More than 50% of all cancers contain a mutation in the p53 gene.

The protein made from

this gene is critical for ridding the body of cells that have developed genetic mistakes that could

lead to cancer. When it stops working, precancerous cells can slip by and a tumor may develop.
1. The transactivation domain or trans-activating domain (TAD) is a transcription
factor scaffold domain which contains binding sites for other proteins such
as transcription coregulators.

2. The proline-rich regions are known to preferentially adopt a polyproline type II helical
conformation. Proline-rich regions often contain serine or threo-nine, which frequently
occur as the residue immediately preceding the proline.

3. Tetramerization domain: In its active conformation, p53 is tetrameric and one domain –
the tetramerization domain – permits the oligomerization of this protein. (The p53
protein is active when it is tetrameric).

4. Tetrameric: a molecule (such as an enzyme or a polymer) that consists of four structural

subunits (such as peptide chains or condensed monomers) tetrameric.
The domain organization of p53 includes two N-terminal transcriptional activation domains
(TADs), a proline-rich domain, a DNA-binding domain, a tetramerization (Tet) domain, and a
basic region. The sequence alignment of the TADs of mouse and human p53 is shown, with the
asterisks indicating the location of the mutations in the TAD mutant knock-in strains. The LW
residues mutated in the p53 knock-in mouse strain correspond to amino acids 25 and 26 in mouse
p53 and 22 and 23 in human p53, and the FF residues mutated in the p53,54 knock-in mouse
correspond to amino acids 53 and 54 in both mouse and human p53 (marked in red). A variety of
protein cofactors that regulate chromatin remodeling and/or transcriptional initiation interact
with p53 via one or both TADs, including the transcriptional regulator proteins TAF6, TAF9,
TBP, TRAP80, TFIIH, and histone acetyltransferases p300, CBP, and GCN5, a component of the
human STAGA [STP3–TAF(II)31–GCN5–L acetylase] complex. Please note that the exact
boundaries of the interaction sites are not precise.
Transactivation (Increased rate of Transcription).

(a) (Left) Transactivation domain 1 (TAD1) and robust (strong) transactivation of

classical p53 target genes are required for responses to acute DNA damage, including
apoptosis and cell-cycle arrest. A p53 TAD1 mutant, p53 25,26, is unable to activate
efficiently the expression of canonical p53 target genes, including p21, Noxa, Perp and
Puma. P53 is also unable to induce apoptosis or cell-cycle arrest in response to acute
DNA damage. (Right) Transactivation by either TAD1 or TAD2 allows p53 responses to
oncogene activation. P53 can efficiently activate expression of only a limited number of
mostly novel target genes, but can promote tumor suppression in a number of mouse
models. The tumor suppressor capability of p53 25,26 can be explained by its ability to
activate robustly a limited set of novel direct p53 target genes (Sidt2, Phlda3, Abhd4,
etc.). The capacity of p53 25,26

to promote very low-level activation of various classical p53 target genes may also
contribute to tumor suppression. The ‘?’ denotes additional, still-unknown genes crucial
for responses to acute DNA damage and oncogene activation. (b) Comparison of gene
expression profiles of p53 wild-type and p53-null HrasV12-MEFs reveals more than
1000 differentially expressed genes. To enrich for genes with specific roles in tumor
suppression, we used gene expression profiling data generated with the p53

mutant, which activates only a small subset of p53 target genes, but is a potent tumor
suppressor. Using transcriptomics analysis of HrasV12-MEFs, we identified genes
induced at least twofold and within 1.5 standard deviations by p53

Wt., p53 25,26 and p53 53,54, which all have tumor-suppressor activity, relative to p53
samples, which lack tumor-suppressor activity. This list of 130 genes was then filtered for
those commonly downregulated in human and mouse tumors according to the EBI Gene
Atlas database. A group of 14 candidate genes with probable roles in p53-mediated tumor
suppression was defined by this analysis.
Mdm2 is an important negative regulator of the p53 tumor suppressor. It is an inhibitor of p53
transcriptional activation.
 Programmed cell death is a cell fate, an odd sort of cell fate but nonetheless one
that is essential.

 Cell death keeps our hands from being webbed, our embryonic tails from
persisting, our immune system from responding to our own proteins, and our brain
from being filled with useless electrical connections.

 In fact, the majority of cells generated during brain development die during
development.

 Programmed Cell Death Occurs Through Apoptosis:

 The demise of cells by programmed cell death is marked by a well-defined

sequence of morphological changes, collectively referred to as apoptosis, a Greek
word that means “dropping off” or “falling off,” as leaves from a tree.

 Dying cells shrink and condense and then fragment, releasing small membrane
bound apoptotic bodies, which generally are engulfed by other cells.
 The nuclei condense and the DNA is fragmented.

 Importantly, the intracellular constituents are not released into the extracellular
milieu where they might have deleterious effects on neighboring cells.

 The highly stereotyped changes accompanying apoptosis suggested to early

workers that this type of cell death was under the control of a strict program.

 This program is critical during both embryonic and adult life to maintain normal
cell number and composition.

The genes involved in controlling cell death encode proteins with three distinct
functions:

1. “Killer” proteins are required for a cell to begin the apoptotic process.

2. “Destruction” proteins do things like digest DNA in a dying cell.

3. “Engulfment” proteins are required for phagocytosis of the dying cell by another
cell.
(B

Death by Injury vs. Death by Suicide

(Necrosis vs. Apoptosis)
At first engulfment seems to be simply an after-death cleanup process, but some evidence
suggests that it is part of the final death decision. For example, mutations in killer genes always
prevent cells from initiating apoptosis, whereas mutations that block engulfment sometimes
allow cells to survive that would normally die. That is, cells with engulfment-gene. Mutations
can initiate apoptosis but then sometimes recover.

In contrast to apoptosis, cells that die in response to tissue damage exhibit very different
morphological changes, referred to as necrosis.

Typically, cells that undergo this process swell and burst, releasing their intracellular contents,
which can damage surrounding cells and frequently cause inflammation.
The two best understood signaling pathways that activate a caspase cascade in
mammalian cells are the extrinsic pathway and the intrinsic pathway.

Each pathway uses its own initiator procaspases and activation complex.
The three major
mammalian factions
of the Bcl-2 family.
The BH3-only proteins
(yellow) are essential
initiators of apoptosis
that primarily
antagonize their pro-
survival relatives
(blue), whereas either
Bax or Bak (red) is
required downstream
of Bcl-2.
In a healthy cell, the outer membranes of its mitochondria display the protein Bcl-2 on
their surface. Internal damage to the cell (e.g., from reactive oxygen species) causes

Bcl-2 to activate a related protein, Bax, which punches holes in the outer mitochondrial
membrane, causing cytochrome c to leak out. The released cytochrome c binds to the
protein Apaf-1 ("apoptotic protease activating factor-1").

Using the energy provided by ATP, these complexes aggregate to form apoptosomes.

The apoptosomes bind to and activate caspase-9.

Caspase-9 is one of a family of over a dozen caspases. They are all proteases. They get
their name because they cleave proteins — mostly each other — at aspartic acid (Asp)
residues).

Caspase-9 cleaves and, in so doing, activates other caspases (caspase-3 and -7). The
activation of these "executioner" caspases creates an expanding cascade of proteolytic
activity (rather like that in blood clotting and complement activation) which leads to
digestion of structural proteins in the cytoplasm, degradation of chromosomal DNA, and
phagocytosis of the cell.
Either excessive or insufficient apoptosis can contribute to disease.
 EPIGENETICS GENETICS

Heritable transmission Heritable transmission

of information in the of information based
absence of changes in on differences in DNA
DNA sequence. sequence.

Alterations
SNP

MTHFR, C677T

GCC→GTC
Received the Nobel Prize in Chemistry in 2015: Tomas Lindahl for base excision repair.
Paul Modrich for mismatch repair. Aziz Sancar for nucleotide excision repair.
Epigenetics modification:
Changing in gene expression without affecting the primary DNA sequence.

So, epigenetic modifications regulate the processes during the transition from genotype to
phenotype.

Epigenetic modification including DNA methylation, histone modification and micro RNAs.
Hypomethylation is a process that leads to gene activation (oncogenes) and is carried out by two
mechanisms.

Passive process: loss or reduction of DNMT enzyme during cell division.

Active process: ten-eleven translocation (TET) enzyme via an enhanced oxidation mechanism
leads to convert methylated cytosine to unmethylated cytosine.

Hypermethylated is a process that leads to gene inactivation (Tumor suppressor genes). which
suggested an increase of DNMTs enzymes activity in cancer cells compared to normal cells.
Histone modification:
Histone proteins (H2A, H2B, H3 and H4) have N- and C- terminals that form histone tails,
where post-translation modifications often take place causing changes of chromatin structure,
including phosphorylation, methylation, and acetylation.

Histone acetylation:
Histone acetyl transferase (HAT): tend to add acetyl group on lysine residue on histone tail,
which leads to chromatin becoming permissive and as a consequence a transcription process
takes place.

Histone deacetylase (HDAC): tend to remove an acetyl group, causing stability of chromatin
structure and expression inhibition.
Histone methylation:
Histone methylation takes place on lysine or arginine residues, and is regulated by:

Histone methyltransferases that transfer methyl groups from SAM to amino groups or
guanidino group of lysine or arginine respectively.

Histone demethylases, which function antagonistically to histone methyltransferases by

removing a methyl group.

MicroRNAs:
MicroRNAs (miRNAs), belong to a non-coding RNA family and consist of 17-25 nucleotides.

miRNAs epigenetically contribute in governing gene expression in various pathways, for

instance miRNA expression may be controlled by epigenetic mechanisms, miRNAs are involved
in inhibition of some epigenetic elements, or cooperation between miRNA and epigenetic
elements is required to achieve a specific task.

In addition, some of 50% miRNAs, that are placed in CpG islands, have been regulated by
DNA methylation and they were abnormally expressed in different tumours including miR-
31 (breast cancer) and miR-124a (colon cancer).
Different types of DNA damage could be fixed by specific repair mechanisms. In BER/SSB repair, the poly (ADP-
ribose) polymerase protein 1 (PARP1) is activated and then recognizes the DNA lesion by the zinc-binding
domain. Subsequently, PARP1 assembles poly (ADP)-ribose (PAR)chains on target proteins, including histone
H1,H2B and PARP1 itself, and the PAR structures act as platforms to recruit multiple protein complexes which
function in DNA repair and chromatin modification.