BIOINFORMATICS ANALYSIS OF THE STRUCTURAL AND...
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BIOINFORMATICS ANALYSIS OF THE STRUCTURAL AND BIOCHEMICAL EFFECT OF THE CHEMICAL WEAPON MUSTARD GAS
ON THE P53 PROTEIN AND CELL DEATH
RITA ESMAILI
A dissertation submitted in partial fulfillment of the
requirements for the award of the degree of
Master of Science (Biotechnology)
Faculty of Bioscience and Medical engineering
Universiti Teknologi Malaysia
JUNE 2013
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ACKNOWLEDGEMENT
I am grateful to all those who have helped and encouraged me during my
journey in pursuing a M.Sc. degree. It is a matter of great pleasure and honor for me
to express my deep sense of gratitude and appreciation to my respected and learned
Research Supervisor Assoc Prof Dr. Mohd Shahir Shamsir Bin Omar for his
constant support during my study at UTM.
Besides, I would like to thank the authority of Universiti Teknologi Malaysia
(UTM) for providing me with a good environment and facilities such as Computer
laboratory to complete this project with software which I need during process. I
would like to express the best thanks to my father, brother, and sister that they
supported and made opportunity for me to further education.
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ABSTRACT
The universal and extensive usage of Sulfur Mustard gas (SM) as a
disqualification chemical warfare potential in the past century has demonstrated its
long-term toxic impacts. Sulfur mustard (SM), also known as mustard gas, is an
alkylating compound used as a chemical weapon in World War I and by Iraqi forces
against Iranians and indigenous Iraqi Kurds during the Iran–Iraq War of the 1980s.
We can notice the carcinogenic effects of exposure to SM gas. The present study
characterizes Bioinformatics analysis of the structural and biochemical effect of the
chemical weapon mustard gas on the P53 protein and cell death. Inactivation of the
p53 gene is essentially due to small mutations (missense and nonsense mutations or
insertions/deletions of several nucleotides. For this study we used from Primary
databases (experimental results directly into database), secondary databases (results
of analysis of primary databases) and aggregate of many databases (Links to other
data items, combination of data and consolidation of data).The database used
includes: IARC TP53, P53web site-free, UniProt, NCBI and PDB format. Also, we
used from some software in methodology such as PYMOL and CROMACS for
visualizing, molecular dynamics simulation and analysis. We assay the most
important of P53 region means that DNA-binding domain(DBD) from the point of
view protein stability after mutation and the its effects on cell cycle arrest (cell
death).The results have shown that all missense mutations selected in this case had
caused remarkable flexibility and stability on DBD of the P53.Structural alterations
had not been observed in DNA-binding domain, so it may be through functional
changes in the certain amino acid residues and bounding linkage to DNA.
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ABSTRAK
Penggunaan berleluasa sulfur mustard sebagai agen kimia berbahaya telah
terbukti membawa kesan tosik yang berpanjangan. Sulfur mustard (SM) juga
dikenali sebagai gas mustar, merupakan bahan beralkali yang digunakan sebagai
senjata di Perang Dunia I dan pihak tentera Iraq dalam menentang pihak Iran juga
puak-puak Iraq Kurdis semasa perang Iran-Iraq dalam tahun 1980. Pendedahan SM
membawa kesan karsinogenik. Kajian semasa mengklasifikasikan analisa
bioinformatik ke atas struktur dan kesan biokimia senjata gas mustard terhadap
protein p53 dan sel mati. Penyahaktifan gen p53 secara asasnya disebabkan oleh
mutasi gen (mutasi tidak logik atau penambahan/pengurangan beberapa
nukleotida).Pangkalan data utama (keputusan eksperimen yang langsung dimasukkan
ke dalam pangkalan data), Pangkalan data sekunder (analisa keputusan dari
pangkalan data utama) dan agregat pangkalan data (menghubungkan ke data
lain,kombinasi data dan konsolidasi data) telah digunakan dalam kajian ini.
Pangkalan data lain yang digunakan termasuklah: IRAC TP53, P53 laman sesawang,
UniProt, NCBI dan format PDB. Selain itu, perisian PYMOL dan CROMACS juga
digunakan bagi tujuan visualisasi, simulasi dinamik molekul dan analisis. Kami
assay yang paling penting P53region bermakna bahawa DNA mengikat
domain(DBD) dari sudut pandangan protein kestabilan selepas mutasi dan kesannya
terhadap kitaran menangkap sel (sel mati). Keputusan telah menunjukkan bahawa
semua mutasi missense dipilih dalam kes ini telah menyebabkan fleksibiliti yang luar
biasa dan kestabilan di DBD daripada P53 yang. Perubahan struktur itu tidak
dipelihara dalam DBD, jadi ia mungkin melalui perubahan berfungsi dalam sisa-sisa
asid amino tertentu dan Mengehad hubungan untuk DNA.
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TABLE OF CONTENTS
CHAPTER TITLE PAGE
DECLARATION iv
DEDICATION v
ACKNOWLEDGMENT vi
ABSTRACT vii
ABSTRAK viii
TABLE OF CONTENTS ix
LIST OF TABLES xii
LIST OF FIGURES xiii
1 INTRODUCTION
1.1 Background of study 1
1.2 Problem statement
1.3 Aim of the study
4
4
1.4 Objectives of the study 5
1.5 Significance of the study 5
1.6 Scope of the study 5
2 LITERATURE REVIEW
2.1 Definition of a chemical weapon 6
2.1.1 History of chemical weapons 7
2.1.2 What is Sulfur Mustard Gas? 7
2.2 Association between Mustard gas and Lung Cancer 11
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CHAPTER TITLE PAGE
2.3 TP53 (tumor protein p53)
13
2.3.1 Gene and protein structure of P53 15
2.3.2 Mutation caused by chemical compounds 19
2.3.3 Mutation on the P53 Domain 20
2.4 Mutation Analysis by software (modeling) P53 25
3 MATERIALS AND METHODS
3.1 Project planning 28
3.2 Sources for data mining 30
3.3 Identification of the P53 sequence in human 32
3.4 UniProt (Universal protein resource) 32
3.5 Finding the P53 PDB file and Doing BLAST 35
3.6 Compile information related to Tp53 and 3D
structure of the p53
36
3.7 Visualization of P53 structure and Doing mutation 38
3.7.1 Visualization of P53 structure 38
3.7.2 PyMol 38
3.7.3 Doing mutation and save the PDB 39
3.8 Structural Simulation the P53 core domain in complex
with DNA by the means of mutant one and wild type
39
3.8.1 GROMACS 39
3.9 Comparison of normal and mutant structures of P53
molecule and analysis of the results
40
3.9.1 RMSD (Root mean square deviation) 40
3.9.2 RMSD (Root mean square fluctuation) 41
4 RESULTS AND DISCUSSION
4.1 Identification of the P53 and Gene (Tp53) sequence in Human 42
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CHAPTER TITLE PAGE
4.1.1 To obtain the FASTA format of the p53 45
4.2 Finding the PDB file for human’s P53 and doing
BLAST
48
4.2.1 Protein BLAST of the P53 49
4.3 Finding the Mutated sequences and specific domain
of the p53 by SM-gas
53
4.4 Visualization of P53 Structure and Doing mutation 55
4.4.1 Visualization of the P53 55
4.4.2 The Execution of the Mutation process 58
4.5 Molecular dynamics simulation of P53 62
4.5.1 Estimative Modeling of Protein Dynamics
with GROMACS
62
4.5.2 The performance of the Molecular Dynamic
simulation to Wild Type P53
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4.5.3 Molecular Dynamic simulation to three
mutations on TP53
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4.5.3.1 RMSD Analysis and Discussion 66
4.5.3.2 RMSF Analysis and Discussion 70
5 DISCUSSION AND CONCLUSION
5.1 Discussion 72
5.2 Conclusion 73
5.3 Future work 74
RFERENCES 75
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LIST OF TABLES
TABLE NO. TITLE PAGE
2.1 Missense mutations found in Tetramerization domain 25
3.1 Databases related to SM gas and p53 31
3.2 distribution of silent and missense mutation the p53 due to
MG
37
4.1 Bio-data, p53 gene expression and distribution numerous
of mutations at 20 Iranian males exposed to sulfur mustard
during War 1982–1988
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LIST OF FIGURES
FIGURE NO. TITLE PAGE
1.1 Chemical structure. Mustard gas (bis (chloroethyl)
thioether) alkylating a DNA amine base. This compound
can eliminate a chloride ion via intermolecular
nucleophilic replacing to create a cyclic sulfonium ion.
Through binding to the amino group of the DNA strand
causes damage to DNA
2
1.2 The sulfur mustard induces DNA damage. A high
concentration of sulfur mustard (SM) over activates
PARP (poly-ADP ribolysation of cellular proteins). The
reducing NAD+ and ATP, leading to necrotic cell death.
Moremoderate SM-induced DNA damage does activate
PARP, but does not the intracellular NAD+ pool.
Activated PARP undergoes auto-modification and can
initiate cell cycle arrest, DNA damage repair and
apoptotic cell death
3
2.1 Structure of mustard gas. Atoms are color-coded:
hydrogen (white), carbon (grey), sulfur (yellow) and
chlorine (green)
8
2.2 Cross-linking of guanine residues by sulfur mustard 10
2.3 Location of the human TP53 Gene 15
2.4 Human p53 protein (Hp53) divides into five domains that
possesses specific functions
16
2.5 Organization of the human p53 gene. 22 000 bp; 11 exons
coding for a 2.2 Kb mRNA. Translation begins in exon 2
17
2.6 Homosapiens; Crystal structure of a P53 complexed 18
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FIGURE NO. TITLE PAGE
2.7 Structure of the p53 DNA binding domain bound to DNA 22
2.8 The P53 mutation in human cancers. Hot spot region 24
2.9 Duplication of GC to AT transitions and GC to TA
transversions. Transitions at a CpG dinucleotide are
depicted in gray
26
2.10 Distribution of mutational events and comparison
between exons (5-8) and (2-11) of the TP53 gene.
27
3.1 General Flow of Methodology 29
3.2 Protein name in UniProtKB (Human) 33
3.3 The sequences of p53 and FAST 34
3.4 The structure of the core of the p53 35
3.5 Schematic representation of the p53 structure 36
4.1 The entry of p53 in human 43
4.2 Isoform 1 of the p53 44
4.3 P53(Human)-NCBI 45
4.4 Search in Uniprot for p53 data 46
4.4.1 The FASTA format of p53-DNAbinding domain(uniprot) 47
4.4.2 The FASTA format of p53-DNAbinding domain(NCBI) 47
4.5.1 The PDB file and crystal structure of p53 complex with
DNA in human(1TSR)
48
4.5.2 The PDB file and crystal structure of p53 complex with
DNA in human(1TUP)
49
4.6.1 Summary of Blast.P53Homosapiens(NCBI) 50
4.6.2 Sequence alignment.p53 core domain complex with DNA
and chain A(NCBI)
50
4.6.3 PDB1TSR. Chain A,P53 core domain with DNA 51
4.7 The Blast result by Uniprot 52
4.8 The wild type p53 exons and nucleotide sequencing
54
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FIGURE NO. TITLE PAGE
4.9 P53 core domain in complex with DNA (A Chain) 55
4.10.a PyMOL. Opening the PDB file 56
4.10.b Visualization of p53 core domain with DNA structure and
residues position
57
4.11 Proline amino acid replacement with leucine at the
position 151
59
4.12 Arginine amino acid replacement with Histidine at the
position 156
60
4.13 Glycine amino acid replacement with Valine at the
position245
61
4.14 The process of performance molecular dynamic
simulation
63
4.15 The flow chart of MD simulation run to 1TSR.pdb of p53 65
4.16 The root-mean-square aberrations (RMSD) of the
backbone atoms to their first Minimized compound
structures as a function for P53-WT, P151L, R156H and
G245V in The DNA-binding domain (A chain)
68
4.17 Residue-based on Cα .RMSF related to the structure of
P53-Wildtype with Mutants p53 in DNA–binding
domain (A-chain)
69
4.18 1TSR A-chain. DNA-binding domain of P53- WT and
Representation of Loops, β-sheets and helices
71
4.19 Representation of structural 1TSR (A- chain) in WT and
mutants of p53 Without any conformational alterations
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1
CHAPTER 1
INTRODUCTION
1.1 Background of study
Today’s civilized world, in spite of international treaties and agreement that
have banned the utilization and accumulation of chemical weapons, some countries
continue to hold a significant amount of stockpile as part of their legacy arsenal or
for deterrent purposes. The relative ease to manufacture these chemical weapons
makes them the “poor man’s weapons of mass destruction”. In recent years, these
weapons are used frequently because of low cost production and lack of antidote to
chemical weapons. During the Iraq–Iran war (1981–1989), vast of chemical weapons
as an example mustard gas has caused serious injuries, morbidity, mortality, toxicity
and chronic side effects in vital organs such as eye, skin and especially the
respiratory system. Mustard gas commonly is a class of relate to cytotoxic and
vesicant chemical warfare with the power to form wide blisters on the exposed skin
and in the lungs (Nishimoto et al., 1983, 1988; Yamakido et al., 1996). Pure sulfur
mustards are colorless, viscous liquids at room temperature. Sulfur Mustard gas (bis,
2-chloroethyl-sulphid) is a strong Alkylation agent like opium/fix. Inhalation of the
mustard gas (MG), a bi-functional alkylating agent will cause severe lung damage
because of the mutagenic and carcinogenic alkylating agents which can targets the
DNA. Mustard gas formula is (SCl2 + 2 C2H4 → (Cl-CH2CH2)2S). It can be
synthesized by treating sulfur dichloride with ethylene in Depretz (Balali, 2005).
Mustard gas creates a very reactive intermediate (Figure1) and tends to
permanently alkylate the guanine nucleotide in DNA strands, which prevents cellular
division and causes the initiation of programmed cell death or apoptosis and the
2
damaged DNA will have caused the development of lung cancer (Khalili, 2009 and
Ghanei, 2010).
Figure1.1: Chemical structure. Mustard gas (bis (chloroethyl) thioether) alkylating a
DNA amine base. This compound can eliminate a chloride ion via intermolecular
nucleophilic replacing to create a cyclic sulfonium ion. Through binding to the amino
group of the DNA strand causes damage to DNA
(www.bt.cdc.gov/agent/sulfurmustard)
Mustard gas is an alkylating compound that is able to exchange hydrogen for
an alkyl group to a specific place on a molecule. Alkylating agents assault the
nitrogen, oxygen or sulfur molecule. Common alkylating agents are: Methyl iodide
(or other haloalkanes), dimethylsulfate and some carboxylic acid alkyl esters. These
chemical compounds link with various nucleophilic groups in nucleic acids and
proteins, causing mutagenic, carcinogenic, or cytotoxic effects associated cancer
(Bignold, 2006). There is evidence that the carcinogenicity of SM gas activates by
genotoxic mechanism of action that complicates DNA alkylation leading to cross-
link configuration and inhibition of DNA synthesis and repair, point mutation.
Mustard gas makes cancer of the lung (Figure 1.2).
The protein 53 or tumor protein 53 (known as human p53) is a tumor
suppressor protein that is encoded by the TP53 gene and is involved in the
pathogenesis of malignant disease. The genetic abnormalities of the TP53 gene (p53)
are the most common cause of lung cancer. This is because of the P53 mechanism
including anticancer function, and has a role in apoptosis, genomic firmness, and
inhibition of angiogenesis (http://www.p53.free.fr) TP53 is used as a marker for early
detection, prognosis and as a therapeutic option.
3
Figure 1.2: The sulfur mustard induces DNA damage. A high concentration of
sulfur mustard (SM) over activates PARP (poly-ADP ribolysation of cellular
proteins). with diminish NAD+ and ATP, it eventuate to necrotic cell death.DNA
damage due to SM does via activation of PARP, but does not the intracellular NAD+
pool. Activated PARP undergoes auto-modification, so it can cause cell cycle arrest,
DNA damage repair and apoptotic cell death (Kehek et al., 2009).
Fundamentally, inactivation of the p53 gene is pro or due to small mutations
(missense and nonsense mutations or insertions/deletions of several nucleotides). The
most common mutations critically damage sequence-specific DNA-binding and
transactivation, especially the TP53 missense mutations in human cancers appertains
with their functional effects (Joerger et al., 2006). In humans, TP53 gene located on
the short arm of chromosome 17 (17p13.1) and has 393 amino acids. Alkylating
agents has been shown to activate the mutant p53 that are used for cancer therapy.
Studies have shown that the MG affects the p53 by causing mutations within exon 5-
8. These mutations were predominately G to A transition. In some cases, there were
multiple p53 point mutations (Khalili, 2009). Other research has shown the presence
of mutations in exon 4-9 or an alteration at codon 278 (CCT→CCA) (Karami, 2007).
4
1.2 Problem statement
Currently, there is limited knowledge on how the alkylating agent of the mustard
gas affects the five domains of p53 3D structures and it potential role as a gene
suppressor affecting the process of apoptosis. Moreover to date, there is no any
bioinformatics analysis of damaged 3D structure of p53 by alkylation agents as which
can demonstrate a precise modeling and simulating of them upon to mutations. In
addition, majority of collected data which came from clinical and laboratory experiments
depicted p53 manipulated by alkylation agents at transcription level with few indication
of these results on post-translation and modification of protein structure. In addition, the
types of mutation that can occur on the p53 domains caused by the alkylation factors are
not well understood. Thus, building a precise model of these manipulations can be useful
for better understanding of p53 mutated structure and designing new effective drugs
where final structure of protein is targeted.
1.3 Aim of the study
The purpose of this study is to investigate the possible effects of
sulfur mustard on the tertiary structure of proteins 53 as well as study the
domains of the Tp53 that are more susceptible to conformation changes due
to alkylation effects. The data for this project will be sourced by both
bioinformatics databases and experimental results available in the public
domain. The data that is gathered from these databases will be analyzed
using bioinformatics software to simulate the effect of alkylation agent.
5
1.4 Objectives of the study
1.3.1 Finding out the effects of SM gas on Tp53 and protein structure
and dynamics
1.3.2 Determining the sequence, domain and conformation of the
mutated of p53 and modeling the type of mutated
1.3.3 Comparing and analyzing the structural dynamics between the
Wild type and mutation of the p53 due to the effect of MG
1.5 Significance of the study
This study will help to understand the MG effects on the p53 structure and
conformational changes via simulation. The obtained output enables to interpret
useful information regarding the alkylating agent pathway on DNA. On the other
hand, data will help us about reverse mutation via simulating and modeling of the
mutation by using bioinformatics’ tools.
1.6 Scope of the study
In this research, the focus will be on Tp53 mutation in lung cancer that
related to effects of MG. The information will be gathered from both bioinformatics
databases and experimental results. The data will be mined, mapped and modeled
using bioinformatics web-based tools and at the end, comparing the protein structure
of the mutated and wild type of Tp53.
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