160_IPSITA SAHOO_THESIS (1).pdf

UNDERSTANDING THE INVOLVEMENT OF
N-TERMINAL DOMAIN OF FATS IN INTERACTION
WITH TUMOROGENIC PROTEINS
A Thesis Submitted to Centurion University of Technology and Management in
partial fulfillment of the requirements for the award of the degree of
MASTER OF SCIENCE
IN
ZOOLOGY
By
IPSITA SAHOO
200705180160
Under the guidance of
DR. GAGAN KUMAR PANIGRAHI
Department of Zoology
School of Applied Sciences
CENTURION UNIVERSITY OF TECHNOLOGY AND MANAGEMENT,
BHUBANESWAR, KHORDHA – 752050, ODISHA, INDIA
www.cutm.ac.in
May 2022

i
DECLARATION
I, declare that the thesis entitled “Understanding the involvement of N-terminal domain of
FATs in interaction with tumorogenic proteins” is my own work conducted under the
supervision of Dr. Gagan Kumar Panigrahi, Assistant Professor, Department of Zoology,
School of Applied Sciences, Centurion University of Technology and Management,
Bhubaneswar, Odisha.
Furthermore, I declare that this thesis does not contain any part of any work submitted for the
award of any degree either in this University or in any other University without proper citation.
IPSITA SAHOO
Registration Number: 200705180160
PLACE- Bhubaneswar
DATE- 10/05/2022

ii
School of Applied Sciences
CENTURION UNIVERSITY OF TECHNOLOGY AND MANAGEMENT,
BHUBANESWAR, ODISHA, INDIA
CERTIFICATE
This is to certify that the thesis entitled “Understanding the involvement of N-terminal domain
of FATs in interaction with tumorogenic proteins” is a bonafide work done by Ipsita sahoo of
M.Sc. (Zoology) bearing Registration No. 200705180160 under the guidance of DR. GAGAN
KUMAR PANIGRAHI, Assistant Professor, Department of Zoology, Centurion University Of
Technology and Management, Bhubaneswar, in partial fulfillment of the requirements for the
award of the degree of Master of Science in Zoology, School of Applied Sciences, Centurion
University of Technology and Management, Bhubaneswar, Odisha, India. The project work has
done and the report satisfies the requirements for the award of the degree mentioned.
Dr. Gagan Kumar Panigrahi
Supervisor
Assistant Professor
Department of Zoology
CUTM, BBSR
Dr. Rukmini Mishra
Domain Coordinator
Associate Professor & Head
Department of Botany
CUTM, BBSR
Dr. Yashaswi Nayak
Dean & Head of the Department
Associate professor
Department of Zoology,
CUTM, BBSR

Acknowledgement
This project thesis entitled “Understanding the involvement of N-terminal domain of FATs in
interaction with tumorogenic proteins” has seen contributions from various individuals. It has
been an honor to work under my guide, Dr. Gagan Kumar Panigrahi, Assistant Professor,
Department of Zoology, Centurion University Of Technology and Management, Bhubaneswar. I
am extremely thankful to him for his support and mentorship throughout the project. I also
extend a heartfelt gratitude towards my Domain Coordinator, Dr. Rukmini Mishra, HOD,
Associate professor, Department of Botany, Centurion University of Technology and
Management, Bhubaneswar. This project would not have had better supervisors than them.
I would also extend my gratitude towards Dr. Yashaswi Nayak, Dean & Head of the Department
Associate professor Department of Zoology, Centurion University of Technology and
Management, Bhubaneswar.
I would also like to thank to entire team who has been a constant moral support. Lastly, I would
like to thank my family and friends for their kind support. I feel grateful to Lord Almighty who
has showered His graces upon me during this period.
PLACE: Bhubaneswar
DATE :10/05/2022
Ipsita Sahoo
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CONTENTS
SL NO TITLE PAGE NO
1. DECLARATION i
2. CERTIFICATE ii
3. ACKNOWLEDGEMENT iii
4. LIST OF FIGURES v
5. LIST OF TABLES vi
6. ABSTRACT 1-2
7. INTRODUCTION 3-5
8. REVIEW OF LITERATURE 6-16
9. METHODOLOGY 17-22
10. RESULTS AND DISCUSSION 23-50
11.
CONCLUSION AND FUTURE PERSPECTIVES
51-54
12.
REFERENCES
55-64
iv

LIST OF FIGURES
SL NO FIGURES
PAGE
NO
1. Drosophila and vertebrate FAT cadherin members 9
2. Association between FAT1 and Hippo signaling pathway 11
3. Overview of Wnt/β-catenin signaling 13
4. Association between FAT1 and EMT 14
5. Methodology infography 22
6. Amino acid sequence of (a) FAT1, (b) FAT2, (c) FAT, and (d) FAT4 24-32
7. Protein sequence homology of FATs 33
8. Domain organizations of (a) FAT1, (b) FAT2, (c) FAT, and (d) FAT4 34
9. 3-D structure of (a) FAT1, (b) FAT2, (c) FAT3, and (d) FAT4 35
10. Putative protein partners (a) FAT1, (b) FAT2, (c) FAT3, and (d) FAT4 36
11. Construction of domain mutants of N-terminal region of FATs 37-40
12.
Interaction of (a) wild-type FAT1, (b) FAT1 (∆1-100), (c) FAT1
(∆101-200), (d) FAT1 (∆201-300), (e) FAT1 (∆301-400), and (f) FAT1
(∆401-500) with TNF-A
41-42
13.
(∆401-500) with HEPHL1
44-45
14.
(∆401-500) with MTNR1B
46-47
15.
(∆401-500) with VANGL2
49-50
v

LIST OF TABLES
SL NO TABLES PAGE NO
1. Interaction of FAT1 with TNF-A 43
2. Interaction of FAT2 with HEPHL1 45
3. Interaction of FAT3 with MTNR1B 48
4. Interaction of FAT4 with VANGL2 50
vi

1
ABSTRACT
Fat family members (FAT1, FAT2, FAT3, and FAT4) are human homologs of Drosophila Fat
and are implicated in tumour suppression and planar cell polarity. Cellular homeostasis is largely
maintained at the cellular level via transcription regulation, which can vary in response to
physiological alterations. FAT atypical cadherin 1 (FAT1), which encodes a protocadherin, is
one of the most frequently mutated genes in human cancer. FATs is thought to play a vital role in
the maintenance of organ and cellular homeostasis, as well as activating a number of signalling
pathways via protein-protein interactions, such as the Wnt/catenin, Hippo, and MAPK/ERK
signaling pathways. Unregulated FATs expression can cause cancer and have a negative impact
on prognosis. In this study, we focused on the structural and functional aspects of various
domains and motifs of FATs. Global bioinformatics databases resulted in streamlining a list of
putative protein associates of FATs.
FAT1 is a huge protein composed of 4588 amino acid residues. By mutational analysis and
further protein-protein docking studies using multiple bioinformatic tools it was confirmed that
the N-terminal 100 amino acids of FAT1 are vital for interaction with TNF-A since the deletion
of initial amino acid residues resulted in weak interaction between FAT1 (∆1-100) and TNF-A
with respect to wild-type FAT1-TNF-A interaction. The docking score for FAT1 (∆301-400)-
TNF-A interaction was slightly elevated to -287.29 suggesting that these residues did not
affected the strength of interaction between two proteins rather it is quite relevant that this
domain might regulate the interaction of FAT1 with TNF-A.
FAT2 acts as the tumour suppressor that slow down cell division, repair DNA mistakes, or tell
cell when to shown apoptosis. It plays important role in molecular mechanism of cancer
pathogenesis. It also acts as the EGF like domain; a common mitogenic factor stimulates call
proliferation of different types of cells. The EGF domain activates by the EGF receptor works in
intracellular signaling. The N-terminal 100 amino acids of FAT2 are vital for interaction with
HEPHL1 since the deletion of initial amino acid residues resulted in weak interaction between
FAT2 (∆1-100) and HEPHL1 with respect to wild-type FAT2- HEPHL1 interaction. The
docking score for FAT2 (∆401-500)- HEPHL1 interaction was drastically elevated to -283.03

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suggesting that these residues affected the strength of interaction between two proteins rather it is
quite relevant that this domain might regulate the interaction of FAT2 with HEPHL1.
FAT3 acts as the hemophilic cell adhesion via plasma membrane adhesion molecules. It also
uses in development of organism from its early stage. The proteins involved in the FAT3 actions
that are gas protein, pair rule protein, segmentation polarity protein. The N-terminal 100 amino
acids deletion changes about 100 score difference to show that affected the interaction between
FAT3 (∆1-100)-MTNR1B. Since the initial amino acid residues resulted in week interaction
between FAT3 (∆1-100)-MTNR1B. The docking score for FAT3 (∆301-400)-MTNR1B
interaction was drastically elevated to -447.31 suggesting these residues affected the strength of
interaction between two proteins rather it is quite relevant that this domain might regulate the
interaction of FAT3 with MTNR1B.
FAT4 acts as heterophilic adhesion molecules which to be shown adhere multiple protein to
make complexes helps in cell signaling pathways. It also acts as the instructional protein in case
of tissues grade. In case of mammal, the neural cell proliferation developed by FAT4 protein.
The docking score for FAT4 (∆1-100)-VANGL2 interaction was slightly elevated to -311.61
these residues did not affected the strength of interaction between two proteins rather it is quite
relevant that this domain might regulate the interaction of FAT4 with VANGL2. The N-terminal
100 amino acids deletion changes about 51 score difference to show that affected the interaction
between FAT4 (∆401-500)-VANGL2. Since the initial amino acid residues resulted in week
interaction between FAT4 (∆401-500)-VANGL2.
Our preliminary results will pave way forward in improving the prognosis and treatment of
patients with cancer.

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INTRODUCTION
FAT1, FAT2, FAT3, and FAT4 are the genes that make up the human FAT family (Schreiner et
al., 2006; Sun and Irvine, 2011). It was found in Drosophila in the 1920s as a result of a deadly
mutation. F2 and Ft2, two FAT cadherein members found in Drosophila, a rethought to tumer
suppressors.“The Ft gene results in a Epithelial overgrowth phenotype in Drosophila larvae, with
mutational modifications affecting the wings, eye antenna, legs, glands, and genital imaging
disc”(Peng et al., 2021). The fact that localises and expresses levels of the Hippo signalling
pathway transcription factors like Yorkie (Yki), Warts (Wts), and another FAT cadherin member
in Drosophila is connected with the expansion of the phenotype found in Ft inactivation in
humans. Fat2 is necessary for the formation and stability of ectodermal tubular structures. Any
Fat2 mutation causes aberrant development of renal tubular structures in humans, including the
absence of the trachea, gastric glands, and salivary glands. The complete coding sequence of
FAT1, FAT2 and partial coding sequence of FAT3 was published (Mariot et al., 2015;
Perugorria et al., 2019; Schwartz et al., 2003). The Complete coding sequence of FAT3 and
FAT4 was reported (Camargo et al., 2007). The most similar to FAT3 is FAT1. Big proteins with
extracellular cadherin repeats, EGF-like domains, and Lamnin G-like domains are encoded by
both human and Drosophila FAT family genes. Codons 275-352 of FAT2 are identical to the 3rd
cadherin repeat of FAT1. However, this region of FAT2 was not anticipated to represent the
cadherin repeats using NCBI's conserved domain search (CDS) algorithms. Because cadherin
repeats and EGF-like domains are defined loosely in CDS algorithms, it is still unclear whether
areas remotely linked to cadherin repeats and EGF-like domains are functional or not. From
Drosophila to vertebrates, the FAT cadherin family has grown from 2-4 members, but its
structure and function have remained consistent. Invertebrates of the FAT family have distinct
configurations in which the amount of laminin motifs and EGF-like motifs leads to the unique
function. FAT1 and FAT4 have extracellular domain cadherin repeats, whereas FAT2 and FAT3
contain 32 and 33 repeats, respectively. “The condition of constant internal, physical, and
chemical circumstances that biological systems maintain is known as homeostasis” (Mariot et
al., 2015). All organisms' metabolic processes can only take place in highly precise physical and
chemical conditions. The circumstances vary depending on the organism, and the chemical
reaction occurs within the cell or in the interstitial fluid that surrounds the cells. “The best-known

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homeostatic systems in humans and other animals are regulators that keep the extracellular fluid
composition constant”(Peng et al., 2021). The alternative homeostatic system, on the other hand,
encompasses many components of human physiological controls and other entities in the body.
Hyper and hypo conditions, such as hypothermia, hypotension, and hypertension, are usually
preceded when the amount of variables is larger or lower than what is necessary by the process
of homeostasis. From the previous reviews found that there are three domains that is helical,
cytoplasmic and extracellular in FAT1 gene. The helical domain is very less contained than
cytoplasmic and extracellular topology in case of FAT1 cascade. The role of different domains is
different in fat1 protein. The helical domain of FAT1 describes the mutational studies discovered
in HNSCC which associated with research of two researchers and from the cancer genomics. The
therapeutic studies pave the way to the result of activation of HNSCC by Notch signaling
pathway. This domain also acts as a cadherin molecule to binds with the protein of fatty acid
binding protein, contains high chain longevity and high affinity. New data from in vitro and
whole animal research suggests that fatty acid transmembrane transport is mediated by proteins.
FABPs appear to have a role in the cell's extranuclear compartments by transporting their ligands
into the cytosol, where they interact with particular proteins. The extracellular domain plays
major role that to consider as cadherin gene superfamily. From the reviews we found the
function of a mammalian FAT cadherin. This domain, which overlaps with dynamic actin
structure, shoes the specific structures of FAT1 at filopodial tips, lamelipodial edges, and cell-
cell borders. Disorganization of cell junction related f- actin and f other actin fibers/cables,
disruption of cell-cell contacts, and suppression of cell polarity development at wound borders
were all seen when FAT1 was knocked down via RNA interference. Ena/vasodilator – activated
phosphor proteins were identified as a possible downstream effector of FAT1. This suggests that
FAT1 modulates cell contacts and polarity through regulating actin cytoskeletal structure at cell
peripheries. The current study emphasizes the importance of FAT1 and TNF-A in maintaining
cellular homeostasis and thus aligns with one of the objectives on United Nations formulated
Sustainable Development Goals (SDGs); SDG3 which ensures healthy lives and promotes well-
being for all at all stages. In this study, we focused on the structural and functional aspects of
various domains and motifs of FAT1, primarily involved in interaction with Tumor necrosis
factor-A (TNF-A).

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REVIEW OF LITERATURE
FAT family gene
FAT family gene consists of FAT1, FAT2, FAT3 and FAT4 found in case of human. It was
found in Drosophila in the 1920s as a result of a deadly mutation. FAT is a gene which is
orthologous of the Drosophila fat gene. It basically encodes the tumour suppressor, which is
essential for controlling cell proliferation during development. This gene is expressed at high
level, fetal to epithelia. The human FAT1 structure was cloned in 1955 from a human T-ALL (T-
CELL ACUTE LEUKEMIA), consists of 27 exons found from chromosome 4q34-35. FAT1 acts
as a biomarker of pancreatic cancer. The FAT cadherin has been described both as putative
tumour suppressor or oncogene in differently. TNF (often called Tumour Necrosis factor
ALPHA) is an adipokine and a cytokine . It may consist of various trans-membrane proteins
with a homologus TNF domain. TNF promotes insulin resistance, and is associated with obesity
induced, FAT2 is also known as atypical cadherin2. This gene is the second identified human
homolog of the Drosophila fat gene, which encodes a tumour suppressor essential for controlling
cell proliferation, during Drosophila development the gene product is a member of the cadherin
superfamily, a group of integral membrane protein characterized by the presence of cadherin
type repeats. In addition to consisting 34 tandem cadherin type repeats, the gene product has two
epidermal growth factor (EGF) like repeats and one laminin-G like domain. This protein most
likely functions as a cell adhesion molecule controlling cell proliferation and playing an
important role in cerebellum development. FAT3 is considered atypical cadherin3. Using
computer based motif trap screening, we have identified a third member of the mammalian fat
family, FAT3, Human and rat FAT3 are also similar to the Drosophila tumour suppressor gene.
Each member of the FAT family is differently expressed in the central nervous system during
development, while both FAT3 m RNA and FAT1 mRNA are abundantly expressed in the fetal
brain (Parsons et al., 2008).
The rat FAT3 gene encodes a large protein of 4555 amino acids with 34 cadherin domains, 4
epidermal growth factor (EGF)-like motifs, a laminin A-G motif, and a cytoplasmic domain.
Each member of the fat family is differentially expressed in the central nervous system during
development (Wang et al., 2014).

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FAT4 is also considered as atypical cadherin4, which is FAT tumour suppressor homologus4.
The FAT4 gene provides instruction for making a protein that is found in most tissues. The
protein spans the membrane surrounding cells so that part of the protein is outside the cell and
part of the protein is inside the cell. The precise function of the FAT4 protein is largely
unknown, however FAT4 protein involved in the components within cells (cell polarity).The
FAT4 protein also thought to function as a tumour suppressor , which is rapidly divided and in
an uncontrolled way (Jiang et al., 2012).
In case of FAT4 some genetically changes mutated genes causes disorders like Hennekam
syndrome. This syndrome causes about at least seven mutations in the FAT4 gene, inherited
disorder resulting from malformation of the lymphatic system. The FAT4 gene mutations that
cause Hennekam syndrome reduces the activity of the FAT4 protein, which seems to impair
normal development of the lymphatic system. It poorly affects the lymphatic vessels that are
abnormally expanded and are prone to break open, puffiness or swelling caused by a buildup of
fluid ( lymphodema).The mutation of FAT4 gene about causes 25% of Hennekam syndrome.
Also mutation of FAT4 gene causes van maldergem syndrome which decreases the function of
FAT4 protein. It also causes the disruption of brain which causes the cell polarity leads to
preriventricular heterotopia. Its symptoms unknown which may lead to the replaces the protein
building block (amino acid) giutamic acid with the amino acid lysine at position 2375 in the
FAT4 protein. FAT4 gene mutations have also been found in many types of cancers. That is
skin cancer called head and neck squamous cell carcinoma stomach cancer and pancreatic
cancer. The FAT4 gene mutations involved somatic mutations, which are found only in cells that
become cancerous and are not inheritated. It is likely prevents mutations that FAT4 protein
acting as a tumour suppressor which is the characteristics of uncontrolled growth and division of
cells (Funato et al., 2006; Scheller et al., 2008; Speer et al., 2009).
The inhibitory role of FAT1 has been widely demonstrated in various tumours. Its effect on the
ability of non-small cell lung cancer (NSCLC) to initiate tumours. Expression of FAT1 mRNA
predicted an increase in overall survival and initial progression-free survival in patients with lung
cancer, especially adenocarcinoma. FAT1 mRNA was found to have lower levels of lung cancer
tissue compared to normal tissue Organization. Functionally, we focused on the effect of FAT1

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on the ability of NSCLC cells to initiate tumours and found that over expression of FAT1
reduced the expression of tumour initiation markers. In addition, FAT1 over expression was
reduced. ALDH1 activity and spherical capacity of NSCLC cells. Mechanically, FAT1 has been
found to promote nuclear cytoplasmic transport of YAP1 (Yes associated protein 1), a key
performer of Hippo signalling, and mutant YAP1. YAP (YAP5SA), which can avoid LATS1 / 2-
mediated phosphorylation, rescued FAT1-mediated inhibition of tumour initiation of NSCLC
cells. This study shows that FAT1 suppresses the ability of NSCLC cells to initiate tumours by
activating hippo signalling (Kawamori et al., 2011; Mao et al., 2011).
Figure 1: Drosophila and vertebrate FAT cadherin members
FAT1 and the Hippo signaling pathway
Salvador-wts is the other name of Hippo Signaling pathway which is refers to an over growth of
the tissues. The growth of particular organ to specific size to be considered as the Hippo
pathway. It basically includes three primary components like upstream regulator, core kinase
cassettes and downstream transcriptional activators (Sadeqzadeh et al., 2014; Schwartz et al.,
2003). All the entire pathways should be governed by the process occurring at cellular level
including cell division and programmed cell death ( or apoptosis). This pathway plays many role
in case of study about human cancer which results the vital role in steamcell and tissue specific
progenitor cell for self renewal and expression. Using mosaic genetic screens, the Hippo

10
pathway is identified in Drosophila. It is also helped to identify many genes that function as
oncogenes or tumour suppressors in mammals. The Hippo pathway is conducted by a core kinase
cassette in which it requires a protein kinase, Wts in case of Drosophila, and Mst1/2 which is the
protein kinase in case of mammal (Königshoff et al., 2009).
In this cellular process the highly conserved groups are considered as serine/thrine kinase,
includes cell proliferation, apoptosis and various stress responses. The kinases are known
regulators of cell cycle progression growth and development.. the invariant proline which is
highly concerned two proteins known to facilitate the activation of Wts Salvador and Mob as
tumour suppressor.Hippo can also phosphorylate and activate Mats to associate with and
strengthen the kinase activity of Wts (Dulak et al., 2013; Lafaille et al., 2012).
Another activators like transcriptional coactivator which considered as Yorkie (Yki). Yki is
unable to bind itself. It mostly binds to transcriptional factors like scalloped(sd) and Yki-sd
complex located at nucleus. cyclinE which is the organ growth promoter allows several gene
expression. In Drosophila the specific protein prevents apoptosis such as diap1(Sadeqzadeh et
al., 2011; Valletta et al., 2014). Yki is also associated with to affect the cell number expresses in
Bantam microRNA. In case of mammal two Yki othologs to YAP (yes associated protein and
transcriptional coactivator with PDZ binding motif such as WWTRI known as TAZ. TAZ and
YAP included with TEADS as transcriptional factor (M. Katoh, 2005; Naishiro et al., 2005).
The transmembrane protein FAT and several membrane associated protein with upstream
regulators of the Hippo/Wts cascade. From the previous reviews we know that FAT act as a
atypical Cadherin protein, associated with the GPI- anchored cell surface protein Glypican-3
(GPC3) (known to interacts with human liver cancer). GPC3 is also shown to modulate YAP
signaling in liver cancer. For whole growth of nuclear tissues the FAT binds to the activator
protein such as Dachsous(DS) for patterning.(Guo et al., 2013; Martin et al., 2014; Nishioka et
al., 2009)
Another important protein complex that is KibraEX-Mer (KEM) apically located involved in
Hippo Signaling pathway.FAT may also regulates Wts independently of Ex/HPO, through the

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inhibition of the unconventional myosin Dachs. Normally it binds to and promotes disregulation
of Wts. The heart is the first organ formed during mammalian development. A properly sized
and functional heart is vital throughout the entire lifespan. Loss of cardiomyocytes because of
injury or diseases leads to heart failure, which is a major cause of human morbidity and
mortality. Unfortunately, regenerative potential of the adult heart is limited (Lahrouchi, n.d.;
Tran et al., 2004) The Hippo pathway is a recently identified signaling cascade that plays an
evolutionarily conserved role in organ size control by inhibiting cell proliferation, promoting
apoptosis, regulating fates of stem/progenitor cells, and in some circumstances, limiting cell size
(Pastushenko et al., 2021; Yaoita et al., 2005). Research indicates a key role of this pathway in
regulation of cardiomyocyte proliferation and heart size. Inactivation of the Hippo pathway or
activation of its downstream effector, the Yes-associated protein transcription coactivator,
improves cardiac regeneration. Several known upstream signals of the Hippo pathway such as
mechanical stress, G-protein-coupled receptor signaling, and oxidative stress are known to play
critical roles in cardiac physiology (Inoue et al., 2001a; Lorenza et al., 2003; Uglow et al., 2003).
In addition, Yes-associated protein has been shown to regulate cardiomyocyte fate through
multiple transcriptional mechanisms (Cappello et al., 2013; Moeller et al., 2004; Saburi et al.,
2012)
Figure 2: Association between FAT1 and Hippo signaling pathway

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FAT1 and the Wnt/beta-catenin signaling pathway
The Wnt signalling system is in charge of human development and tissue homeostasis in adults.
Many disorders, including osteoporosis, Alzheimer's disease, and pigmented diseases, are linked
to Wnt signalling abnormalities (Morris et al., 2013). In a number of malignancies, including
HCC and colon cancer, abnormal activation of the Wnt/beta-catenin signalling pathway is
hypothesised to promote carcinogenesis and cancer cell proliferation (Dunne et al., 1995; Huang
et al., 2019; Y. Katoh & Katoh, 2006). “The Wnt signal/Beta-catenin pathway consists of three
steps: Wnt signal transduction at the membrane, stable control of beta-catenin in the cytoplasm,
and activation of Wnt target genes in the nucleus”(Peng et al., 2021). However, FAT1 protein
may influence Wnt signalling by modulating the last two stages. Classical cadherin, like beta-
catenin, may bind to it and influence its transcriptional activity. FAT1 can also bind to beta-
catenin, preventing it from translocating to the nucleus (Oh & Irvine, 2008). This connects FAT1
to the standard Wnt signalling pathway.
Beta-catenin in human cells bounded to endogenous FAT1 (revealed by Morris et al). FAT1
might express inactively and not to lines beta-catenin at the cell membrane, permitting canonical
Wnt signal transmission and tumour development. Overexpression of beta-catenin enhanced cell
proliferation, cell cycle progression, and BrdU incorporation (a sign of increased proliferation),
all of which were suppressed by FAT1 (Lahrouchi, n.d.). CTNNB1 knockout regulates FAT1
knockdown (encoding Beta-catenin). Cell growth, cell cycle progression, and BrdU
incorporation are all inhibited by it. FAT1 knock-down regulates the Wnt/beta-catenin signalling
pathway. “Myc, cyclin D1, an inhibitor of DNA binding 2, transcription factor 4, claudin, and
zinc finger E BOX binding homeobox 1 are among the Wnt target genes that are upregulated
(ZEB1)” (Bryant et al., 1988; De Bock et al., 2012; Mariot et al., 2015).

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Figure 3: Overview of Wnt/β-catenin signaling
FAT1 and EMT
EMT is a cellular biological process that encourages cancer cells to migrate. In addition,
stemness, invasion, and medication resistance are all characteristics of EMT (Beyer et al., 2013;
Blair et al., 2006). The link between FAT1 and EMT has been shown in various cancer forms,
including ESCC (Beyer et al., 2013), HCC (Bennett & Harvey, 2006) and glioblastoma (Bryant
et al., 1988). “After reducing FAT1 expression, the epithelial marker E cadherin expression
reduced dramatically, but N cadherin, vimentin, and the stromal marker Snail expression rose,
resulting in enhanced cell proliferation, suggesting that FAT1 may be a crucial regulator of
EMT” (Beyer et al., 2013). FAT1 regulates the co-expression of epithelial and mesenchymal
transcription programmes in cancer cells, resulting in the emergence of a hybrid EMT
phenotype, by regulating SOX2 expression levels by affecting the expression levels of EZH2 and
the CaMK2 CD44 SRC YAP ZEB1 axis, and by regulating SOX2 expression levels by affecting
the expression levels of EZH2 and the CaMK2 CD44 SRC (Inoue et al., 2001b; Meng et al., 123
C.E.; Srivastava et al., 2018).

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Figure 4: Association between FAT1 and EMT
Role of the FAT family in human development and diseases
FAT1 is a member of the fat family gene that plays an important role in humans, causing several
genetic disorders. According to an earlier research, the 4q-syndrome is caused by a homozygous
frame-shift mutation in FAT1, which is characterised with or without nephropathy, facial
malformations, cutaneous syndactyly, and ocular anomalies (Nusse & Clevers, 2017). These
illnesses are related to neural tube closure abnormalities, in which the absence of FAT1 function
inhibits epithelial cell adhesion and fusion. FAT1 demonstrates functional deficits in some
circumstances, resulting in decreased epithelial cell adhesion and the elimination of podocytes. It
is mostly responsible for glomerulonephritis (Bryant et al., 1988; Nusse & Clevers, 2017), a kind
of kidney disease. FAT1 has been identified as a BPAD susceptibility gene. Binding with EVH1
and Beta-catenin in the FAT1 region revealed single nucleotide polymorphisms (SNPs). The
human T-BALL cell line was used to clone FAT1. It's a form of tumour that develops in the
haematological system (Bennett & Harvey, 2006). FAT1 is found in 60% of T-ALL cases, 30%
of precursor B-CELL ALL cases, and 10% of acute myeloid leukaemia cases. As a result,
elevated FAT1 mRNA expression is linked to a poor prognosis and a high recurrence rate in T-
ALL patients. FAT1 protein, which is derived from FAT family members, is overexpressed in
human hematopoietic malignancies (Beyer et al., 2013). Changes in post-translational processing

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are linked to FAT1 overexpression in T-ALL (Bennett & Harvey, 2006), resulting in mainly
staining. FAT1 mRNA levels are much higher in ductal carcinoma in situ than in invasive breast
cancer, and knocking it out has been shown to hasten the development of ductal carcinoma in
situ to invasive breast cancer (He et al., 1998). Non-synonymous mutations in the human FAT1,
FAT2, and FAT4 genes were found in one and two out of 24 pancreatic cancer samples,
respectively, using the whole-exome sequencing method. Human FAT2 and FAT4 gene non-
synonymous mutations were found in 1 and 2 instances of head and neck squamous cell
carcinoma, respectively. In transgenic mice expressing wild type Raf1 transgene under the
direction of the human sp-c (surfactant promotes protein c) promoter, the mouse FAT3 is
dramatically down regulated in lung cancer (Bryant et al., 1988; Schwartz et al., 2003; Valletta et
al., 2014). “The absence of a cell-specific target is the mechanism of non-viral gene therapy.
Using microinjection and in situ hybridization, we tested various potential DNA sequences for
their ability to promote plasmid nuclear import in alveolar type-2 epithelial (AT2) cells. Of these,
only a region inside the human sp-promotor was able to induce nuclear localization of plasmid
DNA. Intratracheal DNA transfer leads to electroporation, according to earlier study”(Peng et al.,
2021). The sp-c promoter, and hence AT2 cell-specific nuclear import of DNA, might be a safe
and effective way to produce alveolar type 2 cell-specific genes. Three of the six breast cancer
cell lines had human FAT4 mRNA expression (Hu et al., 2017; Saxena et al., 2020; Staley &
Irvine, 2012). The whole-exome sequencing technique detects a non-synonymous FAT4
mutation in one out of every ten instances of hepatocellular cancer. “The FAT4 gene, which
belongs to the FAT gene family, is often altered in human malignancies, including melanoma
(40%), pancreatic cancer (8%), HNSCC (6%), and gastric cancer (5%)” (Blair et al., 2006).
Functional domains of FAT1
From the previous reviews found that there are three domains that is helical, cytoplasmic and
extracellular in FAT1 gene (Schreiner et al., 2006; Wrighton et al., 2008). The helical domain is
very less contained than cytoplasmic and extracellular topology in case of FAT1 cascade. The
role of different domains be different in fat1 protein. The helical domain of FAT1 describes the
mutational studies discovered in HNSCC which associated with research of two researchers and
from the cancer genomics (Pan, 2010; Perugorria et al., 2019). The therapeutic studies pave the
way to the result of activation of HNSCC by Notch signaling pathway. This domain also acts as

16
a cadherin molecule to binds with the protein of fatty acid binding protein, contains high chain
longevity and high affinity (Britt et al., 2020; Shariff et al., 2009). New data from in vitro and
whole animal research suggests that fatty acid transmembrane transport is mediated by proteins.
FABPs appear to have a role in the cell's extranuclear compartments by transporting their ligands
into the cytosol, where they interact with particular proteins. The extracellular domain plays
major role that to consider as cadherin gene superfamily (Mariot et al., 2015; Morin et al., 2016).
From the reviews we found the function of a mammalian FAT cadherin. This domain, which
overlaps with dynamic actin structure, shoes the specific structures of FAT1 at filopodial tips,
lamelipodial edges, and cell-cell borders (Bray et al., 2018; Puppo et al., 2015) Disorganization
of cell junction related f- actin and f other actin fibers/cables, disruption of cell-cell contacts, and
suppression of cell polarity development at wound borders were all seen when FAT1 was
knocked down via RNA interference. Ena/vasodilator – activated phosphor proteins were
identified as a possible downstream effector of FAT1. This suggests that FAT1 modulates cell
contacts and polarity through regulating actin cytoskeletal structure at cell peripheries (Agrawal
et al., 2012; Peng et al., 2021; Song et al., 2014).

18
METHODOLOY
Determination of protein sequences and putative protein partners
Freely accessible database of protein sequence and functional information is found from uniport.
It contains a large amount of information about the biological function of proteins derived from
the research literature.
Uniport was conducted by several European bioinformatics organizations and a foundation
from Washington,DC, United states. It is also a database of comprises many other databases such
as uniport knowledgebase (UniportKB), the Uniport reference clusters (UniRef) and the Uniport
Archive (UniParc). The Uniport consortium collaborated with the European Bioinformatics
Institute (EBI), the Swiss Institute of Bioinformatics (SIB) and the Protein Information
Resources (PIR). EBI developed large resources of bioinformatics databases and services. SIB is
the founding centre of the swissport group and maintains the EXPASY (Exoert Protein Analysis
System) server a central resource for proteomics databases and tools. Likewise PIR is the oldest
sequence which to be considered as analysis and sequences the structure of protein. Also
classifies the protein sequences in this tool.
Uniport is to provide the scientific community with a comprehensive, high quality and freely
assesible resource of protein sequence and functional information.
The Uniport knowledge base is to be used to pointed particular role to access for extensive
curated protein information, including function, classification and cross reference. UniportKB
comprises two sections such as UniportKB/swissport and UniportKB/TrEMB. In
UniportKB/swissport manually annotated 3D model and is reviewed. In UniportKB/TrEMBL
automatically annotated and not to reviewed.
The Uniport Reference clusters (UniRef) databases provide clustered sets of sequences from the
UniportKB and selected Uniport Archive records to obtain complete coverage of sequence space
at several resolutions while hiding redundant sequences.
The Uniport Archieve (UniParc) is a comprehensive repository used to keep track of sequences
and their identifiers. We use the server https://www.uniport.org/ for opening the Uniport.

19
Then we find the Uniport interface comprises of many datas. By using particular gene name in
the search box the interface shows gene sequence analysis, FASTA sequences molecular and
biological functions, taxonomy, domain organization, string structure etc.
It is the database finds out from uniport shows that how one protein interacts with more than one
protein. It is the functional protein association networks which also open in NCBI by the server
https://string.db.org/. Basically shows crosslinking interaction of particular protein with other
proteins by specific functions which to indicate better analysis of datas about a required protein.
Determination of three dimensional configurations of proteins
Swissport is the central hub for the collection of functional information on protein, which
annotated protein sequences and maintained by the Swiss Institute of Bioinformatics (SIB),
Swithzerland and the European Bioinformatics Institute (EBI). Swissport communicated with
other databases by integratin of their redutance level i.e minimal to high range which shown the
post-translational modification, domain structure, variants etc. in different curated protein
sequence databases. Recently the development shows in case of protein databases, variety of new
documentation files and improvement to TrEMB, a computer annotated supplement to swissport.
TrEMBL contains the entire swissport format derived from the translation of all coding
sequences (CDS) in the EMBL nucleotide sequences database. The swissport is annotated by the
server to https://www.expasy.chi/sprot and https://www.ebi.ac.uk/sport. Basically the annotated
data of swissport be distinguished by different core data, taxonomic data, citation information for
each sequence .the described annotation of data have different function of proteins ,similarities
to other protein, diseases associated with deficienciesin the protein, sequence conflict variants,
different post translational modification for example carbohydrates, phosphorylation, acetylation,
GPI anchor etc. It also shows annoted in case of domain and sites as well as secondary and
quarternary structures also. From the recent development it is found that different model
organisms to provide for annotation. Those are Bacillus subtilis, Drosophila melanogaster,
Escherichia coli, Saccaromyces cereviasiae, Mus musculus, Homo sapiens, Haemophilus
influenza etc.
Collectively the organisms represent ~ 40% of the total number of sequence entries in Swissport.
We currently attempting to finish the integration in to swissprot of all the predicted proteins from

20
E coli., B subtilis and yeast. Swissport is overally used to increased data flow from genome
preojects to the sequence databases. We face a number of challenges to our way of database
annotation.
Swiss model is the part of swissprotKB is used to structural bioinfromatics web server dedicated
to homology modeling of 3D protein structure. Homology modeling is currently the most
accurate methods to generate reliable 3D protein models and is routinely used in many practical
applications. Homology modeling method makes use of experimental protein templates to build
models for evolutionary related targets. It assess in the web server https://swissmodel.expasy.org/
This swissmodel build homology model of a given protein using following steps:
 Identification of structural templates BLAST and HHbits are used to identify templates.
The templates are stored in the swissmodel library which derived from PDB.
 Alignment of target sequence and template structure.
 Model building and energy minimization. Swissmodel implements a rigid fragment
assembly approach for modeling.
 The assessment of the models quality using QMEAN astastical potential of mean force
and sequence identity score.
 The model repository provides access to an date collection of annotated three-
dimensional protein models for a set of model organisms of high general imterest.
New developments of the SWISS-MODEL expert system features that automated modeling of
homo-oligomeric assemblies , modeling of essential metal ions and biologically relevant ligands
in protein structure, local model reliability estimates based on the QMEAN local score function,
mapping of uniport feature to models.

21
Determination of three dimensional configurations of proteins
Pymol is an open source but proprietary molecular visualization system created by Warren
Lyford DeLano. The private software company by DeLano scientific LLC dedicated to creating
useful tools that become universally accessible to scientific and educational communities.
Currently it is commercialized by Schrodinger,inc.as original software license was a permissive
license they were able to remove it, new versions are no longer released under the python
license, but under a custom license and some of the source code is no longer to released. Pymol
can produce high quality 3D images of small molecules and biological macromolecules such as
proteins. According to original author by 2009, almost a quarter of all published images of 3D
protein structures in the scientific literature were made using pymol. It is one of the few mostly
open source model visualization tools available for use in structural biology. It uses openGL
Extension wrangler library (GLEW) and free GLUT and can solve poission Boltzmann equation
using adaptive poisson Boltzmann solver. Anyone can either compile an executable from the
python licensed source code or pay for a subscription to support service to obtain access to
precompile executable.
It has seems that the 3D model as well as docking models. As it is the visualization tool, here it
changes colour as well as size for user’s requirement. The user most use the different ligands and
to delete it as for their requirement. The image be download by notepad model version on the file
menu. Then from the display menu have change the background as white and export the image
as .png from file menu.
Protein-protein interactions
Protein-protein and protein-DNA/RNAinteraction play a fundamental role in a variety of
biological processes. Determining the complex structures of these interactions is valuable, in
which molecular docking has played a important role. HDOCK is the novel web server of our
hybrid docking algorithm of template based modeling and free docking in rescued by the free
docking protocol. The web server is used to retrieve the data such that https://hdock.
phys.hust.edu.in/.

22
Docking play important role in use of therapeustic interventions or drugs targeting. As to prepare
a highly complex drugs. Other complexes like protein-DNA, protein-RNA be used in the
molecular process it may quite easy to found from the docking. But its high cost and technical
difficulties in experimental method, molecular docking which compatainally predicts the
complex from individual structures play important role in the determination of complex
structures. The similar work flow used for CAPRI also ued in case of docking. Compairing the
current docking server, our server accepts not only structures but also sequences as input for
protein and can automatically integrate the binding information from the PDB. HDOCK plays
vital role for the both protein- protein and protein-DNA/ RNA docking which docking score with
their models be result to user by a web page and through an Email notification if provide a valid
Email id.
Protein sequence homology of FATs
Multiple sequence alignment may refer to the process or the result of sequence alignment of
three or more biological sequences, generally protein, RNA or DNA. In many cases , the input
set of query sequences are assumed to have an evolutionary relationship by which they share a
linkage and are descened from a common anscestor. For multiple sequence alignment we use
multalign a tool for protein and nucleic acid sequence created by Florence concept.
Figure 5: Methodology infography

24
RESULTS AND DISCUSSION
Amino acid sequence homology and protein domain organization of FATs
The amino acid sequences of FATs were retrived from uniprot. The total length of amino acid
residues in FAT1 was found to be 4588 (figure 6(a)). The length of amino acid residues in FAT2
was found to be 4349 (figure 6(b)). In FAT3 the length of amino acid residues was found to be
4122 (figure 6(c)) , whereas in FAT4 it was found to be 4466( figure 6(d)).
MGRHLALLLLLLLLFQHFGDSDGSQRLEQTPLQFTHLEYNVTVQENSAAKTYVGHPVKMG
VYITHPAWEVRYKIVSGDSENLFKAEEYILGDFCFLRIRTKGGNTAILNREVKDHYTLIV
KALEKNTNVEARTKVRVQVLDTNDLRPLFSPTSYSVSLPENTAIRTSIARVSATDADIGT
NGEFYYSFKDRTDMFAIHPTSGVIVLTGRLDYLETKLYEMEILAADRGMKLYGSSGISSM
AKLTVHIEQANECAPVITAVTLSPSELDRDPAYAIVTVDDCDQGANGDIASLSIVAGDLL
QQFRTVRSFPGSKEYKVKAIGGIDWDSHPFGYNLTLQAKDKGTPPQFSSVKVIHVTSPQF
KAGPVKFEKDVYRAEISEFAPPNTPVVMVKAIPAYSHLRYVFKSTPGKAKFSLNYNTGLI
SILEPVKRQQAAHFELEVTTSDRKASTKVLVKVLGANSNPPEFTQTAYKAAFDENVPIGT
TVMSLSAVDPDEGENGYVTYSIANLNHVPFAIDHFTGAVSTSENLDYELMPRVYTLRIRA
SDWGLPYRREVEVLATITLNNLNDNTPLFEKINCEGTIPRDLGVGEQITTVSAIDADELQ
LVQYQIEAGNELDFFSLNPNSGVLSLKRSLMDGLGAKVSFHSLRITATDGENFATPLYIN
ITVAASHKLVNLQCEETGVAKMLAEKLLQANKLHNQGEVEDIFFDSHSVNAHIPQFRSTL
PTGIQVKENQPVGSSVIFMNSTDLDTGFNGKLVYAVSGGNEDSCFMIDMETGMLKILSPL
DRETTDKYTLNITVYDLGIPQKAAWRLLHVVVVDANDNPPEFLQESYFVEVSEDKEVHSE
IIQVEATDKDLGPNGHVTYSIVTDTDTFSIDSVTGVVNIARPLDRELQHEHSLKIEARDQ
AREEPQLFSTVVVKVSLEDVNDNPPTFIPPNYRVKVREDLPEGTVIMWLEAHDPDLGQSG
QVRYSLLDHGEGNFDVDKLSGAVRIVQQLDFEKKQVYNLTVRAKDKGKPVSLSSTCYVEV
EVVDVNENLHPPVFSSFVEKGTVKEDAPVGSLVMTVSAHDEDARRDGEIRYSIRDGSGVG
VFKIGEETGVIETSDRLDRESTSHYWLTVFATDQGVVPLSSFIEIYIEVEDVNDNAPQTS
EPVYYPEIMENSPKDVSVVQIEAFDPDSSSNDKLMYKITSGNPQGFFSIHPKTGLITTTS
RKLDREQQDEHILEVTVTDNGSPPKSTIARVIVKILDENDNKPQFLQKFYKIRLPEREKP
DRERNARREPLYHVIATDKDEGPNAEISYSIEDGNEHGKFFIEPKTGVVSSKRFSAAGEY
DILSIKAVDNGRPQKSSTTRLHIEWISKPKPSLEPISFEESFFTFTVMESDPVAHMIGVI
SVEPPGIPLWFDITGGNYDSHFDVDKGTGTIIVAKPLDAEQKSNYNLTVEATDGTTTILT
QVFIKVIDTNDHRPQFSTSKYEVVIPEDTAPETEILQISAVDQDEKNKLIYTLQSSRDPL
SLKKFRLDPATGSLYTSEKLDHEAVHQHTLTVMVRDQDVPVKRNFARIVVNVSDTNDHAP
WFTASSYKGRVYESAAVGSVVLQVTALDKDKGKNAEVLYSIESGNIGNSFMIDPVLGSIK
TAKELDRSNQAEYDLMVKATDKGSPPMSEITSVRIFVTIADNASPKFTSKEYSVELSETV

25
SIGSFVGMVTAHSQSSVVYEIKDGNTGDAFDINPHSGTIITQKALDFETLPIYTLIIQGT
NMAGLSTNTTVLVHLQDENDNAPVFMQAEYTGLISESASINSVVLTDRNVPLVIRAADAD
KDSNALLVYHIVEPSVHTYFAIDSSTGAIHTVLSLDYEETSIFHFTVQVHDMGTPRLFAE
YAANVTVHVIDINDCPPVFAKPLYEASLLLPTYKGVKVITVNATDADSSAFSQLIYSITE
GNIGEKFSMDYKTGALTVQNTTQLRSRYELTVRASDGRFAGLTSVKINVKESKESHLKFT
QDVYSAVVKENSTEAETLAVITAIGNPINEPLFYHILNPDRRFKISRTSGVLSTTGTPFD
REQQEAFDVVVEVTEEHKPSAVAHVVVKVIVEDQNDNAPVFVNLPYYAVVKVDTEVGHVI
RYVTAVDRDSGRNGEVHYYLKEHHEHFQIGPLGEISLKKQFELDTLNKEYLVTVVAKDGG
NPAFSAEVIVPITVMNKAMPVFEKPFYSAEIAESIQVHSPVVHVQANSPEGLKVFYSITD
GDPFSQFTINFNTGVINVIAPLDFEAHPAYKLSIRATDSLTGAHAEVFVDIIVDDINDNP
PVFAQQSYAVTLSEASVIGTSVVQVRATDSDSEPNRGISYQMFGNHSKSHDHFHVDSSTG
LISLLRTLDYEQSRQHTIFVRAVDGGMPTLSSDVIVTVDVTDLNDNPPLFEQQIYEARIS
EHAPHGHFVTCVKAYDADSSDIDKLQYSILSGNDHKHFVIDSATGIITLSNLHRHALKPF
YSLNLSVSDGVFRSSTQVHVTVIGGNLHSPAFLQNEYEVELAENAPLHTLVMEVKTTDGD
SGIYGHVTYHIVNDFAKDRFYINERGQIFTLEKLDRETPAEKVISVRLMAKDAGGKVAFC
TVNVILTDDNDNAPQFRATKYEVNIGSSAAKGTSVVKVLASDADEGSNADITYAIEADSE
SVKENLEINKLSGVITTKESLIGLENEFFTFFVRAVDNGSPSKESVVLVYVKILPPEMQL
PKFSEPFYTFTVSEDVPIGTEIDLIRAEHSGTVLYSLVKGNTPESNRDESFVIDRQSGRL
KLEKSLDHETTKWYQFSILARCTQDDHEMVASVDVSIQVKDANDNSPVFESSPYEAFIVE
NLPGGSRVIQIRASDADSGTNGQVMYSLDQSQSVEVIESFAINMETGWITTLKELDHEKR
DNYQIKVVASDHGEKIQLSSTAIVDVTVTDVNDSPPRFTAEIYKGTVSEDDPQGGVIAIL
STTDADSEEINRQVTYFITGGDPLGQFAVETIQNEWKVYVKKPLDREKRDNYLLTITATD
GTFSSKAIVEVKVLDANDNSPVCEKTLYSDTIPEDVLPGKLIMQISATDADIRSNAEITY
TLLGSGAEKFKLNPDTGELKTSTPLDREEQAVYHLLVRATDGGGRFCQASIVLTLEDVND
NAPEFSADPYAITVFENTEPGTLLTRVQATDADAGLNRKILYSLIDSADGQFSINELSGI
IQLEKPLDRELQAVYTLSLKAVDQGLPRRLTATGTVIVSVLDINDNPPVFEYREYGATVS
EDILVGTEVLQVYAASRDIEANAEITYSIISGNEHGKFSIDSKTGAVFIIENLDYESSHE
YYLTVEATDGGTPSLSDVATVNVNVTDINDNTPVFSQDTYTTVISEDAVLEQSVITVMAD
DADGPSNSHIHYSIIDGNQGSSFTIDPVRGEVKVTKLLDRETISGYTLTVQASDNGSPPR
VNTTTVNIDVSDVNDNAPVFSRGNYSVIIQENKPVGFSVLQLVVTDEDSSHNGPPFFFTI
VTGNDEKAFEVNPQGVLLTSSAIKRKEKDHYLLQVKVADNGKPQLSSLTYIDIRVIEESI
YPPAILPLEIFITSSGEEYSGGVIGKIHATDQDVYDTLTYSLDPQMDNLFSVSSTGGKLI
AHKKLDIGQYLLNVSVTDGKFTTVADITVHIRQVTQEMLNHTIAIRFANLTPEEFVGDYW
RNFQRALRNILGVRRNDIQIVSLQSSEPHPHLDVLLFVEKPGSAQISTKQLLHKINSSVT
DIEEIIGVRILNVFQKLCAGLDCPWKFCDEKVSVDESVMSTHSTARLSFVTPRHHRAAVC
LCKEGRCPPVHHGCEDDPCPEGSECVSDPWEEKHTCVCPSGRFGQCPGSSSMTLTGNSYV
KYRLTENENKLEMKLTMRLRTYSTHAVVMYARGTDYSILEIHHGRLQYKFDCGSGPGIVS
VQSIQVNDGQWHAVALEVNGNYARLVLDQVHTASGTAPGTLKTLNLDNYVFFGGHIRQQG

26
TRHGRSPQVGNGFRGCMDSIYLNGQELPLNSKPRSYAHIEESVDVSPGCFLTATEDCASN
PCQNGGVCNPSPAGGYYCKCSALYIGTHCEISVNPCSSKPCLYGGTCVVDNGGFVCQCRG
LYTGQRCQLSPYCKDEPCKNGGTCFDSLDGAVCQCDSGFRGERCQSDIDECSGNPCLHGA
LCENTHGSYHCNCSHEYRGRHCEDAAPNQYVSTPWNIGLAEGIGIVVFVAGIFLLVVVFV
LCRKMISRKKKHQAEPKDKHLGPATAFLQRPYFDSKLNKNIYSDIPPQVPVRPISYTPSI
PSDSRNNLDRNSFEGSAIPEHPEFSTFNPESVHGHRKAVAVCSVAPNLPPPPPSNSPSDS
DSIQKPSWDFDYDTKVVDLDPCLSKKPLEEKPSQPYSARESLSEVQSLSSFQSESCDDNG
YHWDTSDWMPSVPLPDIQEFPNYEVIDEQTPLYSADPNAIDTDYYPGGYDIESDFPPPPE
DFPAADELPPLPPEFSNQFESIHPPRDMPAAGSLGSSSRNRQRFNLNQYLPNFYPLDMSE
Figure 6: (a) Amino acid sequence of FAT1
MTIALLGFAIFLLHCATCEKPLEGILSSSAWHFTHSHYNATIYENSSPKTYVESFEKMGI
YLAEPQWAVRYRIISGDVANVFKTEEYVVGNFCFLRIRTKSSNTALLNREVRDSYTLIIQ
ATEKTLELEALTRVVVHILDQNDLKPLFSPPSYRVTISEDMPLKSPICKVTATDADLGQN
AEFYYAFNTRSEMFAIHPTSGVVTVAGKLNVTWRGKHELQVLAVDRMRKISEGNGFGSLA
ALVVHVEPALRKPPAIASVVVTPPDSNDGTTYATVLVDANSSGAEVESVEVVGGDPGKHF
KAIKSYARSNEFSLVSVKDINWMEYLHGFNLSLQARSGSGPYFYSQIRGFHLPPSKLSSL
KFEKAVYRVQLSEFSPPGSRVVMVRVTPAFPNLQYVLKPSSENVGFKLNARTGLITTTKL
MDFHDRAHYQLHIRTSPGQASTVVVIDIVDCNNHAPLFNRSSYDGTLDENIPPGTSVLAV
TATDRDHGENGYVTYSIAGPKALPFSIDPYLGIISTSKPMDYELMKRIYTFRVRASDWGS
PFRREKEVSIFLQLRNLNDNQPMFEEVNCTGSIRQDWPVGKSIMTMSAIDVDELQNLKYE
IVSGNELEYFDLNHFSGVISLKRPFINLTAGQPTSYSLKITASDGKNYASPTTLNITVVK
DPHFEVPVTCDKTGVLTQFTKTILHFIGLQNQESSDEEFTSLSTYQINHYTPQFEDHFPQ
SIDVLESVPINTPLARLAATDPDAGFNGKLVYVIADGNEEGCFDIELETGLLTVAAPLDY
EATNFYILNVTVYDLGTPQKSSWKLLTVNVKDWNDNAPRFPPGGYQLTISEDTEVGTTIA
ELTTKDADSEDNGRVRYTLLSPTEKFSLHPLTGELVVTGHLDRESEPRYILKVEARDQPS
KGHQLFSVTDLIITLEDVNDNSPQCITEHNRLKVPEDLPPGTVLTFLDASDPDLGPAGEV
RYVLMDGAHGTFRVDLMTGALILERELDFERRAGYNLSLWASDGGRPLARRTLCHVEVIV
LDVNENLHPPHFASFVHQGQVQENSPSGTQVIVVAAQDDDSGLDGELQYFLRAGTGLAAF
SINQDTGMIQTLAPLDREFASYYWLTVLAVDRGSVPLSSVTEVYIEVTDANDNPPQMSQA
VFYPSIQEDAPVGTSVLQLDAWDPDSSSKGKLTFNITSGNYMGFFMIHPVTGLLSTAQQL
DRENKDEHILEVTVLDNGEPSLKSTSRVVVGILDVNDNPPIFSHKLFNVRLPERLSPVSP
GPVYRLVASDLDEGLNGRVTYSIEDSDEEAFSIDLVTGVVSSSSTFTAGEYNILTIKATD
SGQPPLSASVRLHIEWIPWPRPSSIPLAFDETYYSFTVMETDPVNHMVGVISVEGRPGLF
WFNISGGDKDMDFDIEKTTGSIVIARPLDTRRRSNYNLTVEVTDGSRTIATQVHIFMIAN
INHHRPQFLETRYEVRVPQDTVPGVELLRVQAIDQDKGKSLIYTIHGSQDPGSASLFQLD
PSSGVLVTVGKLDLGSGPSQHTLTVMVRDQEIPIKRNFVWVTIHVEDGNLHPPRFTQLHY

27
EASVPDTIAPGTELLQVRAMDADRGVNAEVHYSLLKGNSEGFFNINALLGIITLAQKLDQ
ANHAPHTLTVKAEDQGSPQWHDLATVIIHVYPSDRSAPIFSKSEYFVEIPESIPVGSPIL
LVSAMSPSEVTYELREGNKDGVFSMNSYSGLISTQKKLDHEKISSYQLKIRGSNMAGAFT
DVMVVVDIIDENDNAPMFLKSTFVGQISEAAPLYSMIMDKNNNPFVIHASDSDKEANSLL
VYKILEPEALKFFKIDPSMGTLTIVSEMDYESMPSFQFCVYVHDQGSPVLFAPRPAQVII
HVRDVNDSPPRFSEQIYEVAIVGPIHPGMELLMVRASDEDSEVNYSIKTGNADEAVTIHP
VTGSISVLNPAFLGLSRKLTIRASDGLYQDTALVKISLTQVLDKSLQFDQDVYWAAVKEN
LQDRKALVILGAQGNHLNDTLSYFLLNGTDMFHMVQSAGVLQTRGVAFDREQQDTHELAV
EVRDNRTPQRVAQGLVRVSIEDVNDNPPKFKHLPYYTIIQDGTEPGDVLFQVSATDEDLG
TNGAVTYEFAEDYTYFRIDPYLGDISLKKPFDYQALNKYHLKVIARDGGTPSLQSEEEVL
VTVRNKSNPLFQSPYYKVRVPENITLYTPILHTQARSPEGLRLIYNIVEEEPLMLFTTDF
KTGVLTVTGPLDYESKTKHVFTVRATDTALGSFSEATVEVLVEDVNDNPPTFSQLVYTTS
ISEGLPAQTPVIQLLASDQDSGRNRDVSYQIVEDGSDVSKFFQINGSTGEMSTVQELDYE
AQQHFHVKVRAMDKGDPPLTGETLVVVNVSDINDNPPEFRQPQYEANVSELATCGHLVLK
VQAIDPDSRDTSRLEYLILSGNQDRHFFINSSSGIISMFNLCKKHLDSSYNLRVGASDGV
FRATVPVYINTTNANKYSPEFQQHLYEAELAENAMVGTKVIDLLAIDKDSGPYGTIDYTI
INKLASEKFSINPNGQIATLQKLDRENSTERVIAIKVMARDGGGRVAFCTVKIILTDEND
NPPQFKASEYTVSIQSNVSKDSPVIQVLAYDADEGQNADVTYSVNPEDLVKDVIEINPVT
GVVKVKDSLVGLENQTLDFFIKAQDGGPPHWNSLVPVRLQVVPKKVSLPKFSEPLYTFSA
PEDLPEGSEIGIVKAVAAQDPVIYSLVRGTTPESNKDGVFSLDPDTGVIKVRKPMDHEST
KLYQIDVMAHCLQNTDVVSLVSVNIQVGDVNDNRPVFEADPYKAVLTENMPVGTSVIQVT
AIDKDTGRDGQVSYRLSADPGSNVHELFAIDSESGWITTLQELDCETCQTYHFHVVAYDH
GQTIQLSSQALVQVSITDENDNAPRFASEEYRGSVVENSEPGELVATLKTLDADISEQNR
QVTCYITEGDPLGQFGISQVGDEWRISSRKTLDREHTAKYLLRVTASDGKFQASVTVEIF
VLDVNDNSPQCSQLLYTGKVHEDVFPGHFILKVSATDLDTDTNAQITYSLHGPGAHEFKL
DPHTGELTTLTALDRERKDVFNLVAKATDGGGRSCQADITLHVEDVNDNAPRFFPSHCAV
AVFDNTTVKTPVAVVFARDPDQGANAQVVYSLPDSAEGHFSIDATTGVIRLEKPLQVRPQ
APLELTVRASDLGTPIPLSTLGTVTVSVVGLEDYLPVFLNTEHSVQVPEDAPPGTEVLQL
ATLTRPGAEKTGYRVVSGNEQGRFRLDARTGILYVNASLDFETSPKYFLSIECSRKSSSS
LSDVTTVMVNITDVNEHRPQFPQDPYSTRVLENALVGDVILTVSATDEDGPLNSDITYSL
IGGNQLGHFTIHPKKGELQVAKALDREQASSYSLKLRATDSGQPPLHEDTDIAIQVADVN
DNPPRFFQLNYSTTVQENSPIGSKVLQLILSDPDSPENGPPYSFRITKGNNGSAFRVTPD
GWLVTAEGLSRRAQEWYQLQIQASDSGIPPLSSLTSVRVHVTEQSHYAPSALPLEIFITV
GEDEFQGGMVGKIHATDRDPQDTLTYSLAEEETLGRHFSVGAPDGKIIAAQGLPRGHYSF
NVTVSDGTFTTTAGVHVYVWHVGQEALQQAMWMGFYQLTPEELVSDHWRNLQRFLSHKLD
IKRANIHLASLQPAEAVAGVDVLLVFEGHSGTFYEFQELASIITHSAKEMEHSVGVQMRS
AMPMVPCQGPTCQGQICHNTVHLDPKVGPTYSTARLSILTPRHHLQRSCSCNGTATRFSG
QSYVRYRAPAARNWHIHFYLKTLQPQAILLFTNETASVSLKLASGVPQLEYHCLGGFYGN

28
LSSQRHVNDHEWHSILVEEMDASIRLMVDSMGNTSLVVPENCRGLRPERHLLLGGLILLH
SSSNVSQGFEGCLDAVVVNEEALDLLAPGKTVAGLLETQALTQCCLHSDYCSQNTCLNGG
KCSWTHGAGYVCKCPPQFSGKHCEQGRENCTFAPCLEGGTCILSPKGASCNCPHPYTGDR
CEMEARGCSEGHCLVTPEIQRGDWGQQELLIITVAVAFIIISTVGLLFYCRRCKSHKPVA
MEDPDLLARSVGVDTQAMPAIELNPLSASSCNNLNQPEPSKASVPNELVTFGPNSKQRPV
Figure 6: (b) Amino acid sequence of FAT2
MDIIMGHCVGTRPPACCLILLLFKLLATVSQGLPGTGPLGFHFTHSIYNATVYENSAART
YVNSQSRMGITLIDLSWDIKYRIVSGDEEGFFKAEEVIIADFCFLRIRTKGGNSAILNRE
IQDNYLLIVKGSVRGEDLEAWTKVNIQVLDMNDLRPLFSPTTYSVTIAESTPLRTSVAQV
TATDADIGSNGEFYYYFKNKVDLFSVHPTSGVISLSGRLNYDEKNRYDLEILAVDRGMKL
YGNNGVSSTAKLYVHIERINEHAPTIHVVTHVPFSLEKEPTYAVVTVDDLDDGANGEIES
VSIVAGDPLDQFFLAKEGKWLNEYKIKERKQIDWESFPYGYNLTLQAKDKGSPQKCSALK
AVYIGNPTRDTVPIRFEKEVYDVSISEFSPPGVVVAIVKLSPEPIDVEYKLSPGEDAVYF
KINPRSGLIVTARPLNTVKKEVYKLEVTNKEGDLKAQVTISIEDANDHTPEFQQPLYDAY
VNESVPVGTSVLTVSASDKDKGENGYITYSIASLNLLPFVINQFTGVISTTEELDFESSP
EIYRFIVRASDWGSPYRHESEVNVTIRIGNVNDNSPLFEKVACQGVISYDFPVGGHITAV
SAIDIDELELVKYKIISGNELGFFYLNPDSGVLQLKKSLTNSGIKNGNFALRITATDGEN
LADPMSINISVLHGKVSSKSFSCRETRVAQKLAEKLLIKAKANGKLNLEDGFLDFYSINR
QGPYFDKSFPSDVAVKEDLPVGANILKIKAYDADSGFNGKVLFTISDGNTDSCFNIDMET
GQLKVLMPMDREHTDLYLLNITIYDLGNPQKSSWRLLTINVEDANDNSPVFIQDSYSVNI
LESSGIGTEIIQVEARDKDLGSNGEVTYSVLTDTQQFAINSSTGIVYVADQLDRESKANY
SLKIEARDKAESGQQLFSVVTLKVFLDDVNDCSPAFIPSSYSVKVLEDLPVGTVIAWLET
HDPDLGLGGQVRYSLVNDYNGRFEIDKASGAIRLSKELDYEKQQFYNLTVRAKDKGRPVS
LSSVSFVEVEVVDVNENLHTPYFPDFAVVGSVKENSRIGTSVLQVTARDEDSGRDGEIQY
SIRDGSGLGRFSIDDESGVITAADILDRETMGSYWLTVYATDRGVVPLYSTIEVYIEVED
VNDNAPLTSEPIYYPVVMENSPKDVSVIQIQAEDPDSSSNEKLTYRITSGNPQNFFAINI
KTGLITTTSRKLDREQQAEHFLEVTVTDGGPSPKQSTIWVVVQVLDENDNKPQFPEKVYQ
IKLPERDRKKRGEPIYRAFAFDRDEGPNAEISYSIVDGNDDGKFFIDPKTGMVSSRKQFT
AGSYDILTIKAVDNGRPQKSSTARLHIEWIKKPPPSPIPLTFDEPFYNFTVMESDRVTEI
VGVVSVQPANTPLWFDIVGGNFDSAFDAEKGVGTIVIAKPLDAEQRSIYNMSVEVTDGTN
VAVTQVFIKVLDNNDNGPEFSQPNYDVTISEDVLPDTEILQIEATDRDEKHKLSYTVHSS
IDSISMRKFRIDPSTGVLYTAERLDHEAQDKHILNIMVRDQEFPYRRNLARVIVNVEDAN
DHSPYFTNPLYEASVFESAALGSAVLQVTALDKDKGENAELIYTIEAGNTGNMFKIEPVL
GIITICKEPDMTTMGQFVLSIKVTDQGSPPMSATAIVRISVTMSDNSHPKFIHKDYQAEV
NENVDIGTSVILISAISQSTLIYEVKDGDINGIFTINPYSGVITTQKALDYERTSSYQLI
IQATNMAGMASNATVNIQIVDENDNAPVFLFSQYSGSLSEAAPINSIVRSLDNSPLVIRA

29
TDADSNRNALLVYQIVESTAKKFFTVDSSTGAIRTIANLDHETIAHFHFHVHVRDSGSPQ
LTAESPVEVNIEVTDVNDNPPVFTQAVFETILLLPTYVGVEVLKVSATDPDSEVPPELTY
SLMEGSLDHFLIDSNSGVLTIKNNNLSKDHYMLIVKVSDGKFYSTSMVTIMVKEAMDSGL
HFTQSFYSTSISENNTNITKVAIVNAVGNRLNEPLKYSILNPGNKFKIKSTSGVIQTTGV
PFDREEQELYELVVEASRELDHLRVARVVVRVNIEDINDNSPVFVGLPYYAAVQVDAEPG
TLIYQVTAIDKDKGPNGEVTYVLQDDYGHFEINPNSGNVILKEAFNSDLSNIEYGVTILA
KDGGKPSLSTSVELPITIVNKAMPVFDKPFYTASVNEDIRMNTPILSINATSPEGQGIIY
IIIDGDPFKQFNIDFDTGVLKVVSPLDYEVTSAYKLTIRASDALTGARAEVTVDLLVNDV
NDNPPIFDQPTYNTTLSEASLIGTPVLQVVSIDADSENNKMVHYQIVQDTYNSTDYFHID
SSSGLILTARMLDHELVQHCTLKVRSIDSGFPSLSSEVLVHIYISDVNDNPPVFNQLIYE
SYVSELAPRGHFVTCVQASDADSSDFDRLEYSILSGNDRTSFLMDSKSGVITLSNHRKQR
MEPLYSLNVSVSDGLFTSTAQVHIRVLGANLYSPAFSQSTYVAEVRENVAAGTKVIHVRA
TDGDPGTYGQISYAIINDFAKDRFLIDSNGQVITTERLDRENPLEGDVSIFVRALDGGGR
TTFCTVRVIVVDENDNAPQFMTVEYRASVRADVGRGHLVTQVQAIDPDDGANSRITYSLY
SEASVSVADLLEIDPDNGWMVTKGNFNQLKNTVLSFFVKAVDGGIPVKHSLIPVYIHVLP
PETFLPSFTQSQYSFTIAEDTAIGSTVDTLRILPSQNVWFSTVNGERPENNKGGIFVIEQ
ETGTIKLDKRLDRETSPAFHFKVAATIPLDKVDIVFTVDVDIKVLDLNDNKPVFETSSYD
TIIMEGMPVGTKLTQVRAIDMDWGANGQVTYSLHSDSQPEKVMEAFNIDSNTGWISTLKD
LDHETDPTFTFSVVASDLGEAFSLSSTALVSVRVTDINDNAPVFAQEVYRGNVKESDPPG
EVVAVLSTWDRDTSDVNRQVSYHITGGNPRGRFALGLVQSEWKVYVKRPLDREEQDIYFL
NITATDGLFVTQAMVEVSVSDVNDNSPVCDQVAYTALLPEDIPSNKIILKVSAKDADIGS
NGYIRYSLYGSGNSEFFLDPESGELKTLALLDRERIPVYSLMAKATDGGGRFCQSNIHLI
LEDVNDNPPVFSSDHYNTCVYENTATKALLTRVQAVDPDIGINRKVVYSLADSAGGVFSI
DSSSGIIILEQPLDREQQSSYNISVRATDQSPGQSLSSLTTVTITVLDINDNPPVFERRD
YLVTVPEDTSPGTQVLAVFATSKDIGTNAEITYLIRSGNEQGKFKINPKTGGISVSEVLD
YELCKRFYLVVEAKDGGTPALSAVATVNINLTDVNDNPPKFSQDVYSAVISEDALVGDSV
ILLIAEDVDSQPNGQIHFSIVNGDRDNEFTVDPVLGLVKVKKKLDRERVSGYSLLVQAVD
SGIPAMSSTATVNIDISDVNDNSPVFTPANYTAVIQENKPVGTSILQLVVTDRDSFHNGP
PFSFSILSGNEEEEFVLDPHGILRSAVVFQHTESLEYVLCVQAKDSGKPQQVSHTYIRVR
VIEESTHKPTAIPLEIFIVTMEDDFPGGVIGKIHATDQDMYDVLTFALKSEQKSLFKVNS
HDGKIIALGGLDSGKYVLNVSVSDGRFQVPIDVVVHVEQLVHEMLQNTVTIRFENVSPED
FVGLHMHGFRRTLRNAVLTQKQDSLRIISIQPVAGTNQLDMLFAVEMHSSEFYKPAYLIQ
KLSNARRHLENIMRISAILEKNCSGLDCQEQHCEQGLSLDSHALMTYSTARISFVCPRFY
RNVRCTCNGGLCPGSNDPCVEKPCPGDMQCVSYEASRRPFLCQCPPGKLGECSGHTSLSF
AGNSYIKYRLSENSKEEDFKLALRLRTLQSNGIIMYTRANPCIILKIVDGKLWFQLDCGS
GPGILGISGRAVNDGSWHSVFLELNRNFTSLSLDDSYVERRRAPLYFQTLSTESSIYFGA
LVQADNIRSLTDTRVTQVLSGFQGCLDSVILNNNELPLQNKRSSFAEVVGLTELKLGCVL
YPDACKRSPCQHGGSCTGLPSGGYQCTCLSQFTGRNCESEITACFPNPCRNGGSCDPIGN

30
TFICNCKAGLTGVTCEEDINECEREECENGGSCVNVFGSFLCNCTPGYVGQYCGLRPVVV
PNIQAGHSYVGKEELIGIAVVLFVIFILVVLFIVFRKKVFRKNYSRNNITLVQDPATAAL
LNKSNGIPFRNLRGSGDGRNVYQEVGPPQVPVRPMAYTPCFQSDSRSNLDKIVDGLGGEH
QEMTTFHPESPRILTARRGVVVCSVAPNLPAVSPCRSDCDSIRKNGWDAGTENKGVDDPG
EVTCFAGSNKGSNSEVQSLSSFQSDSGDDNAYHWDTSDWMPGARLSDIEEVPNYENQDGG
SAHQGSTRELESDYYLGGYDIDSEYPPPHEEEFLSQDQLPPPLPEDFPDQYEALPPSQPV
SLASTLSPDCRRRPQFHPSQYLPPHPFPNETDLVGPPASCEFSTFAVSMNQGTEPTGPAD
SVSLSLHNSRGTSSSDVSANCGFDDSEVAMSDYESVGELSLASLHIPFVETQHQTQV
Figure 6: (c) Amino acid sequence of FAT3
MDLAPDRATGRPWLPLHTLSVSQLLRVFWLLSLLPGQAWVHGAEPRQVFQVLEEQPPGTL
VGTIQTRPGFTYRLSESHALFAINSSTGALYTTSTIDRESLPSDVINLVVLSSAPTYPTE
VRVLVRDLNDNAPVFPDPSIVVTFKEDSSSGRQVILDTATDSDIGSNGVDHRSYRIIRGN
EAGRFRLDITLNPSGEGAFLHLVSKGGLDREVTPQYQLLVEVEDKGEPKRRGYLQVNVTV
QDINDNPPVFGSSHYQAGVPEDAVVGSSVLQVAAADADEGTNADIRYRLQDEGTPFQMDP
ETGLITVREPLDFEARRQYSLTVQAMDRGVPSLTGRAEALIQLLDVNDNDPVVKFRYFPA
TSRYASVDENAQVGTVVALLTVTDADSPAANGNISVQILGGNEQRHFEVQSSKVPNLSLI
KVASALDRERIPSYNLTVSVSDNYGAPPGAAVQARSSVASLVIFVNDINDHPPVFSQQVY
RVNLSEEAPPGSYVSGISATDGDSGLNANLRYSIVSGNGLGWFHISEHSGLVTTGSSGGL
DRELASQIVLNISARDQGVHPKVSYAQLVVTLLDVNDEKPVFSQPEGYDVSVVENAPTGT
ELLMLRATDGDLGDNGTVRFSLQEAETDRRSFRLDPVSGRLSTISSLDREEQAFYSLLVL
ATDLGSPPQSSMARINVSLLDINDNSPVFYPVQYFAHIKENEPGGSYITTVSATDPDLGT
NGTVKYSISAGDRSRFQVNAQSGVISTRMALDREEKTAYQLQIVATDGGNLQSPNQAIVT
ITVLDTQDNPPVFSQVAYSFVVFENVALGYHVGSVSASTMDLNSNISYLITTGDQKGMFA
INQVTGQLTTANVIDREEQSFYQLKVVASGGTVTGDTMVNITVKDLNDNSPHFLQAIESV
NVVENWQAGHSIFQAKAVDPDEGVNGMVLYSLKQNPKNLFAINEKNGTISLLGPLDVHAG
SYQIEILASDMGVPQLSSSVILTVYVHDVNDNSPVFDQLSYEVTLSESEPVNSRFFKVQA
SDKDSGANGEIAYTIAEGNTGDAFGIFPDGQLYIKSELDRELQDRYVLMVVASDRAVEPL
SATVNVTVILEDVNDNRPLFNSTNYTFYFEEEQRAGSFVGKVSAVDKDFGPNGEVRYSFE
MVQPDFELHAISGEITNTHQFDRESLMRRRGTAVFSFTVIATDQGIPQPLKDQATVHVYM
KDINDNAPKFLKDFYQATISESAANLTQVLRVSASDVDEGNNGLIHYSIIKGNEERQFAI
DSTSGQVTLIGKLDYEATPAYSLVIQAVDSGTIPLNSTCTLNIDILDENDNTPSFPKSTL
FVDVLENMRIGELVSSVTATDSDSGDNADLYYSITGTNNHGTFSISPNTGSIFLAKKLDF
ETQSLYKLNITAKDQGRPPRSSTMSVVIHVRDFNDNPPSFPPGDIFKSIVENIPIGTSVI
SVTAHDPDADINGQLSYTIIQQMPRGNHFTIDEVKGTIYTNAEIDREFANLFELTVKAND
QAVPIETRRYALKNVTILVTDLNDNVPMFISQNALAADPSAVIGSVLTTIMAADPDEGAN
GEIEYEIINGDTDTFIVDRYSGDLRVASALVPSQLIYNLIVSATDLGPERRKSTTELTII

31
LQGLDGPVFTQPKYITILKEGEPIGTNVISIEAASPRGSEAPVEYYIVSVRCEEKTVGRL
FTIGRHTGIIQTAAILDREQGACLYLVDVYAIEKSTAFPRTQRAEVEITLQDINDNPPVF
PTDMLDLTVEENIGDGSKIMQLTAMDADEGANALVTYTIISGADDSFRIDPESGDLIATR
RLDRERRSKYSLLVRADDGLQSSDMRINITVSDVNDHTPKFSRPVYSFDIPEDTIPGSLV
AAILATDDDSGVNGEITYIVNEDDEDGIFFLNPITGVFNLTRLLDYEVQQYYILTVRAED
GGGQFTTIRVYFNILDVNDNPPIFSLNSYSTSLMENLPVGSTVLVFNVTDADDGINSQLT
YSIASGDSLGQFTVDKNGVLKVLKALDRESQSFYNLVVQVHDLPQIPASRFTSTAQVSII
LLDVNDNPPTFLSPKLTYIPENTPIDTVVFKAQATDPDSGPNSYIEYTLLNPLGNKFSIG
TIDGEVRLTGELDREEVSNYTLTVVATDKGQPSLSSSTEVVVMVLDINDNNPIFAQALYK
VEINENTLTGTDIIQVFAADGDEGTNGQVRYGIVNGNTNQEFRIDSVTGAITVAKPLDRE
KTPTYHLTVQATDRGSTPRTDTSTVSIVLLDINDFVPVFELSPYSVNVPENLGTLPRTIL
QVVARDDDRGSNSKLSYVLFGGNEDNAFTLSASGELGVTQSLDRETKERFVLMITATDSG
SPALTGTGTINVIVDDVNDNVPTFASKAYFTTIPEDAPTGTDVLLVNASDADASKNAVIR
IIGGNSQFTINPSTGQIITSALLDRETKDNYTLVVVCSDAGSPEPLSSSTSVLVTVTDVN
DNPPRFQHHPYVTHIPSPTLPGSFVFAVTVTDADIGPNSELHYSLSGRNSEKFHIDPLRG
AIMAAGPLNGASEVTFSVHVKDGGSFPKTDSTTVTVRFVNKADFPKVRAKEQTFMFPENQ
PVSSLVTTITGSSLRGEPMSYYIASGNLGNTFQIDQLTGQVSISQPLDFEKIQKYVVWIE
ARDGGFPPFSSYEKLDITVLDVNDNAPIFKEDPFISEILENLSPRKILTVSAMDKDSGPN
GQLDYEIVNGNMENSFSINHATGEIRSVRPLDREKVSHYVLTIKSSDKGSPSQSTSVKVM
INILDENDNAPRFSQIFSAHVPENSPLGYTVTRVTTSDEDIGINAISRYSIMDASLPFTI
NPSTGDIVISRPLNREDTDRYRIRVSAHDSGWTVSTDVTIFVTDINDNAPRFSRTSYYLD
CPELTEIGSKVTQVFATDPDEGSNGQVFYFIKSQSEYFRINATTGEIFNKQILKYQNVTG
FSNVNINRHSFIVTSSDRGKPSLISETTVTINIVDSNDNAPQFLKSKYFTPVTKNVKVGT
KLIRVTAIDDKDFGLNSEVEYFISNDNHLGKFKLDNDTGWISVASSLISDLNQNFFITVT
AKDKGNPPLSSQATVHITVTEENYHTPEFSQSHMSATIPESHSIGSIVRTVSARDRDAAM
NGLIKYSISSGNEEGIFAINSSTGILTLAKALDYELCQKHEMTISAIDGGWVARTGYCSV
TVNVIDVNDNSPVFLSDDYFPTVLENAPSGTTVIHLNATDADSGTNAVIAYTVQSSDSDL
FVIDPNTGVITTQGFLDFETKQSYHLTVKAFNVPDEERCSFATVNIQLKGTNEYVPRFVS
KLYYFEISEAAPKGTIVGEVFASDRDLGTDGEVHYLIFGNSRKKGFQINKKTGQIYVSGI
LDREKEERVSLKVLAKNFGSIRGADIDEVTVNVTVLDANDPPIFTLNIYSVQISEGVPIG
THVTFVSAFDSDSIPSWSRFSYFIGSGNENGAFSINPQTGQITVTAELDRETLPIYNLSV
LAVDSGTPSATGSASLLVTLEDINDNGPMLTVSEGEVMENKRPGTLVMTLQSTDPDLPPN
QGPFTYYLLSTGPATSYFSLSTAGVLSTTREIDREQIADFYLSVVTKDSGVPQMSSTGTV
HITVIDQNDNPSQSRTVEIFVNYYGNLFPGGILGSVKPQDPDVLDSFHCSLTSGVTSLFS
IPGGTCDLNSQPRSTDGTFDLTVLSNDGVHSTVTSNIRVFFAGFSNATVDNSILLRLGVP
TVKDFLTNHYLHFLRIASSQLTGLGTAVQLYSAYEENNRTFLLAAVKRNHNQYVNPSGVA
TFFESIKEILLRQSGVKVESVDHDSCVHGPCQNGGSCLRRLAVSSVLKSRESLPVIIVAN
EPLQPFLCKCLPGYAGSWCEIDIDECLPSPCHSGGTCHNLVGGFSCSCPDGFTGRACERD

32
INECLQSPCKNGAICQNFPGSFNCVCKTGYTGKMCESSVNYCECNPCFNGGSCQSGVDSY
YCHCPFGVFGKHCELNSYGFEELSYMEFPSLDPNNNYIYVKFATIKSHALLLYNYDNQTG
DRAEFLALEIAEERLRFSYNLGSGTYKLTTMKKVSDGHFHTVIARRAGMAASLTVDSCSE
NQEPGYCTVSNVAVSDDWTLDVQPNRVTVGGIRSLEPILQRRGHVESHDFVGCIMEFAVN
GRPLEPSQALAAQGILDQCPRLEGACTRSPCQHGGTCMDYWSWQQCHCKEGLTGKYCEKS
VTPDTALSLEGKGRLDYHMSQNEKREYLLRQSLRGAMLEPFGVNSLEVKFRTRSENGVLI
HIQESSNYTTVKIKNGKVYFTSDAGIAGKVERNIPEVYVADGHWHTFLIGKNGTATVLSV
DRIYNRDIIHPTQDFGGLDVLTISLGGIPPNQAHRDAQTAGFDGCIASMWYGGESLPFSG
KHSLASISKTDPSVKIGCRGPNICASNPCWGDLLCINQWYAYRCVPPGDCASHPCQNGGS
CEPGLHSGFTCSCPDSHTGRTCEMVVACLGVLCPQGKVCKAGSPAGHVCVLSQGPEEISL
PLWAVPAIVGSCATVLALLVLSLILCNQCRGKKAKNPKEEKKPKEKKKKGSENVAFDDPD
NIPPYGDDMTVRKQPEGNPKPDIIERENPYLIYDETDIPHNSETIPSAPLASPEQEIEHY
DIDNASSIAPSDADIIQHYKQFRSHTPKFSIQRHSPLGFARQSPMPLGASSLTYQPSYGQ
GLRTSSLSHSACPTPNPLSRHSPAPFSKSSTFYRNSPARELHLPIRDGNTLEMHGDTCQP
GIFNYATRLGRRSKSPQAMASHGSRPGSRLKQPIGQIPLESSPPVGLSIEEVERLNTPRP
RNPSICSADHGRSSSEEDCRRPLSRTRNPADGIPAPESSSDSDSHESFTCSEMEYDREKP
MVYTSRMPKLSQVNESDADDEDNYGARLKPRRYHGRRAEGGPVGTQAAAPGTADNTLPMK
LGQQAGTFNWDNLLNWGPGFGHYVDVFKDLASLPEKAAANEEGKAGTTKPVPKDGEAEQY
V
Figure 6: (d) Amino acid sequence of FAT4
Multiple sequence alignment of FATs
The amino acid sequences of the FATs were analyzed and were found that there were very few
residues which were highly conserved across different FATs. For instance, the guanine residues
present at the 210th
, 296th,
420th
, 480th, 493rd
, 517th
, 544th
and 587th
positions. The leucine
residues present at the 224th
and 421st
positions. The adenine residues present at the 243rd
, 488th
and 540th
positions. The proline residues present at the 257th
, 345th
, 462nd
, 510th
and 546th
positions. The serine residues present at the residues present at the 378th
position. The valine
residues present at 450th,
483rd
and 587th
positions. The aspargine residues present at the 458th,
495th
and 542nd
positions. The Phenylalanine residues present at the 464th
, 510th
and 570th
positions. The tyrosin residues present at the 469th
, 500th
and 534th
positions. The glutamic acid
residues present at the 475th
and 528th
positions. The aspartic acid residues present at 489th
, 491st
,
526th
and 542nd
positions. All the above amino acid residues were conserved for FAT1, FAT2,
FAT3, and FAT4 (figure 7). This signifies that even the functional role of these FATs may differ

33
but the sequence conservedness at typical residues points out towards their involvement in
certain common physiological events.
Figure 7: Protein sequence homology of FATs

34
Domain organization of FATs
FAT1 has 4588 amino acid residues which is organized by 22-4181 extracellular region, 4182-
4202 helical region and 4303- 4588 cytoplasmic region. FAT2 has 4349 amino acid residues
which is comprised by 19-4048 extracellular region, 4049- 4069 helical region and 4070- 4349
cytoplasmic region.FAT3 has 4122 amino acid residues which is organized by 33-4154
extracellular region, 4155- 4175 helical region and 4176- 4122 cytoplasmic region.
FAT4 has 4466 amino acid residues which is organized by 39-4504 extracellular region, 4505-
4525 helical region and 4526- 4466 cytoplasmic region. The information was retrieved from
Uniprot.(figure 8(a),(b),(c),(d)).
Figure 8: Domain organizations of (a) FAT1, (b) FAT2, (c) FAT, and (d) FAT4
1 4588
4181
22 4202
FAT1
1 4349
4048
Extracellular
19 4069
Helical
Cytoplasmic
FAT2
1
3
4122
4154 4175
FAT3
1 4466
4504
39 4525
FAT4
1 33
(a)
(b)
(c)
(d)

35
The three- dimensional configuration of FATs
The 3-D models of FATs were obtained from the Swiss Model. The 3-D modeling of FATs was carried
out using the Fasta Sequences and the 3-D models were downloaded as below for our further study
(figure 9 (a),(b),(c),(d)).
(a) (b) (c) (d)
Figure 9: 3-D structure of (a) FAT1, (b) FAT2, (c) FAT3, and (d) FAT4
Putative protein partners of FATs
The putative protein partners of FAT1 were found to be TNF, JUN, RELA, MYC, PPARA, RXRA,
NR2F1, NOS2 and NFKBIA out of which TNF-A was taken for our study. The putative protein partners
of FAT2 were found to be HEPHL1, FJX1,PLD5,PCP2, KCNS1 ,GABRA6,GLRB, RCE1, ESPNL and
SLC36A1 out of which HEPHL1 was taken for our study . The putative protein partners of FAT3 were
found to be LAMC1, FJX1, MTNR1A , MTNR1B, CTNNA1, CTNNA2 , RERE, CSMD3, PTPRD and
CMSS1 out of which MTNR1B was considered for our study . The putative protein partners of FAT3
were found to be MPDZ, YAP1, FJX1, ARFGEF2, LAMC1, INVS, FAT1, VANGL2, FRMD6 and
PCDH15 out of which VANGL2 was taken for our study. (Figure 10 (a),(b),(c),(d)).

36
(a) (b)
(c) (d)
Figure 10: Putative protein partners (a) FAT1, (b) FAT2, (c) FAT3, (d) FAT4

37
Construction of domain mutants of N-terminal region of FATs
Domain mutants of N-terminal regions of FATs were retrieved. The Wild type FAT1 has a total of 4588
amino acid residues. In the mutant type of FAT1 (Δ1-100) the first hundred amino acid residues were
deleted. In the mutant type of FAT1 (Δ101-200) the amino acid residues between positions 101-200 were
deleted. Similarly, in the mutant type of FAT1(Δ301-400) the amino acids between positions 301-400
were deleted followed by the deletion of amino acid residues from positions 401-500 in the mutant type
of FAT1(Δ401-500) (Figure 11(a)).
Figure 11: (a) FAT1 domain mutants
101 4588
4181 4202
FAT1
(∆ 1-100)
100 4588
4181 4202
FAT1
(∆ 101-
200)
201
1
4588
4181 4202
FAT1
(∆ 201-
300)
1 200 301
1 4588
4181
22 4202
FAT1
4588
4181 4202
FAT1
(∆ 301-
400)
1 401
300
4588
4181 4202
FAT1
(∆ 401-
500)
1 400 501

38
deleted. Similarly, in the mutant type of FAT2 (Δ301-400) the amino acids between positions 301-400
of FAT2 (Δ401-500) (Figure 11(b)).
Figure 11: (b) FAT2 domain mutants
1 4349
4048
19 4069
FAT2
101 4349
4048 4069
FAT2
(∆ 1-100)
100 4349
4048 4069
FAT2
(∆ 101-
200)
201
1
4349
4048 4069
FAT2
(∆ 201-
300)
1 200 301
4349
4048 4069
FAT2
(∆ 301-
400)
1 401
300
4349
4048 4069
FAT2
(∆ 401-
500)
1 400 501

39
of FAT3 (Δ401-500) (Figure 11(c)).
Figure 11: (c) FAT3 domain mutants
1 4122
4154
22 4175
FAT3
101 4122
41544175
FAT3
(∆ 1-100)
100 4122
41544175
FAT3
(∆ 101-
200)
201
1
4122
41544175
FAT3
(∆ 201-
300)
1 200 301
4122
41544175
FAT3
(∆ 301-
400)
1 401
300
4122
41544175
FAT3
(∆ 401-
500)
1 400 501

40
of FAT4 (Δ401-500) (Figure 11(d)).
Figure 11: (d) FAT4 domain mutants
1 4466
4504
39 4525
FAT4
101 4466
4504 4525
FAT4
(∆ 1-100)
100 4466
4504 4525
FAT4
(∆ 101-
200)
201
1
4466
4504 4525
FAT4
(∆ 201-
300)
1 200 301
4466
4504 4525
FAT4
(∆ 301-
400)
1 401
300
4466
4504 4525
FAT4
(∆ 401-
500)
1 400 501

41
Interaction of FAT1 with TNF-A
Using the H-DOCK module, the docking scores for protein-protein interactions were predicted. The
Wild-type and domain mutants of FAT1 were included in this study. It was observed that the docking
score for Wild-type FAT1-TNF-A was -285.10 (Figure 12 (a)). The docking score for FAT1 (∆1-100)-
TNF-A was -253.92 (Figure 12 (b)). The docking score for FAT1 (∆101-200)-TNF-A was-265.32
(Figure 12 (c)).The docking score for FAT1 (∆201- 300)-TNF-A was -264.72 (Figure 12 (d)). The
docking score for FAT1 (∆301-400)-TNF-A was -287.29 (Figure 12 (e)). The docking score for FAT1
(∆401-500)-TNF-A was -263.42 (Figure 12 (f)). The N-terminal 100 amino acids of FAT1 are vital for
interaction with TNF-A (Table 1).
TNF-A
FAT 1
DOCKING SCORE: -253.92
FAT1(∆1-100)
TNFA
Figure 12: (b) Interaction of FAT1 (∆1-
100) with TNF-A
Figure 12: (a) Interaction of wild-type
FAT1 with TNF-A

42
DOCKING SCORE:-265.32
FAT1
(∆ 101-200)
TNF-A
TNF-A
FAT1
(∆ 201-300)
DOCKING SCORE:- -264.72
FAT1
(∆ 301-400)
TNF-A
DOCKING SCORE:- -287.29
FAT1
(∆ 401-500)
TNF-A
Figure 12: (c) Interaction of FAT1 (∆101-
200) with TNF-A
Figure 12: (d) Interaction of FAT1
(∆201-300) with TNF-A
Figure 12: (e) Interaction of FAT1 (∆ 301-
400) with TNF-A
Figure 12: (f) Interaction of FAT1
(∆401-500) with TNF-A

43
Table 1: Interaction of FAT1 with TNF-A
Interaction of FAT2 with HEPHL1
score for Wild-type FAT2-HEPHL1 was -312.93 (Figure 13(a)). The docking score for FAT2 (∆1-100)-
HEPHL1 was -291.21 (Figure 13(b)). The docking score for FAT2 (∆101-200)-HEPHL1 was -305.00
(Figure 13(c)). The docking score for FAT2 (∆201- 300)-HEPHL1 was -301.28 (Figure 13(d)). The
docking score for FAT2 (∆301-400)-HEPHL1 was -306.30 (Figure 13(e)). The docking score for FAT2
(∆401-500)-HEPHL1 was -283.03 (Figure 13(f)). The docking score for FAT2 (∆401-500)-HEPHL1
was -283.03. The N-terminal 100 amino acids of FAT2 are vital for interaction with HEPHL1. (Table 2)
SL.
NO.
INTERACTION OF FAT1
AND TNF-A
DOCKING
SCORE
1. WT FAT1 and TNF-A -285.10
2. FAT1(∆1-100)and TNF-A -253.92
3. FAT1(∆101-200)andTNF-A -265.32
4. FAT1(∆201-300)andTNF-A -264.72
5. FAT1(∆301-400)andTNF-A -287.29
6. FAT1(∆401-500)andTNF-A -263.42

44
HEPHL1
FAT2
FAT2
(∆ 1-100)
HEPHL1
FAT2
(∆ 101-
200)
HEPHL1 FAT2
(∆ 201-300)
HEPHL1
FAT2 with HEPHL1
Figure 13: (b) Interaction of FAT2
Figure 13: (c) Interaction of FAT2 (∆101-
200) with HEPHL1
Docking score:- -301.28
Docking score :- -291.21

45
Table 2: Interaction of FAT2 with HEPHL1
HEPHL1
FAT2
(∆ 301-400)
FAT2(∆401-
500)
HEPHL1
SL
No
INTERACTION OF FAT2
AND HEPHL1
DOCKIN
G SCORE
1. WT FAT2 AND HEPHL1 -312.93
2. FAT2(∆1-100) AND HEPHL1 -291.21
3. FAT2(∆101-200) AND
HEPHL1
-305.00
4. FAT2(∆201-300) AND
HEPHL1
-301.28
5. FAT2(∆301-400) AND
HEPHL1
-306.30
6. FAT2(∆401-500) AND
HEPHL1
-283.03
Figure 13: (e) Interaction of FAT2 (∆ 301-
400) with HEPHL1
Figure 13: (f) Interaction of FAT1 (∆401-
500) with HEPHL1

46
Interaction of FAT3 with MTNR1B
score for Wild-type FAT3-MTNR1B was -424.30 (Figure 14 (a)). The docking score for FAT3 (∆1-
100)-MTNR1B was -325.14 (Figure 14 (b)). The docking score for FAT3 (∆101-200)-MTNR1B was -
390.99 (Figure 14 (c)). The docking score for FAT3 (∆201- 300)-MTNR1B was -327.89 Figure 14
(d).The docking score for FAT3 (∆301-400)-MTNR1B was -447.31 (Figure 14 (e)). The docking score
for FAT3 (∆401-500)-MTNR1B was -381.18 (Figure 14 (f)). The docking score for FAT3 (∆301-400)-
HEPHL1 was –447.31. The N-terminal 100 amino acids of FAT3 are vital for interaction with MTNR1B
(Table 3).
FAT3
MTNR1B
FAT3
(∆ 1-100)
MTNR1B
FAT3 with MTNR1B
Figure 14: (b) Interaction of FAT3 (∆1-
100) with MTNR1B

47
MTNR1B
FAT3
(∆ 101-200)
FAT3
(∆ 201-300)
MTNR1B
FAT3
(∆ 301-400)
MTNR1B
FAT3
(∆ 401-500)
MTNR1B
(∆201-300) with MTNR1B
Figure 14: (g) Interaction of FAT3 (∆
301-400) with MTNR1B
(∆ 401-500) with MTNR1B
Figure 14: (c) Interaction of wild-type
FAT3 with MTNR1B

48
Table 3: Interaction of FAT3 with MTNR1B
Interaction of FAT4 with VANGL2
score for Wild-type FAT4-VANGL2 was -304.77 (Figure 15 (a)). The docking score for FAT4 (∆1-
100)-VANGL2 was -311.66 (Figure 15 (b)).The docking score for FAT4 (∆101-200)-VANGL2 was -
282.33 (Figure 15 (c)). The docking score for FAT4 (∆201- 300)-VANGL2 was -265.49 Figure 15 (d).
The docking score for FAT4 (∆301-400)-VANGL2 was -270.49 (Figure 15(e)). The docking score for
FAT4 (∆401-500)-VANGL2 was -253.55 (Figure 15 (f)). The docking score for FAT4 (∆401-500)-
VANGL2 was -253.55. The N-terminal 100 amino acids of FAT4 are vital for interaction with VANGL2
since the deletion of initial amino acid residues resulted in weak interaction between FAT4 (∆1-
100) and VANGL2 with respect to wild-type FAT4-VANGL2 interaction. However, it was
SL.
NO.
INTERACTION FAT3 AND
MTNR1B
DOCKING
SCORE
1. WT FAT3 AND MTNR1B -424.30
2. FAT3(∆1-100) AND MTNR1B -325.14
3. FAT3(∆101-200) AND
MTNR1B
-390.99
4. FAT3(∆201-300) AND
MTNR1B
-327.89
5. FAT3(∆301-400) AND
MTNR1B
-447.31
6. FAT3(∆401-500) AND
MTNR1B
-381.18

49
observed that the docking score for FAT4 (∆401-500)-VANGL2 interaction was elevated to -
253.55 suggesting that the amino acid residues spreading from 401- 500 residues affect the
strength of interaction between two proteins rather it is quite relevant that this domain might
regulate the interaction of FAT4 with VANGL2 (Table 4).
FAT4
VANGL2
FAT4
(∆ 1-100)
VANGL2
FAT4
(∆ 101-200)
VANGL2
FAT4
(∆ 201-300)
VANGL2
FAT4 with VANGL2
Figure 15: (b) Interaction of FAT4
Figure 15: (c) Interaction of FAT4

50
Table 4: Interaction of FAT4 with VANGL2
FAT4
(∆ 301-400)
VANGL2 FAT4
(∆ 401-500)
VANGL2
SL.
NO.
INTERACTION FAT4
OF VANGL2
DOCKING
SCORE
1. WT FAT4 AND VANGL2 -304.77
2. FAT4(∆1-100) AND
VANGL2
-311.61
3. FAT4(∆101-200) AND
VANGL2
-282.33
4. FAT4(∆201-300) AND
VANGL2
-265.49
5. FAT4(∆301-400) AND
VANGL2
-270.45
6. FAT4(∆401-500) AND
VANGL2
-253.55S
Figure 15: (e) Interaction of
FAT4 (∆301-400) with VANGL2

51
CONCLUSION
AND
FUTURE
PERSPECTIVES

52
CONCLUSION AND FUTURE PERSPECTIVES
Our results highlight the importance of various functional domains of FATs including the N-
terminus 1-100 amino acid residues for the corresponding proteins, such as for
FAT1TNFA, FAT2 HEPHL1, FAT3 MTNR1B, FAT4 VANGL2.
These evidences widens up the possibility of administering potential peptides when the FATs
expression is inhibited. It is also steps way forward in improving our understanding on the
functional aspects of critical protein factors such as TNF-A, HEPHL1, MTNR1B, and VANGL2.
Using the H-DOCK module, the docking scores for protein-protein interactions were predicted.
The Wild-type and domain mutants of FAT1 were included in this study. It was observed that the docking
score for Wild-type FAT1-TNF-A was -285.10. The docking score for FAT1 (∆1-100)-TNF-A was -
253.92.The docking score for FAT1 (∆101-200)-TNF-A was-265.32. The docking score for FAT1 (∆201-
300)-TNF-A was -264.72. The docking score for FAT1 (∆301-400)-TNF-A was -287.29. The docking
score for FAT1 (∆401-500)-TNF-A was -263.42. The N-terminal 100 amino acids of FAT1 are vital for
interaction with TNF-A. The N-terminal 100 amino acids of FAT1 are vital for interaction with
TNF-A since the deletion of initial amino acid residues resulted in weak interaction between
FAT1 (∆1-100) and TNF-A with respect to wild-type FAT1-TNF-A interaction. The docking
score for FAT1 (∆301-400)-TNF-A interaction was slightly elevated to -287.29 suggesting that
these residues did not affected the strength of interaction between two proteins rather it is quite
relevant that this domain might regulate the interaction of FAT1 with TNF-A.
score for Wild-type FAT2-HEPHL1 was-312.93. The docking score for FAT2 (∆1-100)-HEPHL1 was -
291. The docking score for FAT2 (∆101-200)-HEPHL1 was -305.00. The docking score for FAT2 (∆201-
300)-HEPHL1 was -301.28. The docking score for FAT2 (∆301-400)-HEPHL1 was -306. The docking
score for FAT2 (∆401-500)-HEPHL1 was -283.03. The docking score for FAT2 (∆401-500)-HEPHL1
was -283.03. The N-terminal 100 amino acids of FAT2 are vital for interaction with HEPHL1. The N-
terminal 100 amino acids of FAT2 are vital for interaction with HEPHL1 since the deletion of
initial amino acid residues resulted in weak interaction between FAT2 (∆1-100) and HEPHL1
with respect to wild-type FAT2- HEPHL1 interaction. The docking score for FAT2 (∆401-500)-
HEPHL1 interaction was drastically elevated to -283.03 suggesting that these residues affected

53
the strength of interaction between two proteins rather it is quite relevant that this domain might
regulate the interaction of FAT2 with HEPHL1.
score for Wild-type FAT3-MTNR1B was - 424.30. The docking score for FAT3 (∆1-100)-MTNR1B was
-325.14. The docking score for FAT3 (∆101-200)-MTNR1B was -390.99 (Figure 14 (c)). The docking
score for FAT3 (∆201- 300)-MTNR1B was -327.89. The docking score for FAT3 (∆301-400)-MTNR1B
was -447.31. The docking score for FAT3 (∆401-500)-MTNR1B was -381.18. The docking score for
FAT3 (∆301-400)-HEPHL1 was -447.31. The N-terminal 100 amino acids of FAT3 are vital for
interaction with MTNR1B. The N-terminal 100 amino acids deletion changes about 100 score
difference to show that affected the interaction between FAT3 (∆1-100)-MTNR1B. Since the
initial amino acid residues resulted in week interaction between FAT3 (∆1-100)- MTNR1B. The
docking score for FAT3 (∆301-400)-MTNR1B interaction was drastically elevated to -447.31
suggesting these residues affected the strength of interaction between two proteins rather it is
quite relevant that this domain might regulate the interaction of FAT3 with MTNR1B.
score for Wild-type FAT4-VANGL2 was -304.77. The docking score for FAT4 (∆1-100)-VANGL2 was -
311.66. The docking score for FAT4 (∆101-200)-VANGL2 was -282.33. The docking score for FAT4
(∆201- 300)-VANGL2 was -265.49. The docking score for FAT4 (∆301-400)-VANGL2 was -270.49.
The docking score for FAT4 (∆401-500)-VANGL2 was -253.55. The docking score for FAT4 (∆401-
500)-VANGL2 was -253.55. The N-terminal 100 amino acids of FAT4 are vital for interaction with
VANGL2 since the deletion of initial amino acid residues resulted in weak interaction between
FAT4 (∆1-100) and VANGL2 with respect to wild-type FAT4-VANGL2 interaction. However,
it was observed that the docking score for FAT4 (∆401-500)-VANGL2 interaction was elevated
to -253.55 suggesting that the amino acid residues spreading from 401- 500 residues affect the
strength of interaction between two proteins rather it is quite relevant that this domain might
regulate the interaction of FAT4 with VANGL2.

54
The FATs regulates multiple signalling pathways including Hippo, Wnt, and MAPK/ERK, as
well as EMT, and offers a new tool to investigate. Understanding about FATs are still
insufficient since it covers such a large field of study, thus additional research is required in
order to properly comprehend the expression. Besides, the involvement of FATs in many types
of tumours, as well as research into the relationship between FATs and the Ena/Vasp protein and
the MAPK/ERK signalling pathway, remain unexplored. The functions of FATs are influenced
by transcription factors and target genes, as well as the molecular processes behind unregulated
FATs expression, are the fundamental challenges in homeostasis that have yet to be resolved.
Moving ahead in our understanding of FATs role in specific diseases or infections might
lead to the discovery of new prognostic and/or therapeutic targets.

56
REFERENCES
Agrawal, N., Jiao, Y., Bettegowda, C., Hutfless, S. M., Wang, Y., David, S., Cheng, Y.,
Twaddell, W. S., Latt, N. L., Shin, E. J., Wang, L.-D., Wang, L., Yang, W., Velculescu, V.
E., Vogelstein, B., Papadopoulos, N., Kinzler, K. W., & Meltzer, S. J. (2012). Comparative
genomic analysis of esophageal adenocarcinoma and squamous cell carcinoma. Cancer
Discovery, 2(10), 899–905. https://doi.org/10.1158/2159-8290.CD-12-0189
Bennett, F. C., & Harvey, K. F. (2006). Fat Cadherin Modulates Organ Size in Drosophila via
the Salvador/Warts/Hippo Signaling Pathway. Current Biology, 16(21), 2101–2110.
https://doi.org/10.1016/J.CUB.2006.09.045
Beyer, T. A., Weiss, A., Khomchuk, Y., Huang, K., Ogunjimi, A. A., Varelas, X., & Wrana, J. L.
(2013). Switch Enhancers Interpret TGF-β and Hippo Signaling to Control Cell Fate in
Human Embryonic Stem Cells. Cell Reports, 5(6), 1611–1624.
https://doi.org/10.1016/J.CELREP.2013.11.021
Blair, I. P., Chetcuti, A. F., Badenhop, R. F., Scimone, A., Moses, M. J., Adams, L. J., Craddock,
N., Green, E., Kirov, G., Owen, M. J., Kwok, J. B. J., Donald, J. A., Mitchell, P. B., &
Schofield, P. R. (2006). Positional cloning, association analysis and expression studies
provide convergent evidence that the cadherin gene FAT contains a bipolar disorder
susceptibility allele. Molecular Psychiatry, 11(4), 372–383.
https://doi.org/10.1038/sj.mp.4001784
Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R. L., Torre, L. A., & Jemal, A. (2018). Global
cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36
cancers in 185 countries. CA: A Cancer Journal for Clinicians, 68(6), 394–424.
https://doi.org/10.3322/caac.21492
Britt, K. L., Cuzick, J., & Phillips, K.-A. (2020). Key steps for effective breast cancer
prevention. Nature Reviews Cancer, 20(8), 417–436. https://doi.org/10.1038/s41568-020-
0266-x
Bryant, P. J., Huettner, B., Held, L. I., Ryerse, J., & Szidonya, J. (1988). Mutations at the fat

57
locus interfere with cell proliferation control and epithelial morphogenesis in Drosophila.
Developmental Biology, 129(2), 541–554. https://doi.org/10.1016/0012-1606(88)90399-5
Camargo, F. D., Gokhale, S., Johnnidis, J. B., Fu, D., Bell, G. W., Jaenisch, R., &
Brummelkamp, T. R. (2007). YAP1 Increases Organ Size and Expands Undifferentiated
Progenitor Cells. Current Biology, 17(23), 2054–2060.
https://doi.org/10.1016/J.CUB.2007.10.039
Cappello, S., Gray, M. J., Badouel, C., Lange, S., Einsiedler, M., Srour, M., Chitayat, D.,
Hamdan, F. F., Jenkins, Z. A., Morgan, T., Preitner, N., Uster, T., Thomas, J., Shannon, P.,
Morrison, V., Di Donato, N., Van Maldergem, L., Neuhann, T., Newbury-Ecob, R., …
Robertson, S. P. (2013). Mutations in genes encoding the cadherin receptor-ligand pair
DCHS1 and FAT4 disrupt cerebral cortical development. Nature Genetics, 45(11), 1300–
1308. https://doi.org/10.1038/ng.2765
De Bock, C. E., Ardjmand, A., Molloy, T. J., Bone, S. M., Johnstone, D., Campbell, D. M.,
Shipman, K. L., Yeadon, T. M., Holst, J., Spanevello, M. D., Nelmes, G., Catchpoole, D.
R., Lincz, L. F., Boyd, A. W., Burns, G. F., & Thorne, R. F. (2012). The Fat1 cadherin is
overexpressed and an independent prognostic factor for survival in paired diagnosis-relapse
samples of precursor B-cell acute lymphoblastic leukemia. Leukemia, 26(5), 918–926.
https://doi.org/10.1038/leu.2011.319
Dulak, A. M., Stojanov, P., Peng, S., Lawrence, M. S., Fox, C., Stewart, C., Bandla, S.,
Imamura, Y., Schumacher, S. E., Shefler, E., McKenna, A., Carter, S. L., Cibulskis, K.,
Sivachenko, A., Saksena, G., Voet, D., Ramos, A. H., Auclair, D., Thompson, K., … Bass,
A. J. (2013). Exome and whole-genome sequencing of esophageal adenocarcinoma
identifies recurrent driver events and mutational complexity. Nature Genetics, 45(5), 478–
486. https://doi.org/10.1038/ng.2591
Dunne, J., Hanby, A. M., Poulsom, R., Jones, T. A., Sheer, D., Chin, W. G., Da, S. M., Zhao, Q.,
Beverley, P. C., & Owen, M. J. (1995). Molecular cloning and tissue expression of FAT, the
human homologue of the Drosophila fat gene that is located on chromosome 4q34-q35 and
encodes a putative adhesion molecule. Genomics, 30(2), 207–223.
https://doi.org/10.1006/geno.1995.9884

58
Funato, Y., Michiue, T., Asashima, M., & Miki, H. (2006). The thioredoxin-related redox-
regulating protein nucleoredoxin inhibits Wnt-beta-catenin signalling through dishevelled.
Nature Cell Biology, 8(5), 501–508. https://doi.org/10.1038/ncb1405
Guo, Y., Wang, P., Sun, H., Cai, R., Xia, W., & Wang, S. (2013). Advanced glycation end
product-induced astrocytic differentiation of cultured neurospheres through inhibition of
Notch-Hes1 pathway-mediated neurogenesis. International Journal of Molecular Sciences,
15(1), 159–170. https://doi.org/10.3390/ijms15010159
He, T. C., Sparks, A. B., Rago, C., Hermeking, H., Zawel, L., da Costa, L. T., Morin, P. J.,
Vogelstein, B., & Kinzler, K. W. (1998). Identification of c-MYC as a target of the APC
pathway. Science (New York, N.Y.), 281(5382), 1509–1512.
https://doi.org/10.1126/science.281.5382.1509
Hu, X., Zhai, Y., Kong, P., Cui, H., Yan, T., Yang, J., Qian, Y., Ma, Y., Wang, F., Li, H., Cheng,
C., Zhang, L., Jia, Z., Li, Y., Yang, B., Xu, E., Wang, J., Yang, J., Bi, Y., … Cui, Y. (2017).
FAT1 prevents epithelial mesenchymal transition (EMT) via MAPK/ERK signaling
pathway in esophageal squamous cell cancer. Cancer Letters, 397, 83–93.
https://doi.org/10.1016/J.CANLET.2017.03.033
Huang, P., Yan, R., Zhang, X., Wang, L., Ke, X., & Qu, Y. (2019). Activating Wnt/β-catenin
signaling pathway for disease therapy: Challenges and opportunities. Pharmacology &
Therapeutics, 196, 79–90. https://doi.org/10.1016/J.PHARMTHERA.2018.11.008
Inoue, T., Yaoita, E., Kurihara, H., Shimizu, F., Sakai, T., Kobayashi, T., Ohshiro, K., Kawachi,
H., Okada, H., Suzuki, H., Kihara, I., & Yamamoto, T. (2001a). FAT is a component of
glomerular slit diaphragms. Kidney International, 59(3), 1003–1012.
https://doi.org/10.1046/j.1523-1755.2001.0590031003.x
Inoue, T., Yaoita, E., Kurihara, H., Shimizu, F., Sakai, T., Kobayashi, T., Ohshiro, K., Kawachi,
H., Okada, H., Suzuki, H., Kihara, I., & Yamamoto, T. (2001b). FAT is a component of
glomerular slit diaphragms. Kidney International, 59(3), 1003–1012.
https://doi.org/10.1046/J.1523-1755.2001.0590031003.X
Jiang, L., Zhang, J., Monticone, R. E., Telljohann, R., Wu, J., Wang, M., & Lakatta, E. G.

59
(2012). Calpain-1 regulation of matrix metalloproteinase 2 activity in vascular smooth
muscle cells facilitates age-associated aortic wall calcification and fibrosis. Hypertension
(Dallas, Tex. : 1979), 60(5), 1192–1199.
https://doi.org/10.1161/HYPERTENSIONAHA.112.196840
Katoh, M. (2005). Comparative genomics on Wnt3 - Wnt9b gene cluster. Int J Mol Med, 15(4),
743–747. https://doi.org/10.3892/ijmm.15.4.743
Katoh, Y., & Katoh, M. (2006). Comparative integromics on FAT1, FAT2, FAT3 and FAT4.
International Journal of Molecular Medicine, 18(3), 523–528.
https://doi.org/10.3892/ijmm.18.3.523
Kawamori, H., Tai, M., Sato, M., Yasugi, T., & Tabata, T. (2011). Fat/Hippo pathway regulates
the progress of neural differentiation signaling in the Drosophila optic lobe. Development,
Growth & Differentiation, 53(5), 653–667. https://doi.org/10.1111/j.1440-
169X.2011.01279.x
Königshoff, M., Kramer, M., Balsara, N., Wilhelm, J., Amarie, O. V., Jahn, A., Rose, F., Fink,
L., Seeger, W., Schaefer, L., Günther, A., & Eickelberg, O. (2009). WNT1-inducible
signaling protein-1 mediates pulmonary fibrosis in mice and is upregulated in humans with
idiopathic pulmonary fibrosis. The Journal of Clinical Investigation, 119(4), 772–787.
https://doi.org/10.1172/JCI33950
Lafaille, F. G., Pessach, I. M., Zhang, S.-Y., Ciancanelli, M. J., Herman, M., Abhyankar, A.,
Ying, S.-W., Keros, S., Goldstein, P. A., Mostoslavsky, G., Ordovas-Montanes, J.,
Jouanguy, E., Plancoulaine, S., Tu, E., Elkabetz, Y., Al-Muhsen, S., Tardieu, M., Schlaeger,
T. M., Daley, G. Q., … Notarangelo, L. D. (2012). Impaired intrinsic immunity to HSV-1 in
human iPSC-derived TLR3-deficient CNS cells. Nature, 491(7426), 769–773.
https://doi.org/10.1038/nature11583
Lahrouchi, N. (n.d.). Homozygous frameshift mutations in FAT1 cause a syndrome characterized
by colobomatous-microphthalmia, ptosis, nephropathy and syndactyly.
https://doi.org/10.1038/s41467-019-08547-w
Lorenza, C., Anjla, P., D., A. N., & Charles, ffrench-C. (2003). Mice Lacking the Giant

60
Protocadherin mFAT1 Exhibit Renal Slit Junction Abnormalities and a Partially Penetrant
Cyclopia and Anophthalmia Phenotype. Molecular and Cellular Biology, 23(10), 3575–
3582. https://doi.org/10.1128/MCB.23.10.3575-3582.2003
Mao, Y., Mulvaney, J., Zakaria, S., Yu, T., Morgan, K. M., Allen, S., Basson, M. A., Francis-
West, P., & Irvine, K. D. (2011). Characterization of a Dchs1 mutant mouse reveals
requirements for Dchs1-Fat4 signaling during mammalian development. Development
(Cambridge, England), 138(5), 947–957. https://doi.org/10.1242/dev.057166
Mariot, V., Roche, S., Hourdé, C., Portilho, D., Sacconi, S., Puppo, F., Duguez, S., Rameau, P.,
Caruso, N., Delezoide, A., Desnuelle, C., Bessières, B., Collardeau, S., Feasson, L.,
Maisonobe, T., Magdinier, F., Helmbacher, F., Butler-Browne, G., Mouly, V., &
Dumonceaux, J. (2015). Correlation between low FAT1 expression and early affected
muscle in FSHD. Neuromuscular Disorders, 25, S312.
https://doi.org/10.1016/j.nmd.2015.06.448
Martin, D., Abba, M. C., Molinolo, A. A., Vitale-Cross, L., Wang, Z., Zaida, M., Delic, N. C.,
Samuels, Y., Lyons, J. G., & Gutkind, J. S. (2014). The head and neck cancer cell
oncogenome: a platform for the development of precision molecular therapies. Oncotarget,
5(19), 8906–8923. https://doi.org/10.18632/oncotarget.2417
Meng, P., Zhang, Y.-F., Zhang, W., Chen, X., Xu, T., Hu, S., Liang, X., Feng, M., Yang, X., &
Ho, M. (123 C.E.). Identification of the atypical cadherin FAT1 as a novel glypican-3
interacting protein in liver cancer cells. Scientific Reports |, 11, 40.
https://doi.org/10.1038/s41598-020-79524-3
Moeller, M. J., Soofi, A., Braun, G. S., Li, X., Watzl, C., Kriz, W., & Holzman, L. B. (2004).
Protocadherin FAT1 binds Ena/VASP proteins and is necessary for actin dynamics and cell
polarization. The EMBO Journal, 23(19), 3769–3779.
https://doi.org/10.1038/sj.emboj.7600380
Morin, P. J., Sparks, A. B., Korinek, V., Barker, N., Morin, P. J., Sparks, A. B., Korinek, V.,
Barker, N., Clevers, H., Vogelstein, B., & Kinzlert, K. W. (2016). Activation of β-Catenin-
Tcf Signaling in Colon Cancer by Mutations in β-Catenin or APC Clevers , Bert Vogelstein

61
and Kenneth W . Kinzler Published by : American Association for the Advancement of
Science Stable URL : http://www.jstor.org/stable/2892707 JS. 275(5307), 1787–1790.
Morris, L. G. T., Kaufman, A. M., Gong, Y., Ramaswami, D., Walsh, L. A., Turcan, Ş., Eng, S.,
Kannan, K., Zou, Y., Peng, L., Banuchi, V. E., Paty, P., Zeng, Z., Vakiani, E., Solit, D.,
Singh, B., Ganly, I., Liau, L., Cloughesy, T. C., … Chan, T. A. (2013). Recurrent somatic
mutation of FAT1 in multiple human cancers leads to aberrant Wnt activation. Nature
Genetics, 45(3), 253–261. https://doi.org/10.1038/ng.2538
Naishiro, Y., Yamada, T., Idogawa, M., Honda, K., Takada, M., Kondo, T., Imai, K., &
Hirohashi, S. (2005). Morphological and transcriptional responses of untransformed
intestinal epithelial cells to an oncogenic beta-catenin protein. Oncogene, 24(19), 3141–
3153. https://doi.org/10.1038/sj.onc.1208517
Nishioka, N., Inoue, K., Adachi, K., Kiyonari, H., Ota, M., Ralston, A., Yabuta, N., Hirahara, S.,
Stephenson, R. O., Ogonuki, N., Makita, R., Kurihara, H., Morin-Kensicki, E. M., Nojima,
H., Rossant, J., Nakao, K., Niwa, H., & Sasaki, H. (2009). The Hippo signaling pathway
components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from
inner cell mass. Developmental Cell, 16(3), 398–410.
https://doi.org/10.1016/j.devcel.2009.02.003
Nusse, R., & Clevers, H. (2017). Wnt/β-Catenin Signaling, Disease, and Emerging Therapeutic
Modalities. Cell, 169(6), 985–999. https://doi.org/10.1016/J.CELL.2017.05.016
Oh, H., & Irvine, K. D. (2008). In vivo regulation of Yorkie phosphorylation and localization.
Development, 135(6), 1081–1088. https://doi.org/10.1242/dev.015255
Pan, D. (2010). The Hippo Signaling Pathway in Development and Cancer. Developmental Cell,
19(4), 491–505. https://doi.org/10.1016/J.DEVCEL.2010.09.011
Parsons, D. W., Jones, S., Zhang, X., Lin, J. C.-H., Leary, R. J., Angenendt, P., Mankoo, P.,
Carter, H., Siu, I.-M., Gallia, G. L., Olivi, A., McLendon, R., Rasheed, B. A., Keir, S.,
Nikolskaya, T., Nikolsky, Y., Busam, D. A., Tekleab, H., Diaz, L. A. J., … Kinzler, K. W.
(2008). An integrated genomic analysis of human glioblastoma multiforme. Science (New
York, N.Y.), 321(5897), 1807–1812. https://doi.org/10.1126/science.1164382

62
Pastushenko, I., Mauri, F., Song, Y., de Cock, F., Meeusen, B., Swedlund, B., Impens, F., Van
Haver, D., Opitz, M., Thery, M., Bareche, Y., Lapouge, G., Vermeersch, M., Van Eycke,
Y.-R., Balsat, C., Decaestecker, C., Sokolow, Y., Hassid, S., Perez-Bustillo, A., …
Blanpain, C. (2021). Fat1 deletion promotes hybrid EMT state, tumour stemness and
metastasis. Nature, 589(7842), 448–455. https://doi.org/10.1038/s41586-020-03046-1
Peng, Z., Gong, Y., & Liang, X. (2021). Role of FAT1 in health and disease (Review). Oncology
Letters, 21(5), 1–13. https://doi.org/10.3892/OL.2021.12659
Perugorria, M. J., Olaizola, P., Labiano, I., Esparza-Baquer, A., Marzioni, M., Marin, J. J. G.,
Bujanda, L., & Banales, J. M. (2019). Wnt–β-catenin signalling in liver development, health
and disease. Nature Reviews Gastroenterology & Hepatology, 16(2), 121–136.
https://doi.org/10.1038/s41575-018-0075-9
Puppo, F., Dionnet, E., Gaillard, M.-C., Gaildrat, P., Castro, C., Vovan, C., Bertaux, K., Bernard,
R., Attarian, S., Goto, K., Nishino, I., Hayashi, Y., Magdinier, F., Krahn, M., Helmbacher,
F., Bartoli, M., & Lévy, N. (2015). Identification of Variants in the 4q35 Gene FAT1 in
Patients with a Facioscapulohumeral Dystrophy-Like Phenotype. Human Mutation, 36(4),
443–453. https://doi.org/https://doi.org/10.1002/humu.22760
Saburi, S., Hester, I., Goodrich, L., & McNeill, H. (2012). Functional interactions between Fat
family cadherins in tissue morphogenesis and planar polarity. Development (Cambridge,
England), 139(10), 1806–1820. https://doi.org/10.1242/dev.077461
Sadeqzadeh, E., de Bock, C. E., & Thorne, R. F. (2014). Sleeping Giants: Emerging Roles for
the Fat Cadherins in Health and Disease. Medicinal Research Reviews, 34(1), 190–221.
https://doi.org/https://doi.org/10.1002/med.21286
Sadeqzadeh, E., De Bock, C. E., Zhang, X. D., Shipman, K. L., Scott, N. M., Song, C., Yeadon,
T., Oliveira, C. S., Jin, B., Hersey, P., Boyd, A. W., Burns, G. F., & Thorne, R. F. (2011).
Dual processing of FAT1 cadherin protein by human melanoma cells generates distinct
protein products. Journal of Biological Chemistry, 286(32), 28181–28191.
https://doi.org/10.1074/jbc.M111.234419
Saxena, K., Jolly, M. K., & Balamurugan, K. (2020). Hypoxia, partial EMT and collective

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