 9.3 Graph showing the percentage of aggregation after treatment with different
conc. Of Luteolin( II attempt)

As can be observed from the graph, significant difference was seen between treated
and untreated samples. However, slight difference in protein aggregation was observed
among samples treated with different conc. of Luteolin. Thus, the experiment was repeated
again.



0
5
10
15
20
25
DMSO 50 M 300 M
A
g
g
r
e
g
a
t
i
o
n

P
e
r
c
e
n
t
a
g
e


Varying conc. of Luteolin
50

EFFECT OF TAFAMIDIS ON TTR VARIANTS
The effect of tafamidis was studied on the variant forms of TTR and the table below
shows the percentage of aggregation observed in each transformant.

IST ATTEMPT
Table 9.3 represents the percentage of cells with aggregates for one to three
transformant treated with tafamidis (Attempt I)
Conc. T1 T2 Average Std. Dev Std. Error
DMSO 50 60 55 7.07107 5
5 M 52.3 46.2 49.25 4.31335 3.05
10 M 45.4 50.2 47.8 3.39411 2.4
20 M 46.7 40.7 43.7 4.24264 3
50 M 44.8 49.7 47.25 3.46482 2.45


Fig 9.4 Graph showing the percentage of aggregation after treatment with different
conc. Of Tafamidis

As evaluated by the percentages observed and the graph above, it can be deduced that
no significant difference in protein aggregation was observed in either the untreated and
0
10
20
30
40
50
60
70
DMSO 5 M 10 M 20 M 50 M
A
g
g
r
e
g
a
t
i
o
n

P
e
r
c
e
n
t
a
g
e

Varying Conc. of Tafamidis
51

treated sample or among samples treated with differen conc. of tafamidis. The experiment
was repeated.

IIND ATTEMPT

Table 9.4 represents the percentage of cells with aggregates for one to three
transformant treated with tafamidis (attempt II)
Conc. T1 T2 T3 Average Std. Dev Std. Error
DMSO 73.2 82.7 84.6 80.166667 6.1076455 3.5262508
50 M 50.7 75.2 75 66.966667 14.087701 8.1335382
100 M 53.7 78.1 76.3 69.366667 13.597549 7.8505485



Fig 9.5 Graph showing the percentage of aggregation after treatment with different
conc. Of tafamidis (II attempt)

In the second attempt also, no notable decrease in concentration was observed in
treated samples as compared to the untreated samples. Moreover, no difference in
aggregation percentage was seen among the treated samples.


0
10
20
30
40
50
60
70
80
90
DMSO 50 M 100 M
A
g
g
r
e
g
a
t
i
o
n

P
e
r
c
e
n
t
a
g
e

Varying Conc. of Tafamidis
52

SUMMARY AND CONCLUSION

Huntingtons disease is a lethal progressive neurodegenerative disease caused due to
abnormal expansion of CAG repeats in the HTT gene. Transthyretin amyloidosis is another
neurodegenerative disease that is deadly and causes organ dysfunction and eventually death.
In both these disorders the underlying reason of pathogenesis is the aggregation of these
misfolded proteins. Despite the ongoing research to understand pathogenesis and develop
therapies to ameliorate the symptoms of these disorders, there is no effective treatment for
these diseases. Several natural and synthetic compounds are being screened that reduces the
protein aggregation. Tafamidis, a compound synthezised by Kellys lab helps to stabilize the
TTR tetramer and thus results in reduction of protein aggregation. In this work, the effect of
tafamidis was studied on aggregation of WT and variant TTR-GFP in the yeast model.
Similarly, luteolin, a natural flavonoid, is known to have neuroprotective effects so it was
used to study its effect on HTT-GFP aggregation in yeast model. The following conclusions
were made based on the present study:
1. In the p426 HTT-GFP constructs, approximately 2-fold decrease in aggregate
percentage was seen in the treated samples during the two attempts of experiment.
However, dose-dependent effect of luteolin on treated samples was not observed.
2. In the p426 TTR-GFP variant constructs, no visible effect of tafamidis was seen on
the protein aggregation. In the treated samples, no decrease in protein aggregation was
seen as compared to the untreated samples. And also, no difference in aggregation
pattern was observed in samples treated with increasing conc. of tafamids.
53

REFERENCES
Almeida MR, Macedo B, Cardoso I, Alves I, Valencia G, Arsequell G, Planas A, Saraiva MJ.
(2004). Selective binding to transthyretin and tetramer stabilization in serum from patients
with familial amyloidotic polyneuropathy by an iodinated diflunisal derivative. Biochem J.
381:351-6

Ando E, Ando Y, Okamura R, Uchino M, Ando M, Negi A. (1997) Ocular manifestations of
familial amyloidotic polyneuropathy type I: long-term follow up. Br J Ophthalmol 81:295
298.

Ando Y, Kakamura M, Araki S. (2005) Transthyretin-Related Familial Amyloidotic
Polyneuropathy. Arch Neurol., 62:1057-1062.

Araki S, Yi S. (2000). Pathology of familial amyloidotic polyneuropathy with TTR met 30 in
Kumamoto, Japan. Neuropathology, 20: 4751.

Asif M. (2012). Review: Phytochemical study of polyphenols in Perilla Frutescens as an
antioxidant. Journal of Phytomedicine, 2:169-178

Benson M. (2009). Genetics: Clinical Implications of TTR Amyloidosis. In Recent Advances
in Transthyretin Evolution, Structure and Biological Functions. Springer, 173189.

Benson MD, Kluve-Beckerman B, Zeldenrust SR, Siesky AM, Bodenmiller DM, Showalter
AD, Sloop KW. (2006) Targeted suppression of an amyloidogenic transthyretin with
antisense oligonucleotides. Muscle Nerve. 33:609618.

Benson MD, Uemichi T. (1996). Review: Transthyretin Amyloidosis. Int J Exp Clin Invest.,
3:44-56

Benson MD. (2001). Amyloidosis; Encyclopedia of Life Science.

Blake CC, Geisow MJ, Oatley SJ, Rrat B, Rrat C. (1978). Structure of prealbumin:
Secondary, tertiary and quaternary interactions determined by Fourier refinement at 1.8 . J
Mol Biol., 121:339356.

Blake CC, Swan ID, Rerat C, Berthou J, Laurent A, Rerat B. (1971). An X-ray study of the
subunit structure of prealbumin. J Mol Biol., 61:217-24

Bogov B, Lubomirova M, Kiperova B. (2008). Biopsy of subcutaneus fatty tissue for
diagnosis of systemic amyloidosis. Hippokratia., 12:236-9.

Botstein D, Chervitz SA, Cherry JM. (1997).Yeast as a model organism. Science., 277:1259-
60

Botstein D, Fink GR. (2011). Yeast: an experimental organism for 21st Century biology.
Genetics., 189:695-704

54

Bulawa CE, Connelly S, Devit M, Wang L, et al. (2012). Tafamidis, a potent and selective
transthyretin kinetic stabilizer that inhibits the amyloid cascade. Proc Natl Acad Sci U S A.,
109: 9629-34

Coelho T, Maia LF, da Silva AM, Cruz MW, et al. (2013). Long-term effects of tafamidis
for the treatment of transthyretin familial amyloid polyneuropathy. J Neurol., 260:2802-14.

Come J, Fraser P, Lansbury PJ. (1993). A Kinetic Model for Amyloid Formation in the Prion
Diseases, Importance of Seeding. PNAS USA., 90:595963.

Connelly S, Choi S, Johnson SM, Kelly JW, Wilson IA. (2010). Structure-based design of
kinetic stabilizers that ameliorate the transthyretin amyloidoses. Curr Opin Struct Biol.
20:5462.

Dajas F, Rivera-Megret F, Blasina F, Arredondo F, Abin-Carriquiry JA, Costa G, Echeverry
C, Lafon L, Heizen H, Ferreira M, Morquio A. (2003). Neuroprotection by flavonoids. Braz J
Med Biol Res. 36:1613-1620

Damas AM., Saraiva MJ. (2000). Review: TTR Amyloidosis Structural Features Leading to
Protein Aggregation and Their Implications on Therapeutic Strategies. J Struct Biol.,
130:290-299

de Lartigue J. (2012). Tafamidis for transthyretin amyloidosis. Drugs Today(Barc).,48:331-7.

Dember LM. (2006). Amyloidosis-associated kidney disease. J Am Soc Nephrol.,17:3458-71.

Desport E, Bridoux F, Sirac C, Delbes S, Bender S, Fernandez B, et al. (2012). Review: Al
amyloidosis. Orphanet J Rare Dis., 7:54

Dobson CM. (2003). Protein folding and misfolding. Nature., 426:88490.

Dubrey SW, Hawkins PN, Falk RH. (2011). Amyloid diseases of the heart: assessment,
diagnosis, and referral. Heart., 97:75-84.

Dwulet FE, Benson MD. (1984). Primary structure of an amyloid prealbumin and its plasma
precursor in a heredofamilial polyneuropathy of Swedish origin. Proc. Nadl. Acad. Sci.,
81:694-698.

Ehrnhoefer DE, Bieschke J, Boeddrich A, et al. (2008). EGCG redirects amyloidogenic
polypeptides into unstructured, off -pathway oligomers. Nat Struct Mol Biol., 15: 55866.

Everett CM, Wood NW. (2004). Trinucleotide repeats and neurodegenerative disease. Brain,
127:2385-405.

Falk RH, Comenzo RL, Skinner M. (1997). Review: The Systemic Amyloidoses. The New
England Journal of Medicine, 337:13.

Falk RH. (2005). Diagnosis and management of the cardiac amyloidoses. Circulation,
112:20472060.

55

Fleming CE, Nunes AF, Sousa MM. (2009). Transthyretin: More than meets the eye. Prog
Neurobiol, 89: 266-76.

Foss TR, Wiseman RL, Kelly JW. (2005). The pathway by which the tetrameric protein
transthyretin dissociates. Biochemistry. 44:1552515533.

Fung WP, Thomas T, Dickson PW, Aldred AR, Milland J, Dziadek M, Power B, Hudson P,
Schreiber G. (1988). Structure and expression of the rat transthyretin (prealbumin) gene. J
Biol Chem., 263:480-8.

Gitler AD. (2008). Beer and Bread to Brains and Beyond: Can Yeast Cells Teach Us about
Neurodegenerative Disease?. NeuroSignals, 16:52-62.

Hamilton JA, Benson MD. (2001). Transthyretin: a review from a structural perspective. Cell
Mol Life Sci., 58:1491-521

Harborne JB, Williams CA. (2000). Advances in flavonoid research since 1992.
Phytochemistry, 55:481504.

Hawkins PN. (2002). Serum amyloid P component scintigraphy for diagnosis and monitoring
amyloidosis. Curr Opin Nephrol Hypertens., 11: 649-55.

Hawkins PN. (2003). Hereditary systemic amyloidosis with renal involvement. J Nephrol.,
16:443-8.

Herlenius G, Wilczek HE, Larsson M, Ericzon BG. (2004). Ten years of international
experience with liver transplantation for familial amyloidotic polyneuropathy: results from
the Familial Amyloidotic Polyneuropathy World Transplant Registry. Transplantation,
77:6471.

Hinnen A, Hicks JB, Fink GR. (1978). Transformation of yeast. Proc Natl Acad Sci USA.,
75:1929-33.

Holmgren G, Steen L, Ekstedt J, Groth CG, Ericzon BG, Eriksson S, et al. (1991).
Biochemical effect of liver transplantation in two Swedish patients with familial amyloidotic
polyneuropathy (FAP-Met30). Clin. Genet., 40:242246.

Hou X, Aguilar M, Small DH. (2007). Transthyretin and familial amyloidotic polyneuropathy
Recent progression in understanding the molecular mechanism of neurodegeneration. FEBS
J. 16371650.

Jacobson DR, McFarlin DE, Kane I, Buxbaum, JN. (1992). Transthyretin Pro55, a variant
associated with early-onset, aggressive, diffuse amyloidosis with cardiac and neurologic
involvement. Hum Genet., 89:353356.

Jahn TR, Parker MJ, Homans SW, et al. (2006). Amyloid formation under physiological
conditions proceeds via a nativelike folding intermediate. Nat Struct Mol Biol., 13:195201.

56

Johnson SM, Connelly S, Fearns C, Powers ET, Kelly JW. (2012). The Transthyretin
Amyloidoses: From Delineating the Molecular Mechanism of Aggregation Linked to
Pathology to a Regulatory Agency Approved Drug. J Mol Biol., 421: 185203.

Johnson SM, Wiseman RL, Sekijima Y, Green NS, Adamski-Werner SL, Kelly JW. (2005).
Native state kinetic stabilization as a strategy to ameliorate protein misfolding diseases: a
focus on the transthyretin amyloidoses. Acc Chem Res., 38:911-21

Kyle RA. (2011). Amyloidosis: a convoluted story. Brit. J. Haem., 114:529538.

Lachmann HJ, Goodman HJB, Gallimore J, et al. (2005). Characteristic and clinical outcome
of 340 patients with systemic AA amyloidosis. In: Grateau G, Kyle RA, Skinner M, eds.
Amyloid and Amyloidosis. Boca Raton, Florida: CRC Press, 173-5.

Landles C, Bates GP. (2004) Huntington and the molecular pathogenesis of Huntingtons
disease. Fourth in molecular medicine review series. EMBO Rep 5:958-963.

Lin Y, Shi R, Wang X, Shen HM. (2008). Luteolin, a flavonoid with potentials for cancer
prevention and therapy. Curr Cancer Drug Targets 8:634646.

Loizos S, Shiakalli Chrysa T, Christos GS. (2014). Amyloidosis: review and imaging
findings. Semin Ultrasound CT MR., 35:225-39.

Lpez-Lzaro M. (2009). Distribution and biological activities of the flavonoid luteolin. Mini
Rev Med Chem., 9:3159.

Mell JC, Burgess SM. (2002). Yeast as a model genetic organism. Encyclopedia of life
sciences.

Miller-Flemming L, Giorgini F, Outeiro TF. (2008). Review: Yeast as a model for studying
human neurodegenerative disorders. Biotechnol. J., 3:325 338.

Miroy GJ, Lai Z, Lashuel HA, Peterson SA, Strang C, Kelly JW. (1996). Inhibiting
transthyretin amyloid fibril formation via protein stabilization. Proc Natl Acad Sci USA.,
93:15051-6.

Misrahi AM, Plante V, Lalu T, Serre L, Adams D, Lacroix DC & Said G. (1998). New
transthyretin variants SER 91 and SER 116 associated with familial amyloidotic
polyneuropathy. Hum Mutat., 12:71.

Monaco HL, Rizzi M & Coda A. (1995). Structure of a complex of two plasma proteins:
transthyretin and retinol-binding protein. Science, 268:10391041.

Monaco HL. (2000). The transthyretin-retinol-binding protein complex. Biochim Biophys
Acta., 18:65-72.

Nazari QA, Kume T, Takada-Takatori Y, Izumi Y and Akaike A. (2013). Protective Effect of
Luteolin on an Oxidative-Stress Model Induced by Microinjection of Sodium Nitroprusside
in Mice. J Pharmacol Sci., 122:109 117

57

Nencetti S, Rossello A, Orlandini E. (2013). Tafamidis (Vyndaqel): A Light for FAP
Patients. ChemMedChem., 8:16171619

Pepys M. (2001). Pathogenesis, diagnosis and treatment of systemic amyloidosis. Phil Trans
Soc Lond B., 356:20311.

Pepys MB. (2006). Amyloidosis. Annu. Rev. Med., 57:223-41.

Pepys-Vered ME, Pepys MB. (2014). Targeted treatment for amyloidosis. Isr Med Assoc
J.,16:277-80.

Perrin V, Regulier E, Abbas-Terki T, et al. (2007). Neuroprotection by Hsp104 and Hsp27 in
lentiviral-based rat models of Huntingtons disease. Mol Ther., 15: 90311.

Pfister EL, Zamore PD. (2009). Huntingtons disease: silencing a brutal killer. Exp Neurol.,
220:22629.

Power DM, Elias NP, Richardson SJ, Mendes J, Soares CM, Santos CR. (2000). Evolution of
the thyroid hormone-binding protein, transthyretin. Gen Comp Endocrinol., 119:241-55.

Rambaran RN1, Serpell LC. (2008). Review: Amyloid fibrils: abnormal protein assembly.
Prion, 2:112-7

Rapezzi C, Quarta CC, Riva L, Longhi S, Gallelli I, et al. (2010). Transthyretin-related
amyloidoses and the heart: a clinical overview. Nature Review Cardiology, 7:398-408.

Razavi H, Palaninathan SK, Powers ET, Wiseman RL, Purkey HE, et al. (2003).
Benzoxazoles as transthyretin amyloid fibril inhibitors: synthesis, evaluation, and mechanism
of action. Angew Chem Int Ed Engl., 42:2758-61.

Reixach N, Deechongkit S, Jiang X, Kelly JW, Buxbaum JN (2004) Tissue damage in the
amyloidoses: Transthyretin monomers and nonnative oligomers are the major cytotoxic
species in tissue culture. Proc Natl Acad Sci USA 101:28172822.

Rice-Evans CA, Miller NJ, Bolwell PG, Bramley PM, Pridham JB. (1995). The relative
antioxidant activities of plant-derived polyphenolic flavonoids. Free Radic Res. 22:375- 83.

Richardson SJ. (2007). Cell and molecular biology of transthyretin and thyroid hormones. Int
Rev Cytol. 258:137-93.

Roos RA. (2010). Huntington's disease: a clinical review. Orphanet J Rare Dis. 5:40

Ross CA. (2002). Polyglutamine pathogenesis: emergence of unifying mechanisms for
Huntingtons disease and related disorders. Neuron, 35:819-822.

Ross CA, Tabrizi SJ. (2011). Review: Huntingtons disease: from molecular pathogenesis to
clinical treatment. Lancet Neurol., 10: 8398

Ross JA, Kasum CM. (2002). Dietary flavonoids: bioavailability, metabolic effects, and
safety. Annu Rev Nutr., 22:1934.
58


Said G, Grippon S, Kirkpatrick P. (2012). Tafamidis. Nat Rev Drug Discov., 11:85-186

Sakaki Y, Yoshioka K, Tanahashi H, Furuya H, Sasaki H. (1989). Human transthyretin
(prealbumin) gene and molecular genetics of familial amyloidotic polyneuropathy. Mol Biol
Med. 6:161-8.

Sambamurti K, Greig NH, Utsuki T, Barnwell EL, Sharma E, et al. (2011). Targets for AD
treatment: conflicting messages from gamma-secretase inhibitors. J. Neurochem., 117:359
374.

Sanchorawala V. (2006). Light-chain (AL) amyloidosis: diagnosis and treatment. Clin J Am
Soc Nephrol., 1:1331-41

Saraiva MJ, Almeida MR, Sherman W, Gawinowicz M, et al. (1992). A new transthyretin
mutation associated with amyloid cardiomyopathy. Am J Hum Genet., 50:10271030.

Saraiva MJ. (2002). Hereditary transthyretin amyloidosis: molecular basis and therapeutical
strategies. Expert Rev Mol Med., 4:111.

Saraiva MJ. (2001).Transthyretin amyloidosis: a tale of weak interactions. FEBS Lett.,
498:201-3.

Sasaki H, Yoshioka N, Takagi Y, Sasaki Y. (1985). Structure of the chromosomal gene for
human serum prealbumin. Gene, 37:191-197.

Seelinger G, Merfort I, Schempp C. (2008). Anti-Oxidant, Anti-Inflammatory and Anti-
Allergic Activities of Luteolin. Planta Med., 74: 1667-1677.

Serpell L. (2000). Alzheimers amyloid fibrils: structure and assembly. Biochim Biophys
Acta., 1502:1630

Sikorski RS, Hieter P. (1989). A system of shuttle vectors and yeast host strains designed for
efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics, 122:19-27.

Sipe JD, Benson MD, Buxbaum JN, Ikeda S, Merlini G et al. (2010). Amyloid fibril protein
nomenclature: 2010 recommendations from the nomenclature committee of the International
Society of Amyloidosis. Amyloid, 17:101-4

Sipe JD, Cohen AS. (2000). Review: History of the Amyloid Fibril. J Struct Biol., 130:88-98.

Soto C, Estrada L, Castilla J. (2006). Amyloids, prions and the inherent infectious nature of
misfolded protein aggregates. Trends Biochem Sci., 31:150-155.

Soto C, Estrada LD. (2008). Review: Protein misfolding and neurodegeneration. Arch
Neurol. 65:184-9.

Sousa MM, Saraiva MJ. (2003). Neurodegeneration in familial amyloid polyneuropathy:
from pathology to molecular signaling. Prog Neurobiol., 71:385400.

59

Sousa MM, Yan SD, Fernandes R, Guimares A, Stern D, Saraiva MJ. (2001). Familial
Amyloid Polyneuropathy: Receptor for Advanced Glycation End Products-Dependent
Triggering of Neuronal Inflammatory and Apoptotic Pathways. J Neurosci., 21:7576-7586.

Sunde M, Blake CC. (1998). From the globular to the fibrous state: protein structure and
structural conversion in amyloid formation. Q Rev Biophys., 31:139.

Sunde M, Richardson SJ, Chang L, Pettersson TM, Schreiber G, Blake CC. (1996). The
crystal structure of transthyretin from chicken. Eur J Biochem., 236:491-9.

Sunde M, Serpell L, Bartlam M, et al. (1997). Common core structure of amyloid fibrils by
synchrotron Xray diffraction. J Mol Biol., 273:72939.

Suter B, Auerbach D, Stagljar I. (2006). Review: Yeast-based functional genomics and
proteomics technologies: the first 15 years and beyond. BioTechniques, 40:625-644.

Takahashi K, Sakashita N, Ando Y, Suga M, Ando M. (1997). Late onset type I familial
amyloidotic polyneuropathy: presentation of three autopsy cases in comparison with 19
autopsy cases of the ordinary type. Pathol Int., 47:353359.

Terry CJ, Damas AM, Oliveira P, Saraiva MJ, et al. (1993). Structure of Met30 variant of
transthyretin and its amyloidogenic implications. EMBO J. 12:735741.

Tsuzuki T, Mita S, Maeda S, Araki S, Shimada K. (1985). Structure of the Human
Prealbumin gene. J Biol Chem., 260:12224-12227.

Wellington CL, Ellerby LM, Gutekunst CA, Rogers D, Warby S, et al. (2002). Caspase
cleavage of mutant huntingtin precedes neurodegeneration in Huntingtons disease. J
Neurosci., 22:7862-7872.

Westermark P. (2005). Aspects on human amyloid forms and their fibril polypeptides. FEBS
J 272: 59425949.

Wruck CJ, Claussen M, Fuhrmann G, Rmer L, et al. (2007). Luteolin protects rat PC12 and
C6 cells against MPP+ induced toxicity via an ERK dependent Keap1-Nrf2-ARE pathway. J
Neural Transm Suppl., 57-67.

Yamamoto A, Lucas JJ, Hen R. (2000). Reversal of neuropathology and motor dysfunction in
a conditional model of Huntingtons disease. Cell, 101: 5766

Yokoyama T, Mizuguchi M, Nabeshima Y, et al. (2013). Hydrogen-bond network and pH
sensitivity in human transthyretin. J Synchrotron Rad 20:834837.

Zheng W, Lu YM, Lu GY, Zhao Q, Cheung O, Blaner WS. (2001). Transthyretin,
thyroxine, and retinol-binding protein in human cerebrospinal fluid: Effect of lead exposure.
Toxicol sci., 61: 107-114

Zoghbi HY, Orr HT. (2000). Glutamine repeats and neurodegeneration. Annu Rev Neurosci.,
23:217-247.