p53 Missense Mutation Cancer

Outline

• Disease related to p53 • Role and regulation pathway• Structure of p53 • Missense mutation and consequences• Experiment’s proposal of p53 missense

mutant cancer

I. Diseases cause by mutated p53

• Aneuploidy phenotype• Li-Fraumeni syndrome (rare) • 50% of human cancers• Breast and colonrectum cancers• Bladder cancers• Lung cancer• Hypoxia • Glioblastoma multiforme (GBM)

ROLES AND REGULATIONS IN p53

TP53 Gene

• TP53 encodes p53 protein, located on short arm of chromosome 17 at position 13.1

Roles of p53• p53 before was oncogene • “Guardian of the genome” • p53 gene today—tumor

suppressor or anticancer• P53 functions at all

checkpoints during the cell cycle

Roles of p53

• Functions as transcription factor

Regulations of p53

• P53 has very complicated pathway and network as the tumor suppressor gene

• Activation state: 1. upstream pathways 2. downstream pathways

• Inactivation state

Active state• p53 is always in an off stage• Only activate when cells are stressed or damaged

1. Upstream Regulation of p53

In stressed conditions, p53 is activated by:

2. Downstream Activation of p53

The turn on of p53 lead to:• cell-cycle

regulation, • initiate growth

arrest• induction of

apoptosis• control

development and differentiation

Inactivation state

• In unstressed condition, p53 is inactivate by Mdm2-binding

• Mdm2 is an ubiquitin ligase

Concentration Level of p53 • P53 is always in an off stage• Activation of p53 always results in apoptotic

and cell cycle arrest• Apoptosis induced and leads to increase level of

p53• MDM2 control the level of p53 creating

negative feedback loop• MDM2 is an E3 ligase that targets p53 to

ubiquitination

Stability of p53’s

STRUCTURE OF p53

Structures of p53

• Same protein family of p63 and p73• P53,p63, p73 family of transcription factor• A nuclear phosphoprotein of MW 53kDa• Open reading frame consists of 393 amino acids• Has 7 domains• The central part is where DNA contacts with the protein

• N-teminus (TAD)1. Transactivation 2. Activation domain 2 (AD2)3. Proline rich region

• Central core4. DNA-binding core domain (DBD)– has Zn and several Arginine amino acids

• C-terminus A. Tetramerization:

5. Nuclear localization signaling domain (NLS)6. Homo-oligmerisation domain (OD)

B. Down regulation of DNA binding:7. regulatory domain

Crystal structure of p53 at Core Domain

The DNA binding domain is folding as beta barrel or beta-sandwich

Structure of p53 • p53 forms a tetramer : _ 4 helical bundle fold

_essentials for DNA binding, protein-protein

interactions, post translational modifications, and p53 degradation

_oligomerisation domain consists of a dimer of dimers

Tetramer and DNA

Missense Mutations in p53

p53 Missense Mutation • Majority of p53 mutation occurs at DNA binding core• >50% occurs at single missense mutation• Hydrophobic and unstable• Loss of stability and loss of molecular contact inactivate p53• Destabilize and deformed p53

Distribution between cancer and stability of p53

Distribution of molecular contacts

Residues in Unstable MutationTable 2. Fractions of unstable mutants (>3kT) from codons 96-289 before and after substitution

Residues in Unstable Missense Mutation

• Common mutated residues are R248 and R273. These two residue directly binding to DNA ligand.• R175, R248, and R282 are help stabilizing the

structure of DNA binding interface.

Significant of p53 to Cancer Treatment

• Target the protein and the pathways of the protein might rescue p53 and restore its functions.

EXPERIMENT

PURPOSE OF THE EXPERIMENT1st Goal: Design the input files and the starting structures for p53 cancer mutants.2nd Goal: Using the AMBER software to run the stimulation and to compare the binding affinity between the mutations and the native states of p53, either with or without DNA ligand.

Hypothesis • Missense mutation due to the destabilize and deformation in the

DNA binding domain (DBD) deactivate p53, affecting protein-ligand interaction.

• Missense mutated at R248, R249 and R282 position will prevent the transcription activation and destroy the ability of p53 to bind to its target DNA sequence.

p53’s mutation sites

Methods• Downloading the crystal structure of 1TRS as native state of p53

protein• Creating and designing a series of input files (prmtop and inpcrd) to

run the stimulations for the both the native state and the destabilized cancer mutants at the sites of R248, R249 and R282

• Compare the stimulated stabilities between these two mutants with and without DNA binding by using MM-PBSA in the Amber software

CHAIN B

The End

Crystal Structure of p53

Function of p53P53 has many anticancer mechanisms, and plays a role in apoptosis, genetic stability, and inhibition of angiogenesis. In its anti-cancer role, p53 works through several mechanisms:• It can activate DNA repair proteins when DNA has sustained damage.• It can induce growth arrest by holding the cell cycle at the G1/S regulation point

on DNA damage recognition (if it holds the cell here for long enough, the DNA repair proteins will have time to fix the damage and the cell will be allowed to continue the cell cycle).

• It can initiate apoptosis, the programmed cell death, if DNA damage proves to be irreparable.

Activated p53 binds DNA and activates expression of several genes including WAF1/CIP1 encoding for p21. p21 (WAF1) binds to the G1-S/CDK (CDK2) and S/CDK complexes (molecules important for the G1/s transition in the cell cycle) inhibiting their activity.

Diseases 1. Loss of p53 creates genomic instability that

most often results in the aneuploidy phenotype.[28]

2. Li-Fraumeni syndrome (rare)3. the mutation or loss of the p53 gene can

be identified in more than 50% of all human cancer cases worldwide.

Structure of p53• Encode by TP53 gene, a 20 Kb gene that contains 11 exons and 10 introns• Located on the small arm of chromosome 17• Highly conserved gene