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)

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 Huntington’s 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

2

SC Synthetic complete

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

3

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

4.2Restriction 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

9.2Graph showing the percentage of protein aggregation after treatment with different conc. of

Luteolin

9.3Graph showing the percentage of aggregation after treatment with different conc. Of

Luteolin( II attempt)

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

(attempt I)

9.5Graph showing the percentage of aggregation after treatment with different conc. Of Tafamidis

(attempt II)

4

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)

5

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

6

TABLE OF CONTENTS

SECTION ONE: CLONING OF TTR-GFP IN A YEAST VECTOR TO EXAMINE PROTEIN AGGREGATION AND TOXICITY.....................................................................13

INTRODUCTION................................................................................................................14REVIEW OF LITERATURE..............................................................................................18MATERIALS AND METHODS.........................................................................................25SUMMARY AND CONCLUSION.....................................................................................43

SECTION TWO: SCREENING OF INHIBITORS OF AGGREGATION OF TTR-GFP AND HTT-GFP........................................................................................................................44

INTRODUCTION................................................................................................................45REVIEW OF LITERATURE..............................................................................................46MATERIALS AND METHODS.........................................................................................50RESULTS AND DISCUSSION..........................................................................................52SUMMARY AND CONCLUSION.....................................................................................57

7

SECTION ONE: CLONING OF TTR-GFP IN A YEAST VECTOR TO

EXAMINE PROTEIN AGGREGATION AND TOXICITY

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 19th century, Virchow adopted the term “amyloid”, from botany, meaning

8

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 β 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 Alzheimer’s 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).

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

9

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).

1.3. Amyloid fibril formation

Amyloid fibrils are insoluble, long, straight and unbranching fibres of 70-120Å in

diameter (Serpell, 1999). They specifically bind certain dyes such as Congo red and

thioflavin T, and they demonstrate a characteristic cross-β pattern on X-ray diffraction,

reflecting distances between β-strands (4.7 Å ) and distances between β-sheets (9–11 Å)

(Sipe, 2005; Sunde, 1998).

10

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).

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

11

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 λ- or κ-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

Alzheimer’s disease is to reduce the production of amyloid β (Aβ) by inhibiting the β- or γ-

secretases that generate the Aβ 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).

REVIEW OF LITERATURE

1.

2.

2.1.Transthyretin amyloidosis

12

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).

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

13

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).

14

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

Transthyretin is a tetrameric protein of 55 kDa composed of four identical β-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 β strands named A-H and one α

helix (Yokoyama et al., 2013). The eight β-strands are arranged in a β-sandwich made of two

four stranded β-sheets and one α-helix formed between β-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 β-sheets composed of four β-strands, from each monomer,

into two β-sheets of eight β-strands (Almeida et al., 2004). The dimer–dimer 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).

15

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 β-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 β-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).

16

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

17

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 baker’s 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 Alzheimer’s 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 Alzheimer’s and Parkinson’s 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).

18

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. β-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.

19

MATERIALS AND METHODS

1.

2.

3.

3.1.Materials3.1.1. 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

20

Speed vac Labconco

Vortex Tarson

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 ReagentsReagents 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 DH5α

Saccharomyces cerevisae

21

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 GmsPeptic digest of animal tissue 20.00

Yeast extract 10.00

Dextrose 20.00pH (at 25˚C) 6.5±0.2

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.

22

Ingredients Gms

Casein enzymic 10.00

Yeast extract 5.00

Sodium Chloride 10.00

pH (at 25˚C) 7.5±0.2

Amino acid mix

Table 3.5 Composition of 10X amino acid mix

Amino Acid mgAlanine 80Arginine 40Aspartic acid 200Glutamic acid 200Histidine 40Leucine 120Lysine 60Methionine 40Phenylalanine 100Threonine 400Tyrosine 60Tryptophan 80Valine 300Serine 750Uracil 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%

23

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

3.2 Methods

24

3.2.1 Plasmid isolation from DH5α 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 µL Buffer P1 and was transferred to 1.5 ml

microcentrifuge tube.

c) 250 µL of Buffer P2 was added and mixed thoroughly by inverting the tube 5-6

times.

d) After that, 300 µL 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 µL 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 µL 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 -20°C.

3.2.2 Restriction Digestion

25

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 (µL)

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 37˚C.

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 50˚C 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 µL, 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.

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.

26

j) QIAquick column was placed into a clean 1.5 ml microcentrifuge tube.

k) To elute DNA, 50 µL 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 (µL)

Digested vector 49

rSAP enzyme 2.5

NF water 23.5

Total 75

2. Then, the reaction mixture was incubated at 37°C for 45 mins.

3. Next, the mixture was incubated at 65°C 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:

Table 3.10 Reaction mixture for ligation

27

Volume (µL)

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 16˚C in a thermal

cycler.

c) Then ligated product was transformed in bacteria (DH5α 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 CaCl2 solution

c) Solution of 100mM CaCl2 solution and 20% glycerol

Method:

a) 180 mL Fresh LB broth was inoculated with the saturated primary culture (E.coli

DH5α) at 0.14 O.D.

b) The inoculated media was then incubated on shaker at 37˚C until it reaches 0.5-0.6

O.D. (about 1 hr).

c) The media was then incubated at 4˚C for 30 min.

d) The culture was centrifuged at 4˚C for 10 min to pellet down the cells.

e) The cell pellet was resuspended with autoclaved MilliQ water (chilled) and

centrifuged at 4˚C for 10 min.

f) Then, the cell pellet was washed with 100mM solution of CaCl2 (chilled) and

centrifuged at 4˚C for 10 min.

g) The step 6 was repeated once more.

28

h) The cell pellet was then resuspended in the chilled solution of 100mM CaCl2 and

20% glycerol and stored at -80˚C 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 µL of competent cells were thawed on ice and transferred into 2

microcentrifuge tube (50 µL each).

b) Then 3 µL 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 42˚C and the samples were given a heat-shock at 42˚C for 90

sec.

e) Then, the tubes were spanned chilled on ice for 2 min.

f) After this, 700 µL of fresh LB broth was added to the samples at RT.

g) The samples were then incubated at 37˚C for 45 min on shaker.

h) Then 100 µL of samples were spread plated on LB-amp plates and incubated at

37˚C 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 (µL)

Positive Control Negative

control

Samples

10X Taq buffer 2.5 2.5

25mM MgCl2 1.5 1.5 1.5

10µM dNTPs 0.7 0.7 0.7

29

10µM Forward

primer

1 1 1

10µM 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 10.0˚C 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 (µL)

10X buffer NEB1 2

Plasmid 2

AgeI 1

Nuclease – free water 15

Final volume 20

b) The mixture was then vortexed and was given a short spin to clear the lid.

c) Then the tubes were incubated at 37˚C for 3 hrs

30

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 30°C.

Day 2:

a) The patch was scooped and mixed in about 500 µL 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 µL 1X LiAc solution made by mixing 100mM

LiAc and 10X TE buffer in MQ, and incubated for 30-45 min at 30˚C.

d) After incubation, the samples were centrifuged and supernatant was discarded.

e) Then the pellet was resuspended in (50 X N) µL of 1X lithium acetate solution,

where N is the number of samples.

f) 300µl of PEG solution was taken in a clean microcentrifuge tube and 50µl of

mixture from the previous step was added to it and mixed thoroughly.

g) Then, 9µl of sperm DNA (preheated at 95˚C) was added and mixed thoroughly.

h) Finally, 4µl of plasmid with the insert was added and mixed.

i) Then, the samples were incubated at 30˚C, 200 rpm for 1.5 hrs.

j) After incubation, samples were given heat shock at 42˚C for 8 min and centrifuged.

k) The supernatant was discarded and the pellet was resuspended in 100µl of

autoclaved water and plated onto SC-ura plate and incubated for 48 hrs at 30°C.

31

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 30°C, 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 30°C, 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 30°C, 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 µL of autoclaved water.

c) Then the following dilutions of the cultures were prepared:

5-1, 5-2, 5-3, 5-4

d) Then 10 µL 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 30°C for 48-72 hrs or till

the growth appeared.

32

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

1.

2.

3.

4.

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.

33

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.

34

Vector backbone

Fragment of Interest (TTR gene)

VVector backbone treated with rSAP enzyme

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 DH5α strain on LB-amp selection plate. No

colonies were seen in negative control (without plasmid) and a high number of transformant

colonies (107-108) 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

35

VVector backbone

Purified Insert

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 30°C for 24 hrs. Next, 2 ml of RafGal-

ura was inoculated at 0.2 O.D. and incubated at 30°C 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. 10µl of 10 transformants of one clone of each construct was spotted on

36

SC-ura and RafGal-ura (for induction of toxic protein) agar plates. After 72 hrs incubation at

30°C, no toxicity was observed in neither WT or variant TTR-GFP construct as can been

seen in the fig.4.7.

37

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.

SUMMARY AND CONCLUSION

1.

2.

3.

4.

5.

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.

38

39

SECTION TWO: SCREENING OF INHIBITORS OF AGGREGATION

OF TTR-GFP AND HTT-GFP

40

INTRODUCTION

1.

2.

3.

4.

5.

6.

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)

Huntington’s 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 Huntington’s disease includes the use of small

molecules. One such example is Luteolin, which is a naturally occurring flavonoid abundant

41

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)

1.2.3.4.5.6.

42

REVIEW OF LITERATURE

1.

2.

3.

4.

5.

6.

7.

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).

43

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

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.

7.1.

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

44

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,

which gradually accumulates into intracellular aggregates containing insoluble β-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

45

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).

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

46

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)

MATERIALS AND METHODS

1.

2.

3.

4.

5.

6.

7.

8.

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

47

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 30°C, 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 µL 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:

g) After adding luteolin/ Tafamidis, the culture was incubated for 8 hrs at 30°C, 200

rpm.

h) After 8 hrs, cultures were washed with MilliQ water.

i) Then the cultures were resuspended in 200 µL RafGal-ura media and incubated for 16

hrs at 30°C, 200 rpm.

j) After this, aggregation was observed at 100X on inverted fluorescence microscope by

counting 200-300 cells manually.

48

Blank ControlVarying conc. Of Luteolin/Tafamidis

RESULTS AND DISCUSSION

1.

2.

3.

4.

5.

6.

7.

8.

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

DH5α 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

49

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.

50

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 µM 7.98 9.1 9.4 8.8266667 0.7484206 0.4321008

500 µM 7.78 8.5 7.9 8.06 0.385746 0.2227106

DMSO 300 µM 500 µM0

2

4

6

8

10

12

14

16

Varying conc. of Luteolin

Aggr

egati

on P

erce

ntag

e

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.

51

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)

IIND ATTEMPT

In the 2nd 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 µM 11 17 14 4.2426407 3

300 µM 10 6 8 2.8284271 2

DMSO 50 µM 300 µM0

5

10

15

20

25

Varying conc. of Luteolin

Aggr

egati

on P

erce

ntag

e

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.

52

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 µM 52.3 46.2 49.25 4.31335 3.05

10 µM 45.4 50.2 47.8 3.39411 2.4

20 µM 46.7 40.7 43.7 4.24264 3

50 µM 44.8 49.7 47.25 3.46482 2.45

DMSO 5 µM 10 µM 20 µM 50 µM0

10

20

30

40

50

60

Varying Conc. of Tafamidis

Agg

rega

tion

Perc

enta

ge

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

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

was repeated.

53

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 µM 50.7 75.2 75 66.966667 14.087701 8.1335382

100 µM 53.7 78.1 76.3 69.366667 13.597549 7.8505485

DMSO 50 µM 100 µM60

65

70

75

80

85

Varying Conc. of Tafamidis

Aggr

egati

on P

erce

ntag

e

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.

54

SUMMARY AND CONCLUSION

Huntington’s 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 Kelly’s 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.

55

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