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 iii

Transcript of BIOINFORMATICS ANALYSIS OF THE STRUCTURAL AND...

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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This dissertation is dedicated to:

My decedent Mother

My father

My brother and sister

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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

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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

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2.1 Structure of mustard gas. Atoms are color-coded:

hydrogen (white), carbon (grey), sulfur (yellow) and

chlorine (green)

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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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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

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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).

 

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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.

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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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