FUS PROTEIN INTERACTIONS IN

MOTOR NEURON DISEASE

JIMMY GEORGE

Supervisor: Dr. JONATHAN WOOD

A project dissertation in partial fulfilment of the requirements for the Master of Science

degree in Molecular Medicine of the University of Sheffield

August 2010

Word Count: 11,569 words

ii

Acknowledgements

Foremost, I am eternally grateful to my supervisor Dr. Jonathan Wood (Academic Neurology Unit) for his

guidance in this project. I owe him his time, his enthusiasm and his patience in making this project all

together an amazing experience. Also I am thankful for his constant support and his invaluable

suggestions that he has offered me during the writing up of this thesis .He has been a mentor to me and I

am extremely honoured to have worked under his supervision.

My special thanks to Dr. Andrew Grierson, for his timely and generous help with the mouse brains,

without his support and generosity it would not have been possible to have carried out the research.

I am grateful to Dr Adrian Higginbottom and Mr.Sandeep SundaraRajan for their guidance and expertise

with various techniques.

I would also like to thank Ms. Ashlesha for being there to share the excitements and frustrations of

performing experiments in the lab.

My thanks also go to Mr. Avinash, Mr Saurabh and Mr Sameehan, for their constant support throughout

the past four months.

My sincerely thanks to all the members of the Academic Neurology Unit who have made these past four

months most memorable.

My sincere thanks to my sister Ms Tresa, who has always been there to share my happiness and sorrow

throughout my post graduation.

I am indebted to my parents without whom I wouldn’t be writing this thesis at all. It is their everlasting

love and support that has helped me become the person I am today.

Last but not the least; I am grateful to Prof. Pamela Shaw (Academic Neurology Unit) for letting me be a

part of a world class research in neurodegenerative diseases.

Jimmy George

iii

Summary

Amyotrophic Lateral Sclerosis (ALS) is an adult neurodegenerative disorder characterized by

loss of upper and lower motor neurons. The disease is usually sporadic but 10% cases are

found to be familial in nature. Many factors like excitotoxicity, apoptosis, mitochondrial

dysfunction, glial involvement and oxidative stress are thought to be responsible for the

pathology of the disease. Recently mutations in two genes, namely TAR DNA binding protein

(TARDBP) and Fused in Sarcoma/Translocation in liposarcoma (FUS/TLS) have found to be

responsible for causing ALS. Since both genes encode RNA processing proteins, RNA

processing pathways have been thrust to the forefront of ALS research. FUS/TLS is a 526

amino acid protein encoded on chromosome 16. Most of the mutations in FUS-related ALS,

take place in the13 residues at the C-terminal end of the protein. These residues have been

found to be highly conserved throughout species, suggesting that they have an important

function, possibly involving protein-protein interactions. We hypothesize that mutations in

the FUS C-terminal end disrupt critical protein-protein interactions, thereby causing ALS. To

test this hypothesis we developed an in vitro assay using synthetic peptides for the 17

residues of the C terminal peptide sequence of FUS Wild type, FUS mutant (R521H) and a

phosphorylated form of FUS. Using affinity purification and SDS- PAGE we found that loss

of protein interactions was observed in FUS mutant when compared to the FUS WT. It was

also observed that phosphorylation in FUS led to a gain of protein interactions when

compared to FUS wild type. These findings provoke further studies on the possible protein-

protein interactions in the in vivo condition. Since protein interactions has been found to play

a role in the pathology of fALS, this in vitro assay will help in guiding further research in

identifying FUS protein-protein interactions.

iv

Table of Contents: -

List of Tables...........................................................................................................................vii

List of Figures...........................................................................................................................ix

Abbreviations.............................................................................................................................x

1. Introduction..................................................................................................................1

1.1.Motor Neuron Disease.............................................................................................1

1.1.1. Classification of Motor Neuron Disease......................................................1

1.2.Amyotrophic Lateral Sclerosis.................................................................................3

1.2.1. Genetics of ALS...........................................................................................3

1.2.2. Pathology of ALS.........................................................................................5

1.2.2.1. Mitochondrial dysfunction....................................................................5

1.2.2.2. Apoptosis...............................................................................................6

1.2.2.3. Disruption in axonal transport...............................................................6

1.2.3. SOD1 in sporadic ALS.................................................................................6

1.2.4. Role of environmental factors......................................................................7

1.3. Fused in Sarcoma (FUS).........................................................................................7

1.3.1. Expression and regulation............................................................................9

1.3.2. Functions of FUS.........................................................................................9

1.3.3. FUS in Cancer............................................................................................10

1.3.4. FUS and TARDBP.....................................................................................14

1.3.5. FUS pathology in FALS.............................................................................16

1.3.6. Age of onset...............................................................................................17

1.3.7. Importance of C-terminal region................................................................18

1.3.8. Role of stress granules................................................................................19

v

1.4. FUS protein interactors.........................................................................................20

1.5. Aims and Hypothesis............................................................................................22

1.5.1. Objectives...................................................................................................22

2. Materials and Methods..............................................................................................23

2.1.Materials.................................................................................................................23

2.1.1. Stock solution.............................................................................................23

2.1.2. Working solution........................................................................................23

2.1.2.1. Coupling of peptides to Sulfolink column..........................................23

2.1.2.2.Affinity Purification.............................................................................24

2.1.2.3. Protein precipitation............................................................................24

2.1.2.4. SDS Polyacrylamide gel electrophoresis............................................24

2.1.2.5. Preparation of Nuclear proteins...........................................................25

2.2.Methods.................................................................................................................26

2.2.1. Peptide Immobolization.............................................................................26

2.2.1.1.Preparation of sample for coupling......................................................26

2.2.1.2. Coupling of Peptides to SulfoLink Columns......................................26

2.2.1.3. Blocking of non-specific binding sites................................................26

2.2.2. Process of Affinity Purification of proteins...............................................27

2.2.2.1. Homogenization of brain tissue and preparation of extract................27

2.2.2.2. Purification of proteins........................................................................27

2.2.2.2.1. Purification from cytoplasmic extracts.....................................27

2.2.2.2.2. Preparation of nuclear extracts.................................................28

2.2.3. SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE............................28

2.2.4. Staining......................................................................................................29

2.2.4.1. Coomassie staining..............................................................................29

vi

2.2.4.2.Silver staining.......................................................................................29

3. Results.........................................................................................................................30

3.1.Peptide design........................................................................................................30

3.2. Affinity purification of proteins using wild type and Spastin peptides

from cytoplasmic extract, without salt wash..........................................................33

3.3.Affinity purification of proteins from wild type and Spastin peptides

from cytoplasmic extract with the use of Salt wash...............................................35

3.4. Affinity purification of proteins using wild type and Spastin peptides

from Nuclear extract...............................................................................................37

3.5. Affinity purification of proteins using FUS mutant and Spastin peptides

from cytoplasmic extracts......................................................................................39

3.6. Repeat affinity purification with FUS mutant and Spastin peptides

using cytoplasmic extracts.....................................................................................39

3.7. Affinity purification with FUS wild type and FUS mutant peptides

using cytoplasmic extract.......................................................................................42

3.8. Affinity purifications with FUS wild type and FUS mutant peptides

from nuclear protein extract...................................................................................44

3.9. Affinity purification with FUS wild type and phosphorylated FUS peptides.......46

3.10. Comparison of the results obtained with the comparisons of mutant

and phosphorylated peptides with wild type..........................................................48

3.11. Analysis of specific proteins through mass spectroscopy................................50

4. Discussion....................................................................................................................51

4.1. Optimum binding conditions.................................................................................51

4.2. Mutation in the C-terminal region of FUS causes loss of protein interactions.....52

4.3. Protein interactions seen in cytoplasmic and nuclear fractions.............................52

vii

4.4. Phosphorylation of the C-terminal region of FUS may affect

protein interactions.................................................................................................53

4.5. Protein interaction in C-terminal FUS with respect to ALS.................................54

4.5.1. Splicing Factor-35......................................................................................55

4.5.2. Y Box protein-1..........................................................................................56

4.6. Limitations............................................................................................................57

4.7. Future work..........................................................................................................57

5. References..................................................................................................................59

viii

List of tables:

Table 1: Classification of various types of motor neuron disorders..................2

Table 2: Table describing the different subtypes of ALS based on their

genetic mutations associated with fALS.............................................4

Table 3: Table describing the DNA binding domains fusing with

FUS/TLS causing different types of cancer.......................................12

ix

List of Figures:-

Figure 1: Structure of FUS gene, describing the various domains and

some of the amino acid changes causing ALS in the 15th

exon…………………8

Figure 2 Schematic representation of fusion involving the N-terminal region

of FUS and CHOP/DDIT3 transcription factor………………………………..13

Figure 3: Figure describing some of the interacting partners of FUS protein…………...21

Figure 4: A software generated prediction of phosphorylation sites in FUS protein…….31

Figure 5: A 10% SDS PAGE analyzing the proteins bound to Spastin and wild type

peptides in the absence of a blocking step or high salt washes………………...34

Figure 6: SDS PAGE gel depicting the proteins bound to and eluted from Spastin

and FUS wild type peptides……………………………………………………36

Figure 7: A 10% SDS PAGE showing the nuclear proteins affinity purified using

Spastin and FUS wild type peptides…………………………………………...38

Figure 8a: 10% SDS PAGE gel comparing the proteins bound to Spastin

and FUS mutant (M) peptides………………………………………………….40

Figure 8b: A 10% SDS PAGE depicting the proteins bound to Spastin

and FUS mutant peptides………………………………………………………41

Figure 9: Silver stained 12% SDS-polyacrylamide gel showing the proteins

interacting with FUS wild type and mutant peptides…………………………..43

Figure 10: A silver stained 12% SDS polyacrylamide gel, showing the nuclear

proteins interacting with FUS WT and Mutant peptides………………………45

Figure 11: A 10% SDS-polyacrylamide gel showing the proteins affinity purified

with FUS WT and phosphorylated FUS peptides……………………………...47

Figure 12: A 12% gel comparing the fractions obtained from Wild type and mutant,

and Wild type and phosphorylated peptide affinity purifications……………...49

x

ABBREVIATIONS:-

DDIT3: DNA damage inducible transcript-3

EWS: Ewing’s sarcoma

ERG: V-ets avian erythroblastosis virus e26

oncogene homolog

EDTA: Ethylenediaminetetraacetic acid

FTLD: Frontotemporal lobar dementia

FUS: Fused in sarcoma

FGFR-1: Fibroblast growth factor receptor-1

GFP: Green fluorescent protein

HEPES: 4-2-hydroxyethyl-1-piperazineethanesulfonic

acid

HCL: Hydrochloric acid

LMN: Lower motor neuron

MND: Motor neuron disease

ALS: Amyotrophic lateral sclerosis

AML: Acute myeloid leukaemia

ANG : Angiogenin

APS: Ammonium persulphate

ATF-1:

Cyclic AMP-dependent transcription factor 1

ATP: Adenosine triphosphate

BSA: Bovine serum albumin

CHOP: C/EBP homologous protein

xi

MAPT: Microtubule associated protein tau

NLS: Nuclear localization signal

NaN3: Sodium azide

PLS: Primary lateral sclerosis

PY: Proline rich motif

PAGE: Polyacrylamide gel electrophoresis

PBS: Phosphate buffered saline

RRM: RNA recognition motif

SOD-1: Superoxide dismutase-1

SETX: Senataxin

SC-35: Splicing factor-35

SDS: Sodium dodecyl sulphate

TARDBP: TAR DNA binding protein

TET: Thymic epithelial tumours

TLS: Translocated in liposarcoma

TFIID: Transcription factor IID

TASR1/2: TLS-asscociated serine arginine protein1/2

TCEP: Tris-2-carboxyethyl-phosphine

TCA: Trichloroacetic acid

UMN: Upper motor neuron

VAPB: Vesicle associated membrane protein B

YB-1: Y box binding protein-1

1

1. INTRODUCTION

1.1 Motor Neuron Disease: - Motor Neuron Disease (MND) refers to a group of disorders

affecting the motor neurons, resulting in the loss of voluntary and involuntary functions of

the body. In these types of disorders, degeneration of the upper and/or lower motor neurons is

observed. This degeneration ultimately leads to weakness of the limbs, bulbar and respiratory

muscles. Dementia, ataxia and spasticity can also occur due to progression of the disorder.

Though there are ranges of disease falling in this category, all seem to exhibit similar

pathological mechanisms. Symptoms such as muscle weakness, difficulty in movement and

breathing, loss of normal day to day functions like walking, talking and coughing are looked

for diagnosing MND in an individual.

Diagnosis of MND is done clinically as no definitive diagnostic test is available. Since the

disease mechanism is not fully understood, a cure for any kind of MND has not been possible

to date.

1.1.1.Classification of Motor Neuron Disease:- In the United Kingdom, Motor Neuron

Disease is classified into three forms: Amyotrophic lateral sclerosis(ALS), progressive

muscular atrophy (PMA) and primary lateral sclerosis (PLS). ALS involves the loss of both

the upper and the lower motor neurons and is the most common of the three. PMA is

characterized primarily by loss of the lower motor neurons (LMN), seen in adult stage of life,

while PLS is basically loss of the upper motor neurons (UMN) and is the rarest of the three

(reviewed in Wood-Allum and Shaw, 2010). Although these three forms are known to affect

the upper and lower motor neurons, the extra-ocular motor neurons and neurons of the pelvic

floor seem to be spared in these three forms. Different types of MND have been classified in

the following table.

2

Disease Motor neuron

involvement

Age of onset Clinical features

Amyotrophic

lateral sclerosis

(ALS)

Upper and lower

motor neurons

Between 45 to

60

Progressive muscle weakness

atrophy and spasticity

Hereditary

spastic

paraplegia

(HSP)

Upper motor

neurons

Early childhood

to 70 yrs.

Progressive spasticity in lower

limbs

Primary lateral

sclerosis (PLS)

Upper motor

neurons

35-60 yrs. Bulbar and spinal spasticity

Lethal

Congenital

contracture

syndrome

Upper and lower

motor neurons

Fetal Early akinesia, degeneration of

anterior horns and muscle atrophy

Spinal muscular

atrophy (SMA)

Lower motor

neurons

6 to 18 months Muscle weakness and atrophy

Spinal bulbar

muscular

atrophy

(SBMA)

Lower motor

neurons

30 to 50 yrs. Progressive muscle weakness and

atrophy

Table 1:- Classification of various types of motor neuron disorders

(Adapted from Dion et al, 2010)

3

1.2 Amyotrophic Lateral Sclerosis:-Amyotrophic lateral sclerosis is a fatal

neurodegenerative disorder which affects both the upper and the lower motor neurons of the

central nervous system. Loss of the motor neurons leads to muscle paralysis, atrophy and

spasticity. This causes the individual to lose control of voluntary and involuntary functions

like walking, movement, speaking and in adverse cases even breathing. ALS, or motor

neuron disease as it is often called, leads to death in around 3 to 5 years of the onset of

disease, usually due to respiratory distress. Some patients with ALS have also been known to

suffer from frontotemporal dementia (FTLD) (Blair et al, 2010).

Various epidemiological studies suggest that around 5-10% of the ALS cases are familial in

nature while the rest are sporadic (Chio et al, 2008). Although many genetic defects have

been found to be associated with the pathogenesis of the disease, its etiology is poorly

understood. But it is clear that motor neuron death seen in ALS is due to combined effects of

many biological processes like mitochondrial dysfunction, impaired glutamate and calcium

homeostasis and oxidative stress. Since non-neuronal cells like astrocytes and glial cells are

also involved in ALS pathogenesis, this disease may be thought of as an unbiased process

affecting both types of cells (Strong, 2010).

1.2.1. Genetics of ALS:- Many genes have been implicated in amyotrophic lateral sclerosis.

Mutations in the SOD-1 gene, located on chromosome 21, account for around 10-20% of the

familial form of the disease (Rosen et al, 1993). SOD-1 encodes Cu/Zn superoxide

dismutase-1. Nearly 150 mutations have been associated with SOD-1 related ALS. Of these

12-23% is familial, while 2-3% is sporadic in nature (Andersen, 2006). Mutations in other

genes like ALS2, SETX, VAPB, and ANG are also known to be responsible, leading to

ALS/MND (Pasinelli et al, 2006). Recently two genes, TARDBP (TAR DNA binding

protein) and FUS (Fused in Sarcoma), have been discovered to play a role in the

pathogenesis of the disease (Kwiatkowski et al 2009, Vance et al, 2009).

4

NAME GENE PROTEIN LOCUS INHERITANCE ONSET

ALS1 SOD1 Superoxide dismutase 1 21q22.1 Dominant/

Recessive

Adult

ALS2 ALSIN Alsin 2q33 Recessive Juvenile

ALS3 Unknown Unknown 18q21 Dominant Adult

ALS4 SETX Senataxin 9q34 Dominant Juvenile

ALS5 Unknown Unknown 15q15.1-

21.1

Recessive Adult

ALS6 FUS/

TLS

Fused in Sarcoma 16q12 Dominant Adult

ALS7 Unknown Unknown 20p13 Dominant Adult

ALS8 VAPB Vesicle associated

membrane protein

20q13.33 Dominant Adult

ALS9 ANG

Angiogenin 14q11 Dominant Adult

ALS10 TARDBP TAR DNA Binding Protein 1p36.2 Dominant Adult

FTD-

ALS

MAPT Microtubule Associated

protein

17q21.1 Dominant Adult

Table 2:- Table describing the different subtypes of ALS based on their genetic mutations

associated with fALS (Adapted from Strong, 2010).

5

Though only 5 -10% of the familial cases are caused by mutations in these two genes, their

products may have an important role in the pathogenesis of the majority of ALS cases,

including the sporadic ones. Both TARDBP and FUS also seem to play a role in the

pathogenesis of an ALS related disorder known as frontotemporal lobar degeneration with

ubiquitin positive inclusions (FTLD). Since both the genes have been reported to be mutated

in both ALS and FTLD cases, a relationship between both diseases has been established

(Neumann et al, 2009)

1.2.2 Pathology of ALS: - The basic understanding of the pathogenesis of ALS started with

studies done on SOD1 mutations. Many factors like mitochondrial dysfunction, calcium

homeostasis dysregulation and impairment of axonal transport came to prominence.

1.2.2.1 Mitochondrial dysfunction: - Mitochondria are one of the critical organelles of the

cell. They are termed as the powerhouse of the cell as they regulate ATP production,

apoptosis and maintain calcium homeostasis among other functions. Mitochondrial

dysfunction has been discovered to be one of the key factors in ALS pathogenesis (Bruijn et

al, 2007). SOD1, an ubiquitinous protein is mainly localized in the cytoplasm but is also seen

in the mitochondria, nucleus and the endoplasmic reticulum (Okado Matsumoto and

Fridovich, 2001). Mutant SOD1 has been reported to be present on the outer membrane of the

mitochondria leading to loss of mitochondrial membrane potential leading to mitochondrial

swelling. This impairs the mitochondrial respiratory complex, decreasing ATP production

(Borthwick et al, 1999).

Buffering of intracellular Ca2+

ions is also one of the functions of the mitochondria. Cells of

ALS patients were seen to have less buffering capacity as compared to that of normal cells.

An increased concentration of Ca2+

ions accompanied by mitochondrial damage was

6

observed in ALS cases (Kruman et al, 1999). Increased Ca2+

ions in cells, especially motor

neurons, is known to cause oxidative stress which ultimately leads to apoptosis.

1.2.2.2. Apoptosis:- Early studies of ALS cases indicated that mitochondria-mediated

apoptosis was responsible for motor neuron degeneration. An increase in the levels of pro-

apoptotic proteins like Bcl-2 and Bcl-xl was observed along with release of cytochrome c in

studies conducted on G93A SOD1 transgenic mice (Pasinelli et al, 2004). These proteins

when released in to the cytoplasm, initiate the process of apoptosis. Caspase 1 and caspase 3

were also seen to be activated in mice with G93A SOD1 mutation, indicating the advent of

apoptosis (Li et al, 2000).

1.2.2.3 Disruption of axonal transport:- Disruption of Axonal transport is also seen in to be

one of the hallmarks of ALS patients. Since transport of signals and materials across cells is

important for their survival, its disruption can prove to be harmful. This disruption was seen

to be one of the first pathological events in mice with mutant SOD1 (Williamson and

Cleveland, 1999). Axonal and synaptic function is governed by the axonal transport of

membrane-bound organelles like mitochondria and is mediated by kinesin and dynein-

dynactin motor complexes. Mutations in these motor complexes can cause motor neuron

degeneration in humans (Hafezparast et al, 2003). Although there is evidence of motor

neuron degeneration caused by defects in axonal transport, no detailed information regarding

its mechanism has been identified.

1.2.3. SOD1 in sporadic ALS:- A recent study on SOD1 toxicity suggested that oxidation

mediated misfolding of proteins results in a modified SOD1 acquiring toxic properties that

are similar to that of mutant SOD1 (Ezzi et al, 2007).This misfolded SOD1 protein could be

an underlying cause for a fraction of sporadic ALS cases without any known mutation.

7

Recent developments in antibodies specific to misfolded forms of SOD1 could shed more

light regarding the pathogenesis behind this.

1.2.4. Role of Environmental factors:- Various environmental factors are believed to

contribute in the pathogenesis of neuronal degeneration in ALS. It was observed that in

Chamorra population of Guam, individuals were affected with ALS associated with

Parkinsonism and progressive dementia (Khabazian et al, 2009). Neurotoxins like sterol

glucosides and β-methyl amino-L-alanine present in the flour consumed by the population

were suggested to be responsible for the pathology. Increased exposure to electromagnetic

fields, certain insecticides and pesticides have also found to contribute to the risk of

developing ALS (reviewed in Dion et al, 2009). Other factor like excessive physical activity,

head injuries, exposure to formaldehyde and cigarette smoking have also being implicated in

ALS pathogenesis (reviewed in Dion et al, 2009).

1.3 FUS:- Fused in Sarcoma:- FUS, also known as TLS (translocated in liposarcoma), is an

oncogene, discovered previously as one part of a t(12;16) translocation product, combining

the N-terminal region of FUS with the C-terminal region of the CHOP protein, leading to the

formation of round cell like liposarcomas (Crozat et al, 1993). FUS/TLS is a part of the TET

protein family which also includes the EWS protein, the TATA binding protein-associated

factor (TAF15/TAFII68), and the Drosophila cabeza /SARF protein (Morohoshi et al, 1998).

The FUS gene is present on chromosome 16, consists of 15 exons, with mutations in ALS

cases being mainly present in the last exon, i.e. the C- terminal end of the protein. FUS

protein contains various domains and regions responsible for a number of functions. It is a

526 amino acid long protein, containing an N-terminal end, a region rich in serine, tyrosine,

glycine and glutamine, an RNA recognition motif (RRM), a zinc finger motif, multiple RGG

repeat regions and a Nuclear localization signal (NLS) at the C terminal end of the protein

(Figure 1).

8

Figure 1: Structure of FUS gene, describing the various domains and some of the amino acid

changes causing ALS in the 15th

exon (Adapted from Kwiatkowski et al, 2009)

9

The zinc finger motif helps in trafficking through the nuclear membrane and also binds

GGUG containing RNA (Blitterswikj and Landers, 2010). The specificity of RNA interaction

is controlled by the RGG and RRM regions. The zinc finger motif helps in regulating

receptor mediated transport between the nucleus and cytoplasm. As shown in Figure 1,

various base substitution mutations in the C-terminal RGG region of FUS protein have been

identified in ALS pathology (Kwiatkowski et al, 2009, Vance et al, 2009)

1.3.1 Expression and regulation:- FUS/TLS is known to be ubiquitously expressed in

human tissues including heart, spleen, lung, kidney, pancreas, thymus and prostrate. It is not

present in the cardiac endothelium and the cardiac muscle cells (Aman et al, 1996). The

expression levels of FUS vary in different tissues. It is upregulated in peripheral blood

samples of patients suffering from acute myeloid leukaemia (AML), while it is down

regulated in human embryonic stem cells (Mills et al, 2000). This shows that FUS/TLS may

have a role in the promotion and maintenance of cell proliferation. FUS/TLS is mainly a

nuclear protein, but in some cases it is also present in the cytoplasm of cells, especially in

pathological conditions. (Andersson et al, 2008). FUS co-localizes with stress granules,

which are regulators of post–transcriptional events. This indicates that FUS/TLS may be

required for the functioning of stress granules (Andersson et al, 2008).

1.3.2 Functions of FUS: - The C-terminal part of the protein is considered to be important

for RNA binding. FUS/TLS carries out many functions like pre-mRNA splicing, microRNA

processing, mRNA transport from the nucleus to the cytoplasm and initiation of cell

spreading centres (Yang et al, 2010). The Carboxy terminus of FUS is known to constitute

motifs for RNA processing, the targets of which could include motor proteins, cytoskeletal

proteins and proteins necessary for the growth of neurons (Sleegers and van Broeckhoven,

2009). It was earlier found out that inhibition of RNA polymerase II caused cytoplasmic

10

accumulation of FUS/TLS suggesting the product of RNA polymerase II is a part of the

complex that FUS forms with RNA molecules (Zinszner et al, 1997).

FUS/TLS was found in RNA transporting granules which translocate to the dendritic spines

(Fujii et al, 2005). It was also observed that hippocampal cells taken from FUS/TLS

knockout mice had abnormal spine formation and density. These results indicate that

FUS/TLS may be essential for regulating neuronal plasticity by altering mRNA transport and

local translation in neurons (Fujii et al, 2005).

Several studies conducted reveal that FUS/TLS plays a role in maintaining the genome

integrity through DNA double strand repair (Fiesel et al, 2010, Bertrand et al, 1999).

FUS/TLS has been found to promote the process of annealing of homologous DNA and for

formation of DNA D loops, which is known to be an essential step in DNA repair. (Fiesel et

al, 2010)

It is also a known fact that FUS binds to a number of gene specific transcription factors and

plays a role as a gene expression modulator. FUS is known to have high affinity to a number

of nuclear hormone receptors like steroid and thyroid hormone receptors (Law et al, 2006).

FUS binds to these receptors which in turn bind to specific DNA sequences and activate

transcription. FUS is also an interaction partner with other factors like apoptosis regulators

(NF-KB), spi-1/PU.1 oncogene and transcription factor IID among others (Law et al, 2006).

It is also known to play a role in transcription initiation with RNA polymerase II and TFIID

complex (Lagier-Tourenne et al, 2010)

1.3.3 FUS in Cancer:- The role of FUS/TLS in cancer was reported as a component of the

fusion gene TLS-CCAAT enhancer binding homologous protein (CHOP), resulting from

chromosomal translocation t(12:16)(q13.3;p11.2), creating a FUS/TLS-CHOP fusion,

observed in myxoid liposarcomas in 1993 by Crozat et al. Liposarcoma is a common form of

11

soft tissue malignancy. The fusion of CHOP and FUS/TLS result in the, basic Leucine zipper

domain of CHOP replacing the RNA binding domain of FUS. Other fusion proteins with

FUS have also been implicated in various forms of Cancer (table 3). In each of the fusions,

the N-terminal of FUS, a transcriptional activation domain is fused with the DNA binding

domain of a transcription factor resulting in another transcription factor. These fusions are

found to be a major cause of cancer, as a high percentage of these fusions are observed in

cancer cells (Law et al, 2006).

12

Binding Domain Disease Chromosomal

translocation

References

ERG Acute myeloid

leukaemia

t(16;21)(p11;q22) Panagopoulos

et al, 1994

CHOP Myxoid

liposarcoma

t(12;16)(q13;p11) Crozat et al,

1993

FUS/TLS ATF-I Angiomatoid

fibrous

hystiocytoma

t(12;16)(q13;p11) Waters et

al,2000

ERG Ewing’s

sarcoma family

of tumours

t(16;21)(p11;q22) Shing et al,

2003

CREB312/BBF2H7 Low-grade

fibromyxoid

sarcoma

t(7;16)(q33;p11) Storlazzi et al

2003

Table 3:-Table describing the DNA binding domains fusing with FUS/TLS causing different

types of cancer (Adapted from Law et al, 2006).

13

Figure 2:- Schematic representation of the fusion involving the N-terminal region of FUS and

CHOP/DDIT3 transcription factor (adapted from Sanchez-Martin et al, 2004)

14

As seen in Figure 2 the N-terminal of the FUS protein, which is the activation domain fuses

with the DDIT3 protein forming the FUS-DDIT3 gene, known to cause Human myxoid

liposarcoma (Sanchez-Martin et al, 2004)

FUS/TLS is also known to fuse with erythroblastosis virus E26 oncogene homologue, caused

by the translocation t(16;21)(p11;q22) observed in acute myeloid leukaemia (Law et al,

2006). The FUS part present in this fusion is similar to the fusion protein FUS-ERG known to

cause acute myeloid leukaemia (Shimizu et al, 1993). Introns 5, 7 and 8 were observed to be

the translocation breakpoints in both diseases (Morohoshi et al, 1998).

The importance of the FUS domain known to fuse in both the cases was studied by Perez-

Losada et al in 2000. According to their studies conducted on transgenic mice, high levels of

expression of CHOP lacking the FUS domain do not cause tumours as compared to the FUS-

CHOP fusion. This inability of CHOP to produce tumours without the FUS domain proved

that the amino acid composition of the FUS N-terminal domain was critical in the

pathogenesis of the disease. It was also shown that the FUS-CHOP fusion deregulated gene

expression and protein activity in liposarcoma individuals (Perez-Loasada et al, 2000).

Although the C-terminal region implicated in ALS has not been found to be important in the

etiology of cancer, the role of FUS in tumour pathogenesis has helped in providing increased

understanding of the basic biology of FUS prior to the discovery of its role in ALS.

1.3.4. FUS and TARDBP- The process of pathogenesis of FUS in ALS is poorly understood.

FUS is known to have a similar structure to that of another protein known as TDP-43, a

43Kda protein derived from TARDBP gene. Both FUS and TDP-43 share similar regions like

RNA recognition domains and glycine rich regions (Lagier-Tourenne and Cleveland, 2009).

15

Since 2008, several mutations, both sporadic and familial have been discovered in TDP-43

(Lagier-Tourenne et al,, 2010). It was also observed that since these mutations were present

only in ALS cases and not in other neurodegenerative diseases such as Alzheimer’s or

Parkinson’s disease, these mutations were specific to ALS (Sreedharan et al, 2008). Silencing

TDP-43 was shown to down-regulate cyclin dependant kinase 6, which is necessary in

regulating gene expression (Ayala et al, 2008). Like FUS, TDP-43 protein has also been

shown to play an important role in RNA processing among other functions. Studies have

shown that TDP-43, interacts with heterogeneous nuclear ribonucloproteins (hnRNP), which

play a role in splicing (Buratti et al,2005).Various other studies (Wang et al, 2008) have

confirmed the role of TDP-43 in RNA transport and localization and thus proved that it is

essential for RNA processing. TDP-43 is known to be associated with stress granules and

processing bodies in neuronal cells when they are under stress or injury. FUS is also known

to be present in stress granules and so alterations in RNA metabolism could be considered a

reason for ALS (Strong et al, 2010). It is possible that either a gain or a loss of function due

to FUS/TLS mutations could lead to ALS. Although FUS and TDP-43 are structurally

similar, the absence of TDP-43 inclusions/mislocalization in FUS/TLS related ALS cases,

shows that the disease mechanism might be different in cases caused by FUS and TARDBP

mutations (Blair et al, 2010)..

One of the important steps in understanding the disease mechanism is to understand how the

mutations change the normal protein stability. Ling et al (2010), with the use of isogenic

stable cell lines found that mutant TDP-43 protein had surprisingly longer half-life than that

of the wild type protein. They suggested that this longer half life, may be the cause for the

abnormal stability of the protein in mutant cases, leading to pathological conditions. Ling and

colleagues also discovered that FUS/TLS was associated with TDP-43 more in mutant than in

16

wild type cases. So there is a possibility that mutant TDP-43 may affect normal FUS/TLS

protein, thus linking the pathways of both FUS and TDP-43 in ALS cases.

1.3.5. FUS pathology in FALS:- To date around 30 ALS causing mutations have been

identified in the FUS/TLS gene which relate to around 4% of the familial cases of ALS, along

with a small number of sporadic cases (Hewitt et al, 2010). The mutations which have been

reported are usually present in the 15th

exon, although some have been found to be in the 3rd

,

5th

, 6th

and the 14th

exons (Lagier-Tourenne et al, 2010).The mutations which take place in

the C-terminal end of the FUS protein are usually base substitution changes. These base pair

changes are seen to take place in the conserved region of the C-terminal end of the protein.

For example, the amino acid arginine is found to be replaced with histidine at the 521

position (Vance et al, 2009; Kwiatkowski et al, 2009). Other residue changes like arginine to

glycine and arginine to cysteine also take place in some of the cases.

Post-mortem studies on samples taken from brain and spinal cord of FUS cases found

abnormal cytoplasmic inclusions of FUS protein in neurons and glial cells (Kwiatkowski et

al, 2009). Another study conducted by Vance et al in 2009 found a similar result, where

patients with FUS mutations were found to have developed cytoplasmic FUS inclusions in

the motor neurons of the spinal cord, absent in normal cases. These cytoplasmic inclusions

might be a result of defects in RNA processing caused by mutations in the FUS gene, but

nothing conclusive has been found out. As the inclusions are mainly seen in the cytoplasm,

increased aggregation of these inclusions may link to defects in nuclear-cytoplasmic transport

(Dormann et al, 2010). The spinal cord of patients affected with ALS showed severe loss of

anterior horn cells in the spinal cord, as well as loss of pigmented neurons and reactive gliosis

in sections from the midbrain. (Blair et al, 2010)

17

The presence of FUS/TLS inclusions is also seen in other disorders. A proteomic study

identified FUS/TLS to be a major component of polyglutamine aggregates in cellular models

of Huntington’s disease and Spinocerebellar ataxia (Doi et al, 2008). Another study

conducted by Woulfe et al in 2010 through post-mortem analysis confirmed the presence of

FUS/TLS in neuronal intranuclear inclusions and in some cases also in cytoplasmic

inclusions in Huntington patients.

1.3.6. Age of Onset: - Studies conducted by Kwiatkowski and Vance et al showed different

results with respect to age of onset of the disease. According to Kwiatkowski and co-workers,

their study showed that the average age of disease onset in cases with arginine to glycine

mutations at 521st base, was found to be 37.5 yrs. Vance and colleagues found that the

disease age onset with the same mutations was around 60.7 yrs, on average. These varied

results indicate that there is a possibility that genetic factors other than FUS mutations govern

the onset and progression of the disease. Identification of these genetic factors could prove to

be therapeutically useful in preventing or delaying the disease pathogenesis (Sleegers and van

Broeckhoven, 2009). Other studies regarding the average onset age have been conducted, but

none with consistent results.

Most of the mutations found by Kwiatkowski et al and Vance et al in 2009 were in arginine

residues of the C-terminal of FUS protein (a highly conserved sequence). Since the mutations

of the C-terminal FUS lead to ALS pathologies, it appears that these arginine residues have a

crucial role in the normal functioning of the protein (Sleegers and van Broeckhoven 2009).

18

1.3.7 Importance of C–terminal region: - The C terminal region, as mentioned before is

highly conserved and has been seen in multiple organisms throughout species.

HOMOSAPIENS: - GPGKMDSRGEHRQDRRERPY

MOUSE: - GPGKMDSRGEHRQDRRERPY

BOVINE: - GPGKMDSRGEHRQDRRERPY

RAT: - GPGKMDSRGEHRQDRRERPY

As seen above the residues of the C terminal end are conserved throughout species. This

conservation might indicate that the C-terminal end has an important function and when

mutated results in pathological effects.

The role of the nuclear localization signal (NLS) in the pathology of FUS related ALS has

also been studied and it has been found that the NLS-like region, homologous to the NLS of

EWS, contains a PY motif, is present in the C-terminal part of FUS and plays an important

role in its transport to the nucleus (Dormann et al, 2010). When the C-terminal region was re-

arranged in a random order i.e. scrambled, FUS could not be transported to the nucleus

(Dormann et al, 2010). This proves that FUS requires a particular C-terminal sequence, in a

particular order which acts as a NLS, to be transported to the nucleus. Deletion of seventeen

residues from the C terminal region led to the cytoplasmic localization of FUS (Gal et al,

2010). This was supported by the study done on the C-terminal residues and their localization

to the nucleus. It was found that GFP tagged with the seventeen residues of the C-terminal

localized to the nucleus, thus strengthening the role of the C terminal residues in protein

localization. Thus it could be indicated that the C-terminal 17 residues of FUS could act as an

NLS for the protein (Gal et al, 2010). Further studies conducted by Dormann et al showed

that mutations such as arginine to histidine at the 521st position and others described by

Vance et al and Kwiatkowski et al, affected the proteins major NLS and this impaired the

19

proteins nuclear localization. Thus the importance of the C-terminal part of FUS can be

strengthened.

Along with the C-terminal part of FUS, another factor known as Transportin is required for

the nuclear import of FUS. The NLS like region of FUS bears similarity with other NLS

sequences having PY motifs, which have been shown to bind with Transportin, a nuclear

transport receptor (Lee et al, 2006). Transportin, a 102 kDa protein has previously known to

interact with the C terminal part of FUS in an in vitro pull down assay (Guttinger et al, 2006),

so its role in vivo was studied. Dormann and colleagues found that when Trp homologues

Trp1 and Trp2 (transportin) were knocked out in transfected cell lines it led to high

accumulation of FUS protein in the cytoplasm, compared to controls. It was also found that

R521G mutation interfered with the Trp pathway leading to the cytosolic accumulation of the

FUS protein (Dormann et, al 2010). These findings suggested that transportin plays a major

part in the functioning of FUS and impaired transportin can cause pathological conditions.

1.3.8 Role of Stress granules: - Stress granules are complexes which contain stalled

mRNA’s, translation initiation factors, ribosomal subunits, and RNA binding proteins like

Poly A binding protein (Andersson and Kedersha, 2008). Some RNA binding proteins are

known to associate with only stress granules and not with other cytoplasmic granules, thus

acting as stress granule markers (Kiebler and Baseell, 2006). Co-staining with antibodies

specific to these proteins showed that FUS localized to stress granules from the cytoplasm

when Trp transport was inhibited (Dormann et al, 2010). Since Trp inhibition can cause

conditions like cellular stress, this mislocalization meant that FUS localization and cellular

stress had a direct correlation. Further studies showed that the presence of mutant FUS in

stress granules had a direct relationship with the degree of cytoplasmic mislocalization and

inversely with the age of onset of the disease. Thus it indicated that FUS mislocalization to

the cytoplasm led to the formation of FUS positive stress granules (Dormann et al, 2010). In

20

studies done with brain sections taken from ALS patients, it was observed that FUS was co-

deposited with stress granules. Thus it is possible that stress granules do play a role in FUS

pathology in ALS.

1.4. FUS protein interactors:- FUS/TLS is known to interact with other proteins and

transcriptional factors. Some of them interact with specific domains while some appear to

interact with the whole protein.

As shown in Figure 3, FUS can be divided into two domains i.e. the transcriptional domain

which comprises of the N-terminal region, and the RNA binding domain which encompasses

the C-terminal region. The transcriptional domain interacts with factors like RNA polymerase

II, transcriptional factor IID and other nuclear hormone receptors. These interactions are

important in fusions with DNA-binding domains, like those of CHOP and EWS which cause

various types of cancer (Law et al, 2006) (Table 3).

The C terminal part of FUS contains the RNA binding domain. As mentioned above

mutations in this RNA binding domain cause ALS. Various factors/proteins like YB-1(Y-

box binding protein-1), SC-35 (splicing factor-35) are known to interact with the domain.

(Law et al, 2006). Each interacting partner has been found to play an important role in the

functioning of FUS/TLS.

21

Figure 3:- Figure describing some of the interacting partners of FUS protein

(Adapted from Law et al, 2006)

22

1.5. Aims and Hypothesis:- In this project we hypothesize that-

• FUS mutations cause ALS by disrupting critical protein interactions involving the

extreme Carboxyl terminus of FUS

The primary objective of this research project was to develop an in vitro assay to study the

protein interactions mediated by the C-terminus of FUS protein, using wild type, mutant and

phosphorylated forms of FUS produced as synthetic peptides.

The first step was to immobilize the synthetic peptides corresponding to the C-terminal

region of the wild type mutant and phosphorylated FUS protein. The next stage was to

establish conditions for specific binding of other proteins to the FUS peptides. The proteins

bound to the peptides were analysed using SDS- PAGE and the specific bound proteins were

then to be identified using mass spectroscopy.

1.5.1 Objectives:-Through this research we intended to determine:-

The protein interactions taking place at the C-terminal of FUS protein using wild type,

mutant and phosphorylated forms of synthetic peptides. As the hypothesis states that

mutations in FUS may disrupt the protein interactions, we intended to identify proteins which

do not interact with mutant FUS peptides. Also, by using a phosphorylated synthetic peptide,

we intended to identify whether phosphorylation in the C-terminal peptide sequence causes

either loss or gain of protein interactions as compared to the wild type FUS protein.

23

2 MATERIALS AND METHODS

2.1. Materials:-

2.1.1. Stock Solutions:- All Solutions were made with deionised water unless stated

otherwise

1) 5M NaCl- 29.2g in 100 ml. Autoclaved and stored at room temperature.

2) 0.1M Glycine (pH 2.5):- 0.72 g in 100 ml. Stored at 4ºC.

3) 1M Tris base:- 12.1 g in 100 ml. Autoclaved and stored at room temperature

4) 1M Hepes (4-2-hydroxyethyl-1-piperazineethanesulfonic acid) pH 7.5:- 47.66 g in

100 ml.

5) Phosphatase inhibitors ( 25X stock):-1mM sodium orthovanadate, 50mM β-

glycerophosphate, 50mM sodium fluoride and 2mM sodium pyrophosphate in

100 ml.

2.1.2. Working Solutions

2.1.2.1. Coupling of peptides to the Sulfolink column:- Sulfolink® Immobolization kit for

peptides (Thermo Scientific Catalog No. 44999)

SulfoLink Column:- 3 x 2 ml, 6% cross linked beaded agarose supplied as a 50%

slurry in storage buffer (10 mM EDTA-Na, 0.05% NaN3, 50% glycerol)

SulfoLink Coupling buffer (120 ml):- 50 mM Tris,5 mM EDTA-Na; pH8.5

Wash Solution (120 ml):-1 M NaCl, 0.05% NaN3

L-Cysteine.HCL :- 50 mM

Bond Breaker® TCEP solution, Neutral pH:- 0.5ml of stabilized aqueous 0.5M

TCEP

24

2.1.2.2. Affinity Purification:-

Homogenisation Buffer:-25 mM Hepes (From 1M stock), 100mM NaCl (from 5M

stock), 0.5% Triton X 100 (v/v) (from 10% Stock), 10% Glycerol, made up to 500

ml using deionised water.

Protease Inhibitor: - 1 Protease Inhibitor Cocktail Tablet (Complete) in 50 ml of

Homogenisation buffer.

0.1%BSA- 50 mg of BSA in 50 ml of homogenisation buffer (w/v).

1M salt wash buffer:- 2.6g in 50 ml of homogenisation buffer

0.5M salt wash buffer:- 1.3 g in 50 ml of homogenisation buffer.

2.1.2.3. Protein Precipitation:-

50% (w/v) Trichloroacetic Acid (TCA):- 50 g in 100 ml

Acetone

2.1.2.4. SDS Polyacrylamide Gel Electrophoresis:-

30% Acrylamine-bis acrylamide (37.5:1) solution (Protogel™)

10% (w/v) Sodium Dodecyl Sulphate (SDS)

4X Resolving gel buffer (pH 8.8): 1.5 M Tris base and 0.4 % (w/v) SDS.

4X Stacking gel buffer (pH 6.8): 0.5 M Tris base and 0.4 % (w/v) SDS

10% (w/v) Ammonium persulphate (APS)

Resolving gel (~10ml; for 2 gels): 4.0 ml water along with 3.3 ml 30 %

Acrylamine-bis acrylamide solution, 2.5 ml of resolving gel buffer (pH 8.8), 100

μl each of 10% SDS and 10% APS and 4 μl of TEMED.

Stacking gel (~10ml; for 2 gels): 3.35 ml water along with 1 ml 30 % Acrylamide-

bisacrylamide solution, 1.5 ml of stacking gel buffer (pH 6.8), 60 μl each of 10%

SDS and 10% APS and 6 μl of TEMED.

25

1X Electrophoresis running buffer: 25 mM Tris, 250 mM glycine and 0.1% (w/v)

SDS

Coomassie stain R250:- 0.2% coomassie blue,7.5% acetic acid, 50% methanol in

1000 ml distilled water.

Colloidal Coomassie blue stain G ( BioRad®)

Destain:- 30% methanol (v/v),10% glacial acetic acid (v/v).

Owl Silver Stain™:- made according to manufacturer’s instructions.

2.1.2.5. Preparation of nuclear proteins:-

1% (v/v)Triton X 100:- 0.5 ml in 50 ml

EDTA free Protease Inhibitor cocktail:- 1 EDTA free Protease Inhibitor cocktail

tablet in 1 ml of deionised water.

DNase I (RNase free) and DNAse buffer (BioLabs.Inc)

0.5% (w/v) Sodium Dodecyl Sulphate:- 0.25g in 50 ml

26

2.2. METHODS:-

2.2.1. Peptide Immobilization:- The synthetic peptides were obtained from Peptide

Synthetics, Peptide protein research Ltd, Hampshire. (All Centrifugations at 1000x g

for 1 min at room temperature).

2.2.1.1. Preparation of sample for Coupling:-1 mg of each peptide (FUS WT, FUS Mutant,

FUS Phosphorylated and Spastin was dissolved in 2 ml of coupling buffer. To this 0.1 ml of

TCEP (Tris, 2 Carboxyethyl phosphine) solution was added to each peptide. TCEP treatment

efficiently reduces the peptide and other disulfide containing molecules. The samples were

then incubated at room temperature for 30 min.

2.2.1.2. Coupling of Peptides to SulfoLink Columns:- SulfoLink resin was re-suspended in

the columns provided by mixing and then centrifuged at 1000 g for 1 min with the caps

removed to remove the storage buffer. Two ml of coupling buffer was added to each column

and centrifuged as before. This equilibration step was repeated two times then the bottom

caps replaced. Two ml of each sulfhydryl containing peptide was added to each column. Both

the top and bottom caps were replaced and the resin and peptides were mixed by rolling them

on a roller, at room temperature for 15 minutes. The columns were then placed upright and

allowed to stand for 30 minutes without mixing. Both the top and bottom caps were then

removed to drain off the unbound excess peptide. The columns were then washed with 2 ml

of coupling buffer and centrifuged s above. This wash step was repeated three times.

2.2.1.3. Blocking of non-specific binding sites: - The bottom cap was replaced and 2 ml of

50mM cysteine solution (containing 15.8 mg of L Cysteine.HCL) was added. The columns

were mixed for 15 minutes and then incubated for 30 minutes without mixing. The buffer was

then allowed to drain off from the column. Two ml of phosphate buffered saline (PBS) was

27

added to each column which was then centrifuged as before. This wash step was repeated

three times. The columns were stored by adding 2ml of PBS containing 0.05% sodium azide

for long term storage.

2.2.2. Process of Affinity Purification of proteins:-

2.2.2.1.Homogenization of brain tissue and preparation of extract:- The matrices were

suspended and equilibrated in homogenisation buffer. Ten ml of 0.1% BSA was added to the

columns, for blocking non specific binding, and allowed to mix by end over end mixing for 1

hour at 4ºC. Around 2 g of mouse brain was taken and coarsely chopped using a clean blade

and homogenised in ice cold homogenisation buffer containing protease inhibitors

(Complete® protease inhibitor cocktail), using a tissue homogeniser and then centrifuged at

10000 g for 10 min at 4ºC. Triton X-100 present in the homogenisation buffer lyses the

plasma membrane and cytoplasmic organelles but not the nucleus, allowing the soluble

cytoplasmic proteins to be extracted. The protease inhibitor cocktail inhibits the serine and

cysteine proteases thus protecting the proteins from degradation.

2.2.2.2 Purifcation of proteins:- Two different protein extracts were used:- a cytoplasmic

and a nuclear extract.

2.2.2.2.1 Purification from cytoplasmic extracts:-The supernatant, containing the

cytoplasmic proteins, was collected and added to the immobilised peptide columns after

draining off the 0.1% BSA block solution. The columns were mixed by rolling them

overnight at 4ºC. The columns were then placed in an upright position and the supernatant

was allowed to flow through the column. The columns were then washed sequentially with

10 ml each of homogenisation buffer containing protease inhibitors, 0.5M/1M NaCl wash

buffer containing homogenisation buffer, and homogenisation buffer to remove off any

unbound proteins. During each wash the columns were mixed on a roller at 4ºC for 15

28

minutes. After the final homogenisation buffer was drained off, 10 ml of 0.1M glycine pH 2.5

was added to the columns. Ten fractions, each of 1ml, were collected in 1.5ml eppendorf

tubes. The fractions were then neutralised using 1M Tris Base. For phosphorylated peptide

purification phosphate inhibitor solution is added to homogenisation buffer (1:25 dilution),

and used similarly as mentioned above.

2.2.2.2.2 Preparation of nuclear extracts: - The insoluble protein pellet containing nuclear

and cytoskeletal proteins obtained from the initial 10,000 g centrifugation of brain

homogenates was used for affinity purification experiments. The pellet was re-suspended in 2

ml of 20 mM HEPES pH7.5, 1% triton X-100, 1 Complete® (EDTA-Free) protease inhibitor

cocktail, DNase-1 (200 units/ml), DNAse buffer (200 u/ml) . This suspension was then mixed

gently on a rotary shaker for 1 hour. The solution was then centrifuged at 3000 x g for 1 min

and the pellet was re-suspended in 1ml of 0.5% Sodium Dodecyl Sulphate (SDS) solution

and kept on ice for 10 minutes. SDS lyses the nuclear membrane and releases soluble nuclear

proteins into solution. DNAse buffer was added to degrade the DNA released in this process.

This was followed by the addition of 20 ml of 20 mM HEPES pH 7, 1% Triton X -100 and

Complete® protease inhibitor cocktail. This solution dilutes the SDS 20-fold for efficient

binding of the proteins to the peptide. The solution was then added to the beads in the column

which was then mixed by rolling overnight. This was followed by the washes mentioned in

the previous section.

2.2.3. SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE):- SDS Polyacrylamide gel

Electrophoresis is a technique for separating proteins based on their molecular mass. Ten and

twelve percent polyacrylamide gels were mainly used in this study. Ten ml of 10% separating

gel and 6 ml of 4% stacking gel were prepared for two gels of 1.00 mm thickness. Five μl of

unstained molecular weight standards (Sigma Aldrich®, MW-6500-200,000 Da) or

Invitrogen-Novex® Sharp (MW-3.5kDa to 260 kDa) were loaded. The gels were initially

29

electrophoresed at 60 volts for 45 minutes to allow stacking of the proteins at the gel interface

and then run at 120 volts for 1.5 to 2 hours. The protein gels were then stained using

Coomassie stain or silver staining.

2.2.4 Staining:-

2.2.4.1. Coomassie staining:- The Gel was then carefully removed from the plates and was

stained using Coomassie stain G or Coomassie colloidal blue G. The gel was stained for two

hours on a rotator shaker for uniform staining throughout the gel. The coomassie stain was

then drained off and destain was added to the gel and allowed to incubate on the rotator

shaker for thirty minutes. The destain solution was drained off and the gel was visualized for

protein bands.

2.2.4.2. Silver staining ( Owl Silver Stain):-

(All incubations were carried out on a rotary shaker)

Gels were stained in polypropylene containers. Fifty ml of fixing solution 1 was added to

each gel and incubated for 10 min. The fixing solution was drained off and 50 ml fixing

solution 2 was added to each gel and incubated for 15 minutes. After draining off the fixing

reagent, 50 ml of pretreatment solution was added to each gel and incubated for 10 minutes.

The gels were then washed with deionized water for 5 minutes. Silver stain solution was

added to each gel and incubated for 15 minutes. The gels were then washed 2-3 times with

deionized water before adding 50 ml of developer solution. Three ml of stopper solution was

added to each gel when the appropriate band intensity was reached. The gels were stored in

20% ethanol and its image was captured using a Scanner or a Digital Camera.

30

3. RESULTS:-

3.1 Peptide Design:- As mentioned previously, three FUS synthetic peptides were designed

for the purpose of the experiments. The C-terminal of FUS protein was chosen as the vast

majority of mutations reported in FUS related ALS cases are found in the 16th

exon encoding

the C-terminal end of the protein (Kwiatkowski et al, 2009). The 17 residues at the C-

terminal end of the protein were chosen as this sequence was found to be conserved in all the

species, including human, mouse and rat. The synthetic peptide sequence was as follows.

Wild Type: - (CG)PGKMDSRGEHRQDRRERPY

The second peptide designed was a mutant form of FUS protein. Since arginine to histidine

mutation at the 521st base pair was one of the common mutations reported in FUS related

ALS cases, it was chosen and the following synthetic peptide was synthesised:-

Mutant:- (CG)PGKMDSRGEHRQDHRERPY

As shown the arginine residue was replaced by histidine at the 521st position

The third peptide designed was a phosphorylated form of FUS C-terminal protein. Protein

phosphorylation has can alter the function of a protein by either inhibiting or allowing protein

interaction. With the use of Net Phos2.0 software we identified possible phosphorylation sites

at the C-terminal. From the software it was predicted that serine present on 513th

residue of

the C-terminal sequence had 0.99% chance of getting phosphorylated.

31

Figure 4:- A software generated prediction of phosphorylation sites in FUS protein.

Arrow indicates the potential of C-terminal Serine (513th residue) getting phosphorylated.

32

Since the phosphorylation potential of serine was high compared to any other C-terminal

peptide, it was decided to be phosphorylated on the synthetic peptide. The sequence of the

synthetic phosphorylated peptide was as follows.

Phosphorylated:- (CG)PGKMDS*RGEHRQDRRERPY

The * added between serine and arginine indicates a phosphor-serine residue.

Phosphorylation of serine/arginine proteins is known to disrupt some protein-protein

interactions (Yang et al, 1998) and thus this phosphorylated peptide was used to study

whether this modification affects the C-terminal of FUS binding with any other protein.

It should be noted that all three peptide sequences contain additional cysteine and glycine

residues, indicated by (CG) added to their N-terminus. The Sulfolink® agarose bead used for

immobilization reacts with the sulfhydryl group of the cysteine residue and forms a covalent

bond, while the glycine atom was added as a spacer molecule to minimize stearic hindrance

between the agarose bead and the peptide sequence.

Spastin was used as control, in this experiment. The sequence of Spastin used was as

follows:-

Spastin:- (C)IRELKPEQVKNMS

Since the sequence of Spastin peptide does not match with FUS C-terminal used, it was

selected as a non specific control.

33

3.2. Affinity purification of proteins using wild type and Spastin peptides from

cytoplasmic extract, without salt wash:- Initially, affinity purifications were performed

using cytoplasmic mouse brain extracts with the immobilised FUS wild type and Spastin

peptides. The different protein fractions which were obtained were then analysed using SDS-

PAGE. No salt washes or blocking reagent such as Bovine serum albumin (BSA) or glycerol

was used in the initial experiments. A 10% polyacrylamide gel was used to analyze the

proteins eluted from the peptide columns. Each number denotes the corresponding fraction

for that particular peptide e.g. WT 2 denotes 2nd

fraction from wild type peptide.

As observed in Figure 5, the amount of proteins eluted from both the synthetic peptides was

high in number. No specific protein bands were observed in either the Spastin or FUS wild

type lanes. All the bands in the corresponding lanes look similar indicating non specific

binding of numerous proteins to the peptide columns. Each fraction was compared to its

similarly numbered fraction for binding specificity.

34

Figure 5:- A 10% SDS PAGE analysing the proteins bound to Spastin and Wild type (WT)

peptides in the absence of a blocking step or high salt washes. The gel was stained with

Coomiase Blue R 250.

Marker Spastin 2 WT 2 Spastin 3 WT3 Spastin 4 WT 4

90 kDa

60 kDa

40 kDa

30 kDa

35

3.3 Affinity purification of proteins from wild type and spastin peptides from

cytoplasmic extract with the use of Salt wash: -

In order to reduce the non-specific binding of proteins to the peptide columns as observed in

Figure 5, 0.1% Bovine serum albumin (BSA) and 10% glycerol was added to the

homogenisation buffer as blocking agent. The remaining procedure was carried out as

mentioned before, with the exception of Salt washes (0.5M or 1M NaCl) which were

introduced in the washing step to remove proteins non-specifically bound to the peptides or

matrix. The protein fractions obtained after the use of salt washes were then analysed using

SDS-PAGE. Since the salt washes removed a lot of non specific protein, the proteins could

not be visualized with Coomassie stain and so silver staining (Owl silver Stain Kit) was used

to stain the gels.

As observed in Figure 6, the use of BSA and glycerol blocking buffer, and a high salt wash

greatly reduced the amount of protein eluted from the columns by decreasing non-specific

binding to both the Spastin and FUS WT peptide columns. Far fewer proteins and much less

total protein content was observed when compared to the gel with no blocking and salt wash

steps (Figure 5). No proteins were specifically seen to bind to either the Spastin or FUS wild

type fractions indicating that some non-specific binding was still present. But since the

protein content in the eluted fractions was greatly decreased, use of BSA and glycerol in the

solutions and a salt wash step was continued.

36

Figure 6:- SDS PAGE gel depicting the proteins bound to and eluted from Spastin and FUS

WT peptides. The marker used was Sigma Aldrich protein standard. The gel was stained

using silver staining.

Spastin2 WT2 Spastin3 WT3 Spastin4 WT4 Marker

200 kDa 116 kDa

97 kDa

BSA-66 kDa

55 kDa

36 kDa

37

3.4 Affinity purification of proteins using wild type and Spastin peptides from Nuclear

extract :- To prepare nuclear extracts the pellet which was obtained from the initial

centrifugation step was digested with DNase I and 1X DNase reaction buffer containing

CaCl2 and MgCl2. The nuclear proteins were extracted with SDS which was then diluted

twenty fold into mixed micelles with Triton X-100 prior to affinity purification. These

proteins were than purified and elutes with the process of affinity purification. The protein

binding was then analysed with a 10% SDS- PAGE.

Figure 7 shows that similar proteins were purified from the nuclear protein extract using both

Spastin and FUS wild type peptides. This indicates the presence of non-specific binding of

proteins to the peptides. The bands seen in fractions 2, 3and 4 are similar in both Spastin and

FUS Wild type eluates, indicating no specific differences. Note that a different set of non-

specific bands was observed in the samples purified from nuclear extracts compared to with

cytoplasmic proteins (Compare Figure 6 with Figure 7).

38

Figure 7:- A 10% SDS PAGE showing the nuclear proteins affinity purified using Spastin

and FUS wild type peptides. The marker used was Novex® Sharp protein standard.

39

3.5 Affinity purification of proteins using FUS Mutant and Spastin peptides from

cytoplasmic extracts:- Affinity purifications were performed using FUS mutant and Spastin

peptides from cytoplasmic extract and the eluates were analysed using SDS PAGE for protein

interactions. A 1 M salt wash was used to remove any non specific binding.

As seen in the Figure 8a specific band of approximately 40 kDa was seen in the Spastin

peptide eluate fraction 8 that was not present in the corresponding FUS mutant peptide eluate.

The band was seen only in one fraction (Spastin 8) and not in any other Spastin fraction. To

check whether this 40 kDa protein represented a mutant peptide specific interacting protein,

the experiment was repeated, this time using a 0.5 M salt wash as used in previous

experiments.

3.6 Repeat affinity purification with FUS mutant and Spastin peptides using

cytoplasmic extracts:- To compare protein interactions between FUS mutant and Spastin

peptide, affinity purification was again performed, but this time with a 0.5 M salt wash. The

eluted fractions were then analysed using a 10% SDS-PAGE gel.

As observed in Figure 8b, a 40 kDa protein band was seen in one FUS mutant peptide

fraction (M8), but not in any other FUS mutant or Spastin fraction. This result contradicted

the previous result, where a 40 kDa protein was only present in single Spastin fraction. This

contrasting result could be due to the use of salt washes of different molar concentrations, but

also suggests that the 40 kDa protein is not a specific FUS mutant peptide interacting protein.

40

Figure 8a:-10% SDS PAGE gel comparing the proteins bound to the Spastin and FUS

mutant (M) peptides. The Figure shows a specific band of approximately 40 kDa present in

the Spastin fraction 8.

41

Figure 8b:- A 10% SDS PAGE depicting the proteins bound to Spastin and FUS

mutant. A specific band is seen on FUS mutant (M 8), approximately 40 k Da, not

present in any of the Spastin lanes.

42

3.7. Affinity purification with FUS wild type and FUS mutant peptides using

cytoplasmic extract:- The cytoplasmic proteins in mouse brain extracts interacting with the

FUS wild type and FUS mutant were then compared for protein-protein interactions.

Interacting proteins were isolated by affinity purification using a 1M salt wash and then

analyzed by electrophoresis in a 12% SDS-PAGE gel (Figure 9).

As seen in Figure 9, a protein band was observed specifically binding to FUS wild type and

not to FUS mutant peptide supporting the hypothesis of loss of protein interactions with the

mutant form of FUS protein. The same protein band was clearly observed in the FUS wild

type fractions 2 and 3 lane (WT 2 and WT 3) and faintly in fraction 4 (WT 4), but not in any

of the corresponding mutant fractions. The molecular weight of the protein was

approximately 32 to 35 kDa when compared to the molecular weight marker proteins. The

experiment was repeated and the same result was obtained.

43

Figure 9:- Silver stained 12% SDS-polyacrylamide gel showing the proteins interacting with

FUS wild type and mutant peptides. The arrow indicates a protein band observed only in FUS

wild type and not FUS Mutant peptide eluates.

.

44

3.8. Affinity purifications with FUS wild type and FUS mutant peptides from nuclear

protein extract:- Nuclear proteins interacting with the FUS wild type and Mutant peptides

were analysed using affinity purification methods. Nuclear extracts were prepared from the

pellet obtained after the initial centrifugation of the brain extracts by DNase I digestion and

extraction with SDS. Affinity purification was done and the proteins were purified. A 1 M

salt wash was employed in the affinity purification protocol. The proteins purified were

analyzed by electrophoresis in a 12 % SDS-PAGE gel.

As can be seen in Figure 10, no specific protein band was observed in the eluates obtained

from either the Wild type or Mutant peptides. No apparent differences were seen between

corresponding wild type and mutant fractions, suggesting non-specific binding of these

proteins to the peptides/matrix. The difference in the protein bands when compared to the

previous experiment (Figure 9) represents the presence of different proteins in the

cytoplasmic and nuclear extracts.

45

Figure 10: - A silver stained 12% SDS polyacrylamide gel, showing the nuclear proteins

interacting with FUS WT and Mutant peptides. No specific protein band was observed in the

eluates from either the Wild type or Mutant peptide columns.

46

3.9. Affinity purification with FUS wild type and phosphorylated FUS peptides:-

FUS wild type and phosphorylated FUS peptides were used for affinity purifications from

cytoplasmic mouse brain extracts. A 0.5 M salt wash was used in this experiment and the

purified proteins were analysed using SDS-PAGE (Figure 11). WT represents wild type and

P represents Phosphorylated peptide.

Figure 11 shows that a faint band was observed in the Phosphorylated FUS extract fraction 3

which was not present in any of the FUS wild type fractions. The band was also faintly

visible in Phosphorylated extract fraction 2. This indication of a specific band in the

phosphorylated FUS fractions suggests that phosphorylation of the FUS C-terminal region

could promote protein interactions. It is of note that the putative interacting proteins

identified in this and the experiment shown in Figure 9 were both found to be around 35 kDa.

47

Figure 11: - A 10% SDS-polyacrylamide gel showing the proteins affinity purified with FUS

WT and phosphorylated FUS peptides. The arrow indicates a faint protein band of around 35

kDa specifically present in the 3rd fraction of Phosphorylated peptide eluate.

38 kDa

48

3.10. Comparison of the results obtained with the comparisons of mutant and

phosphorylated peptides with wild type :- To compare the results obtained in the

experiments shown in Figure 8 and Figure 10, a SDS-polyacrylamide gel was loaded and

electrophoresed with proteins from both experiments to enable direct comparison. This 12%

gel was run to determine whether the putative FUS-interacting proteins identified were of the

same molecular weight. The samples which showed specific protein binding (from Figures 9

and 11) were analysed on the same gel to determine any similarity between both the protein

bands obtained from both the experiments. The marker is not shown in the Figure as it did not

separate properly. The first four lanes labelled WT2 M2 WT3 M3 were fractions from FUS

wild type and mutant study, while the rest four labelled P2 WT2 P3 WT3 were fractions from

FUS wild type and FUS phosphorylated comparison.

Figure 12 clearly shows that the protein bands obtained were different from each other in

molecular mass. The protein binding to FUS wild type in FUS wild type and FUS mutant

study was found to be around 35 kDa, while the protein band observed in Phosphorylated

FUS from the FUS wild type and Phosphorylated FUS comparison was higher than 35 i.e.

around 38 kDa. Thus both the protein bands observed were not similar in molecular mass

49

Figure 12: - A 12% gel comparing the fractions obtained from Wild type and mutant, and

Wild type and phosphorylated peptide affinity purifications. The gel was stained using Mass

Spectroscopy compatible silver stain from Invitrogen®.

50

3.11. Analysis of specific proteins through mass spectroscopy:- Due to the presence of

less amount of proteins in the gel the same SDS gel (Figure 12) was repeated again and this

time stained with Colloidal Coomassie Blue G (Figure not shown). The colloidal stain G is

known to be more sensitive than the other coomassie stain R. The gel after de-staining was

placed on a clean glass plate and the two specific bands were excised using a clean scalpel

blade. They were frozen in eppendorf tubes and sent for Mass Spectroscopic analysis at the

University of York.

Due to less amount of protein present in the band excised from Phosphorylated FUS, a

correct analysis could not be made. Mass Spectroscopy analysis of FUS wild type protein

band indicated the presence of a mixture of protein among them being abundant proteins like

actin and tubulin. Single peptide matches to myelin basic protein and microtubule associated

protein was also observed. Along with the proteins, presence of keratin contamination was

also observed in the protein bands. No conclusive result indicating the presence of a single

particular protein was obtained.

51

4.Discussion:-

The aim of the project was to develop an in vitro assay for the analysis of protein interactions

mediated by the C-terminal region of FUS. The hypothesis to be tested was that mutations in

the C-terminal region of FUS lead to disruption of critical protein interactions. To test this,

we established :-

a) Optimum protein binding conditions to immobilised synthetic

FUS C-terminal peptides.

b) Whether mutation in the C-terminal region of FUS causes loss

of protein interactions.

c) Protein interactions with cytoplasmic and nuclear proteins

d) Whether Phosphorylation of the C-terminal region affects

protein interactions.

4.1. Optimum binding conditions:- In order to fulfil the first objective, i.e. to determine

optimum binding conditions, we had to optimise conditions for protein affinity purification.

We found that just with homogenisation buffer washes, we were not able to obtain specific

binding of proteins (Figure 5). A high background was observed with lots of non-specific

binding. We found out that by including 10% glycerol to disrupt hydrophobic interactions,

using BSA as a blocking reagent (reviewed in Johnson et al, 1984) and performing 0.5M/1M

salt washes after protein binding, reduced non specific binding to a large extent (Figure 6).

It was observed that salt washes of different molar concentrations gave varying results, with

respect to the presence and intensity of the bands which could be seen in Figure 8a and 8b

where using different concentration salt wash resulted in presence of a similar band in

52

different peptides. In Figure 8a, the gel with proteins obtained from 0.5 M salt wash,

indicates a band of around 40 kDa observed in Spastin which was not present in wild type.

Using the same peptides, when 1M wash was used the band was seen to visible in wild type

and absent in Spastin. Two different concentrations gave two different contradictory results.

This indicates that use of salt wash with different concentrations, specific proteins could also

get washed away.

4.2. Mutation in the C-terminal region of FUS causes loss of protein interactions:-

To test this hypothesis, we compared the proteins that bound to the FUS wild type and FUS

mutant peptides. Using the previously established binding and washing conditions, we

purified the proteins which were bound to the two different peptide sequences. On SDS-

PAGE analysis (Figure 9) it was consistently found that one protein was bound to FUS wild

type peptide and not to the FUS mutant peptide. This result supports our hypothesis that FUS

mutations result in a loss of protein interactions in the C-terminal region. Since FUS protein

mislocalizes to the cytoplasm in ALS cases, due to the mutations (Kwiatkowski et al, 2009,

Vance et al, 2009), this result indicates that the protein that fails to bind (or binds much more

weakly) to the FUS mutant peptide could play an important role in transporting FUS protein

to the nucleus. Thus when this protein fails to bind due to a mutation, in this case Arginine to

Histidine at the 521st residue, it could result into a pathological mechanism which causes FUS

protein mislocalization and subsequent aggregation in the cytoplasm.

4.3. Protein interactions seen in cytoplasmic and nuclear fractions: - Both cytoplasmic

and nuclear extracts were analysed to identify proteins that interact with FUS. Triton X 100, a

surfactant was used in the homogenisation buffer to lyse the plasma membrane and

cytoplasmic organelles (Schnaitman, 1971). Nuclear proteins were extracted from the Triton-

insoluble fraction with Sodium Dodecyl Sulphate, a strong ionic detergent, after DNase I

53

digestion. Affinity purifications were performed on both cytoplasmic and nuclear protein

extracts and the results were analysed separately by SDS-PAGE. After electrophoresis and

staining, it was seen that the gels loaded with samples prepared from the different extracts

had different protein banding patterns. As can be seen in Figure 6 and Figure 7, the

cytoplasmic and nuclear proteins of the mouse brain had different banding arrangements

when purified from FUS wild type and Spastin peptides. A similar result was obtained when

FUS wild type and mutant were compared. The cytoplasmic and nuclear proteins isolated

using the same concentration of salt wash from both the peptides, differed in banding patterns

(Figure 9 and Figure 10). This difference in protein banding arrangements in both the

cytoplasmic and nuclear proteins suggests the presence of different proteins in both the

cytoplasm and the nucleus. Moreover, a specific band was obtained in the cytoplasmic

fraction (Figure 9) and not the nuclear (Figure 10), inspite of using the same protein purifying

conditions, indicating that the nuclear protein-interactions is less or not at all affected by the

mutation on the C-terminal peptide.

This result points out that the cytoplasmic proteins interacting with FUS may be different

from the nuclear proteins and that nuclear protein interactions may have a less important role

in the pathology of FUS related ALS. These results may also shed some light on the

importance of cytoplasmic proteins in the patho-physiology of ALS, since they suggest that a

key protein may be present mainly in the cytoplasm and not (or to a lesser extent) in the

nucleus.

4.4. Phosphorylation of the C-terminal region of FUS may affect protein interactions:-

Protein phosphorylation is one of the most important post-translational modifications. It is

known to regulate many cellular processes in eukaryotes and can induce or prevent the

functioning of a cellular process (Hunter 1995). FUS/TLS is known to undergo tyrosine

phosphorylation by the fibroblast growth factor receptor-1 (FGFR1) kinase. This

54

phosphorylation causes FUS/TLS to re-localize to the cytoplasm and aggregate (Klint et al,

2004). Serine/Arginine protein phosphorylation is also known to affect protein-protein

interactions and thus controlling intracellular localization of splicing factors (Yang et al,

1998). Since the probability of serine to get phosphorylated is high (0.99) compared to other

residues of the C-terminal peptide, its role, (when phosphorylated) in protein-interaction was

studied.

To check whether the serine phosphorylation in the C-terminal region could affect protein

interactions, proteins binding to both FUS wild type and phosphorylated FUS peptides were

purified and analysed by SDS-PAGE. A protein band specific to phosphorylated FUS was

observed, which was not found to be present in the FUS wild type peptide column eluates

(Figure 11). This result implies that there may be a gain of protein interactions due to

phosphorylation of the C-terminal region of FUS. Since this protein appeared not to interact

with the wild type, and, it has been shown that serine phosphorylation of FUS leads to FUS

mislocalization in the cytoplasm (Klint et al, 2004), it is possible that this protein could play

an important role in the pathology of the disease. Further studies on protein interactions with

phosphorylated form of FUS mutant peptide will help increase our understanding with

respect to the role played by phosphorylation in FUS related ALS

4.5. Protein interaction in C-terminal FUS with respect to ALS:- According to the

comparison study done between FUS wild type and mutant (Figure 9), a protein with a

molecular weight between 30 and 40 kDa is present in the wild type and absent in the mutant.

From the result obtained from mass spectroscopic analysis, various abundant proteins were

obtained. These include Actin, tubulin and myelin basic protein (MBP). Other proteins found

to be present were microtubule associated protein, 14-3-3 protein and contaminant keratin.

Since the proteins like actin, tubulin and MBP are abundant proteins, and could be a result of

55

background binding, a strong association of either of the proteins with FUS cannot be

established.

On the other hand, if we look at the list known FUS C-terminal protein interactors, we do get

an idea about the protein which could be present in the excised band. The band excised was

around 35 kDa, between 30 and 40 kDa and from the known protein interactors, either of the

two proteins, namely; Splicing factor 35 or Y box binding protein-1, could be present in the

protein. Moreover both the proteins have molecular weight of 35 kDa, indicating one of them

to be the right candidate interactors.

4.5.1 Splicing Factor-35: - Splicing factor 35 or SC-35 is a member of serine arginine (SR)

rich protein family, and is known to interact with the C-terminal region of FUS (Yang et al,

1998). It is a 35 kDa protein encoded by 1703 base pairs on chromosome 17. SC-35 has been

associated with processes like transcription and splicing (McCraken et al, 1997). It was also

shown that removal of SC35 from the cell results in dysregulation of the splicing mechanism.

According to studies done on SC-35, the interaction between FUS and SC-35 rich protein

complexes is important for the development and maintenance of the cell (Yang et al, 1998).

Yang et al (1998) using the yeast 2 hybrid system found that SC-35 binds to the C-terminal

region of FUS, and, that the FUS-SC-35 complex plays an important role in RNA processing

and splicing. Inability to form a complex could lead to inhibition of the processes. Various

base-substitution mutations like Arginine to Histidine could prevent the interaction and thus

lead to alteration in RNA processing.

Moreover, SC-35 proteins are a class of Serine/Arginine rich proteins and it seems logical

that base mutation of arginine to histidine can affect its interaction with the C-terminal FUS.

Since alteration of RNA processing is a major cause for FUS related ALS, the role of SC53 in

ALS pathology cannot be denied.

56

4.5.2. Y box binding protein-1:- Y box binding protein-1, a 35 kDa protein, is a

multifunctional protein known to play a role in the regulation of transcription and mRNA

processing. It is activated by cytotoxic stress, where it binds to the Y-box (single stranded

response element). This nuclease sensitive element binding protein-1, as it is sometimes

called, is known to regulate translation by governing the interaction between eukaryotic

initiation factors and mRNA (reviewed in Law et al, 2006). Thus a role of YB-1 in protein

synthesis has been established.

Studies done by Rapp et al in 2001 using the yeast two hybrid system showed that FUS

interacted with YB-1 through its C-terminal domain. This was supported by studies done by

Chansky et al (2001), who also found that YB-1, along with other factors like SC-35,

interacts with the C- terminal domain of FUS, through a yeast two hybrid screen from a

mouse haematopoietic cell line library. As can be seen in Figure 9, the 35 kDa band

(potentially SC-35 or YB-1) did not bind to the mutant peptide indicating a loss of FUS

interaction with it. Both the proteins in complexes with FUS regulate RNA splicing and

protein synthesis. Alterations in such complexes could lead to many abnormalities like

impaired mRNA splicing, altered protein synthesis and inhibition of RNA processing. Such

alterations could potentially be related to FUS protein mislocalization and accumulation in

the cytoplasm, as seen in ALS. Western blot analysis using anti SC-35 and anti YB-1

antibodies could help in determining the presence of either of the proteins.

Though there have not been any documented cases directly linking SC-35 or YB-1 protein to

pathology of ALS, their absence in the FUS mutant peptide clearly indicates that they do

interact with FUS in the normal functioning of the protein, and that mutation like arginine to

histidine does lead to its inability to interact with the C-terminal peptide of FUS.

57

4.6. Limitations:- Inspite of the positive results, there are many limitations to this study. The

first and the foremost is that this was an in vitro study of the protein interaction and that

suggestive and conclusive result could only be obtained through a conformational in vivo

study. Another drawback of this experiment is that only synthetic C-terminal peptide was

used, and not the whole intact protein. A study with an intact FUS protein would be a good

follow up to this experiment to study the protein interactions taking place in presence of an

entire protein. The amount of proteins eluted each time through affinity purification was less

and leading to insufficient protein being analysed through mass spectroscopy. Though TCA

precipitation to scale up the proteins didn’t work (data not shown) other methods can be

employed to scale up the proteins. Immunoblotting can also be undertaken for the proteins

observed from mass spectroscopic analysis or for known protein interactors. The mass

spectroscopy analysis indicated the presence of lot of proteins like actin tubulin and myelin

basic protein, so gel slices of the corresponding mutant peptides of the wild type and mutant

peptide page analysis should also be analysed for subtraction of the background proteins.

Since both splicing factor 35 and Y box binding protein are nuclear proteins, their presence in

the cytoplasmic excised protein band could be argued. On the contrary it could also be said

that they might be present in the cytoplasm in less amount and due to their weak presence,

they could not be identified through mass spectroscopy.

4.7. Future work:- Identification of the interactors through Western blot analysis with the

use of data obtained from Mass Spectroscopy analysis. The next step would be to validate the

results obtained from the short synthetic peptides with an intact protein. Co-

immunoprecipitation studies from transfected cells or ideally from brain extracts could be

done to compare the interactions in vivo. Furthermore, immunocytochemistry studies should

be performed to confirm whether any protein identified co-localizes with FUS in either the

cytoplasm or the nucleus. The preliminary results obtained also justify using an alternative

58

approach such as the yeast two-hybrid system to identify proteins that interact with the C-

terminal region of wild type but not mutant FUS. Future studies based on the C-terminal

interaction of FUS could prove to be a positive step in a better understanding of ALS.

59

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