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UNIT-1

CELL CYCLE, CELL SIGNALING AND CELL DIFFERENTIATION

CONTENTS:

Cell cycle

Various check points during cell cycle (G1 to S, progression of S phase, G2 to M phase)

Cyclins (Role of cyclins, Synthesis and degradation of cyclins, Structural features)

CDKs (Role and functions)

Activation and inactivation of cyclin dependent kinases

Role of RBs, E2Fs, and DP proteins

P53

Different types of Cyclin dependent kinases-CDKs

CDC25, CAKs, Wee1 proteins, nim-proteins

SCF and APC complexes (cyclosomes)

Licensing factors

Cellular differentiation

Epigenetic control & mechanisms of epigenetic regulation

Yamanaka factors, Oct4, Sox2, Nanog

DNA methylation

Histone acetylation and methylation

Role of signaling in epigenetic control

mTOR pathway

Apoptosis & Necrosis

Cell signaling mechanisms: MAPK/ERK pathway

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Cell cycle

The cell cycle, or cell-division cycle, is the series of events that take place in a cell that
cause it to divide into two daughter cells. These events include the duplication of its
DNA (DNA replication) and some of its organelles, and subsequently the partitioning of
its cytoplasm and other components into two daughter cells in a process called cell
division.

In cells with nuclei (eukaryotes), (i.e., animal, plant, fungal, and protist cells), the cell
cycle is divided into two main stages: interphase and the mitotic (M) phase (including
mitosis and cytokinesis). During interphase, the cell grows, accumulating nutrients
needed for mitosis, and replicates its DNA and some of its organelles. During the mitotic
phase, the replicated chromosomes, organelles, and cytoplasm separate into two new
daughter cells. To ensure the proper replication of cellular components and division,
there are control mechanisms known as cell cycle checkpoints after each of the key
steps of the cycle that determine if the cell can progress to the next phase.

Phases of cell cycle

The eukaryotic cell cycle consists of four distinct phases: G1 phase, S phase (synthesis),
G2 phase (collectively known as interphase) and M phase (mitosis and cytokinesis). M
phase is itself composed of two tightly coupled processes: mitosis, in which the cell's
nucleus divides, and cytokinesis, in which the cell's cytoplasm divides forming two
daughter cells. Activation of each phase is dependent on the proper progression and
completion of the previous one. Cells that have temporarily or reversibly stopped
dividing are said to have entered a state of quiescence called G0 phase.

Table 1: Phases of cell cycle

State Phase Abbreviation Description

A phase where the cell has left the cycle and has stopped
Resting Gap 0 G0
dividing.

Cells increase in size in Gap 1. The G1 checkpoint control

Gap 1 G1
mechanism ensures that everything is ready for DNA synthesis.

Interphase
Synthesis S DNA replication occurs during this phase.

Gap 2 G2 During the gap between DNA synthesis and mitosis, the cell will
continue to grow. The G2 checkpoint control mechanism ensures
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that everything is ready to enter the M (mitosis) phase and

divide.

Cell growth stops at this stage and cellular energy is focused on

Cell the orderly division into two daughter cells. A checkpoint in the
Mitosis M
division middle of mitosis (Metaphase Checkpoint) ensures that the cell
is ready to complete cell division.

After cell division, each of the daughter cells begin the interphase of a new cycle.
Although the various stages of interphase are not usually morphologically
distinguishable, each phase of the cell cycle has a distinct set of specialized biochemical
processes that prepare the cell for initiation of cell division.

G0 phase

G0 is a resting phase where the cell has left the cycle and has stopped dividing. The cell
cycle starts with this phase. Non-proliferative (non-dividing) cells in multicellular
eukaryotes generally enter the quiescent G0 state from G1 and may remain quiescent
for long periods of time, possibly indefinitely (as is often the case for neurons). This is
very common for cells that are fully differentiated. Some cells enter the G0 phase semi-
permanently and are considered post-mitotic, e.g., some liver, kidney, and stomach cells.
Many cells do not enter G0 and continue to divide throughout an organism's life, e.g.,
epithelial cells.

The word "post-mitotic" is sometimes used to refer to both quiescent and senescent
cells. Cellular senescence occurs in response to DNA damage and external stress and
usually constitutes an arrest in G1. Cellular senescence may make a cell's progeny
nonviable; it is often a biochemical alternative to the self-destruction of such a damaged
cell by apoptosis.

Interphase

Interphase is a series of changes that takes place in a newly formed cell and its nucleus
before it becomes capable of division again. It is also called preparatory phase or
intermitosis. Typically interphase lasts for at least 91% of the total time required for the
cell cycle. Interphase proceeds in three stages, G1, S, and G2, followed by the cycle of
mitosis and cytokinesis. The cell's nuclear DNA contents are duplicated during S phase.

G1 Phase (First Gap)

The first stage of interphase is called the G1 phase (first gap) because, from a
microscopic aspect, little change is visible. However, during the G1 stage, the cell is quite
active at the biochemical level. The cell is accumulating the building blocks of
chromosomal DNA and the associated proteins as well as accumulating sufficient energy
reserves to complete the task of replicating each chromosome in the nucleus.

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S phase (DNA replication)

The ensuing S phase starts when DNA synthesis commences; when it is complete, all of
the chromosomes have been replicated, i.e., each chromosome consists of two sister
chromatids. The centrosome is duplicated during the S phase. The two centrosomes will
give rise to the mitotic spindle, the apparatus that orchestrates the movement of
chromosomes during mitosis. At the center of each animal cell, the centrosomes of
animal cells are associated with a pair of rod-like objects, the centrioles, which are at
right angles to each other. Centrioles help organize cell division. Thus, during this phase,
the amount of DNA in the cell has doubled, though the ploidy and number of
chromosomes are unchanged. Rates of RNA transcription and protein synthesis are very
low during this phase. An exception to this is histone production, most of which occurs
during the S phase.

G2 phase (growth)

G2 phase occurs after DNA replication and is a period of protein synthesis and rapid cell
growth to prepare the cell for mitosis. During this phase microtubules begin to
reorganize to form a spindle (preprophase). Before proceeding to mitotic phase, cells
must be checked at the G2checkpoint for any DNA damage within the chromosomes.
The G2 checkpoint is mainly regulated by the tumor protein p53. If the DNA is damaged,
p53 will either repair the DNA or trigger the apoptosis of the cell. If p53 is dysfunctional
or mutated, cells with damaged DNA may continue through the cell cycle, leading to the
development of cancer.

Mitotic phase (chromosome separation)

The relatively brief M phase consists of nuclear division (karyokinesis). It is a relatively

short period of the cell cycle. M phase is complex and highly regulated. The sequence of
events is divided into phases, corresponding to the completion of one set of activities
and the start of the next. These phases are sequentially known as:

Prophase, prometaphase , metaphase, anaphase, telophase

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Table: 2 Various stages of Mitotic phase

Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell
nucleus into two identical sets in two nuclei. During the process of mitosis the pairs of
chromosomes condense and attach to microtubules that pull the sister chromatids to
opposite sides of the cell.

Mitosis occurs exclusively in eukaryotic cells, but occurs in different ways in different
species. For example, animal cells undergo an "open" mitosis, where the nuclear
envelope breaks down before the chromosomes separate, while fungi such as
Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis,
where chromosomes divide within an intact cell nucleus.

Karyokinesis, also known as mitosis, is divided into a series of phases—prophase,

prometaphase, metaphase, anaphase, and telophase—that result in the division of the
cell nucleus . Karyokinesis is also called mitosis.

Cytokinesis phase (separation of all cell components)

Mitosis is immediately followed by cytokinesis, which divides the nuclei, cytoplasm,

organelles and cell membrane into two cells containing roughly equal shares of these
cellular components. Mitosis and cytokinesis together define the division of the mother
cell into two daughter cells, genetically identical to each other and to their parent cell.
This accounts for approximately 10% of the cell cycle.

Because cytokinesis usually occurs in conjunction with mitosis, "mitosis" is often used
interchangeably with "M phase". However, there are many cells where mitosis and
cytokinesis occur separately, forming single cells with multiple nuclei in a process called
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endoreplication. This occurs most notably among the fungi and slime molds, but is
found in various groups. Even in animals, cytokinesis and mitosis may occur
independently, for instance during certain stages of fruit fly embryonic development.
Errors in mitosis can result in cell death through apoptosis or cause mutations that may
lead to cancer.

Fig. 1 Cytokinesis

Cell cycle exit

Some types of cells divide rapidly, and in these cases, the daughter cells may
immediately undergo another round of cell division. For instance, many cell types in an
early embryo divide rapidly, and so do cells in a tumor. Other types of cells divide
slowly or not at all. These cells may exit the G1 phase and enter a resting state called G0
phase. In G0, a cell is not actively preparing to divide, it’s just doing its job. For instance,
it might conduct signals as a neuron (like the one in the drawing below) or store
carbohydrates as a liver cell. G0 is a permanent state for some cells, while others may
re-start division if they get the right signals.

Not all cells adhere to the classic cell cycle pattern in which a newly formed daughter
cell immediately enters the preparatory phases of interphase, closely followed by the
mitotic phase. Cells in G0 phase are not actively preparing to divide. The cell is in a
quiescent (inactive) stage that occurs when cells exit the cell cycle. Some cells enter G0
temporarily until an external signal triggers the onset of G1. Other cells that never or
rarely divide, such as mature cardiac muscle and nerve cells, remain in G0 permanently.

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Various check points during cell cycle (G1 to S, progression of S phase,

G2 to M phase)

Fig. 2 Checkpoints

A checkpoint is a stage in the eukaryotic cell cycle at which the cell examines internal
and external cues and "decides" whether or not to move forward with division.

There are a number of checkpoints, but the three most important ones are:

 The G1 checkpoint, at the G1/S transition.

 The G2 checkpoint, at the G 2/M transition.
 The spindle checkpoint, at the transition from metaphase to anaphase.

Fig. 3 Diagram of cell cycle with major 3 checkpoints

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G1 checkpoint is near the end of G1 (close to the G1/S transition). G2 checkpoint is near
the end of G2 (close to the G2/M transition). Spindle checkpoint is partway through M
phase, and more specifically, at the metaphase/anaphase transition.

The G1 checkpoint
The G1 checkpoint is the main decision point for a cell – that is, the primary point at
which it must choose whether or not to divide. Once the cell passes the G1 checkpoint
and enters S phase, it becomes irreversibly committed to division. That is, barring
unexpected problems, such as DNA damage or replication errors, a cell that passes the
G1 checkpoint will continue the rest of the way through the cell cycle and produce two
daughter cells.

Fig. 4 The G1 checkpoint

The G1 checkpoint is located at the end of G1 phase, before the transition to S phase. If
cells don't pass the G1 checkpoint, they may "loop out" of the cell cycle and into a resting
state called G0, from which they may subsequently re-enter G1 under the appropriate
conditions.

At the G1 checkpoint, cells decide whether or not to proceed with division based on
factors such as:

Cell size, Nutrients, Growth factors, DNA damage

At the G1 checkpoint, a cell checks whether internal and external conditions are right
for division. Here are some of the factors a cell might assess:

 Size. Is the cell large enough to divide?

 Nutrients. Does the cell have enough energy reserves or available nutrients to divide?

 Molecular signals. Is the cell receiving positive cues (such as growth factors) from
neighbours?
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 DNA integrity. Is any of the DNA damaged?

These are not the only factors that can affect progression through the G1 checkpoint,
and which factors are most important depend on the type of cell. For instance, some
cells also need mechanical cues (such as being attached to a supportive network called
the extracellular matrix) in order to divide.

If a cell doesn’t get the go-ahead cues it needs at the G1 checkpoint, it may leave the cell
cycle and enter a resting state called G0 phase. Some cells stay permanently in G0,
while others resume dividing if conditions improve.

The G2 checkpoint

Fig. 5 Cell cycle with the G2 checkpoint marked

At the G2 checkpoint, the cell checks for: DNA damage, DNA replication completeness

To make sure that cell division goes smoothly (produces healthy daughter cells with
complete, undamaged DNA), the cell has an additional checkpoint before M phase, called
the G2 checkpoint.

At this stage, the cell will check:

 DNA integrity. Is any of the DNA damaged?

 DNA replication. Was the DNA completely copied during S phase?

If errors or damage are detected, the cell will pause at the G 2 checkpoint to allow for
repairs. If the checkpoint mechanisms detect problems with the DNA, the cell cycle is
halted, and the cell attempts to either complete DNA replication or repair the damaged
DNA.

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If the damage is irreparable, the cell may undergo apoptosis, or programmed cell death.
This self-destruction mechanism ensures that damaged DNA is not passed on to
daughter cells and is important in preventing cancer.

The spindle checkpoint

Fig. 6 Cell cycle with the spindle checkpoint marked

At the spindle checkpoint, the cell checks for: Chromosome attachment to spindle at the
metaphase plate.

The M checkpoint is also known as the spindle checkpoint: here, the cell examines
whether the entire sister chromatids are correctly attached to the spindle microtubules.
Because the separation of the sister chromatids during anaphase is an irreversible step,
the cycle will not proceed until all the chromosomes are firmly attached to at least two
spindle fibers from opposite poles of the cell.

How does this checkpoint work?

It seems that cells don't actually scan the metaphase plate to confirm that all of the
chromosomes are there. Instead, they look for "straggler" chromosomes that are in the
wrong place (e.g., floating around in the cytoplasm). If a chromosome is misplaced, the
cell will pause mitosis, allowing time for the spindle to capture the stray chromosome.

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Cyclins

Cyclins are among the most important core cell cycle regulators. Cyclins are a group of
related proteins, and there are four basic types found in humans and most other
eukaryotes: G1 cyclins, G1/S cyclins, S cyclins, and M cyclins.

As the names suggest, each cyclin is associated with a particular phase, transition, or set
of phases in the cell cycle and helps drive the events of that phase or period. For
instance, M cyclin promotes the events of M phase, such as nuclear envelope breakdown
and chromosome condensation.

Fig. 7 the cyclin expression cycle. This is a graph showing how concentrations of
the various cyclins change in a cell over the course of the cell cycle.

G1 cyclin: low in G1, rising slowly to a peak in mid-S phase, then dropping slowly back
down to zero at the end of M phase.

G1/S cyclin: very low for most of the cell cycle, with a sharp, symmetrical peak at the
G1/S transition.

S cyclin: low in early G1, rising slowly through late G1 and S, peaking in early G2 and
dropping sharply back to zero in early M phase.

M cyclin: very low through all of G1, rising slowly through, peaking at the G2/M
transition, and dropping sharply to zero in the middle of M phase.

The levels of the different cyclins vary considerably across the cell cycle, as shown in the
diagram at right. A typical cyclin is present at low levels for most of the cycle, but
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increases strongly at the stage where it's needed. M cyclin, for example, peaks
dramatically at the transition from G2 to M phase. G1 cyclins are unusual in that they
are needed for much of the cell cycle.

Cyclin-dependent kinases

In order to drive the cell cycle forward, a cyclin must activate or inactivate many target
proteins inside of the cell. Cyclins drive the events of the cell cycle by partnering with a
family of enzymes called the cyclin-dependent kinases (Cdks). A lone Cdk is inactive,
but the binding of a cyclin activates it, making it a functional enzyme and allowing it to
modify target proteins. Cyclin-dependent kinases are the families of protein kinases first
discovered for their role in regulating the cell cycle. They are also involved in regulating
transcription, mRNA processing, and the differentiation of nerve cells.

Fig. 8 CDKs linked to cyclins

Cdks are kinases, enzymes that phosphorylate (attach phosphate groups to) specific
target proteins. The attached phosphate group acts like a switch, making the target
protein more or less active. When a cyclin attaches to a Cdk, it has two important
effects: it activates the Cdk as a kinase, but it also directs the Cdk to a specific set of
target proteins, ones appropriate to the cell cycle period controlled by the cyclin. For
instance, G1/S cyclins send Cdks to S phase targets (e.g., promoting DNA replication),
while M cyclins send Cdks to M phase targets (e.g., making the nuclear membrane break
down).

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Fig. 9 Simplified diagram showing how cyclins modify activity of Cdks.

Left panel (no cyclin): no cyclin is present, Cdk is inactive, and targets specific to the
G1/S transition are not phosphorylated. Nothing happens, and S phase factors remain
"off."

Right panel (+G1/S cyclin): the G1/S cyclin is present and binds to the Cdk. The Cdk is
now active and phosphorylates various targets specific to the G1/S transition. The
phosphorylated targets cause the activation of DNA replication enzymes, and S phase
begins.

In general, Cdk levels remain relatively constant across the cell cycle, but Cdk activity
and target proteins change as levels of the various cyclins rise and fall. In addition to
needing a cyclin partner, Cdks must also be phosphorylated on a particular site in order
to be active (not shown in the diagrams in this article), and may also be negatively
regulated by phosphorylation of other sites.

Cyclins and Cdks complex

Cyclins and Cdks are very evolutionarily conserved, meaning that they are found in
many different types of species, from yeast to frogs to humans. The details of the system
vary a little: for instance, yeast has just one Cdk, while humans and other mammals
have multiple Cdks that are used at different stages of the cell cycle. The basic principles
are quite similar, so that Cdks and the different types of cyclins can be found in each
species.

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Fig. 10 Various cyclin-CDK complex at different stages of cell cycle

Most of the known cyclin-CDK complexes regulate the progression through the cell
cycle. Animal cells contain at least nine CDKs, four of which, CDK1, 2, 3, and 4, are
directly involved in cell cycle regulation. In mammalian cells, CDK1, with its partners
cyclin A2 and B1, alone can drive the cell cycle. Another one, CDK7, is involved
indirectly as the CDK-activating kinase. Cyclin-CDK complexes phosphorylate substrates
appropriate for the particular cell cycle phase. Cyclin-CDK complexes of earlier cell-
cycle phase help activate cyclin-CDK complexes in later phase.

Table 3: CDK with its cyclin partner

CDK Cyclin partner Function

Cdk1 Cyclin B M phase

Cdk2 Cyclin E G1/S transition

Cdk2 Cyclin A S phase, G2 phase

Cdk3 Cyclin C G1 phase ?

Cdk4 Cyclin D G1 phase

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Maturation-promoting factor (MPF)

A famous example of how cyclins and Cdks work together to control cell cycle
transitions is that of maturation-promoting factor (MPF). The name dates back to the
1970s, when researchers found that cells in M phase contained an unknown factor that
could force frog egg cells (stuck in G2 phase) to enter M phase. This mystery molecule,
called MPF, was discovered in the 1980s to be a Cdk bound to its M cyclin partner.

MPF provides a good example of how cyclins and Cdks can work together to drive a cell
cycle transition. Like a typical cyclin, M cyclin stays at low levels for much of the cell
cycle, but builds up as the cell approaches the G2/M transition. As M cyclin accumulates,
it binds to Cdks already present in the cell, forming complexes that are poised to trigger
M phase. Once these complexes receive an additional signal (essentially, an all-clear
confirming that the cell’s DNA is intact), they become active and set the events of M
phase in motion.

The MPF complexes add phosphate tags to several different proteins in the nuclear
envelope, resulting in its breakdown (a key event of early M phase), and also activate
targets that promote chromosome condensation and other M phase events. The role of
MPF in nuclear envelope breakdown is shown in simplified form in the diagram below.

Fig. 11 Simplified diagram showing how Cdk and M cyclin combine to form MPF.

Left panel: The MPF complex phosphorylates various targets specific to M phase, and the
phosphorylated targets cause spindle formation, chromosome condensation, nuclear
membrane breakdown, and other events of early M phase.

Right panel: Specific example of MPF triggering nuclear envelope breakdown. The MPF
complex phosphorylates proteins in the nuclear envelope, resulting in the fragmentation
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of the nuclear membrane into vesicles (and release of some of the proteins from the
membrane).

Rb protein
The retinoblastoma protein is a tumor suppressor protein that is dysfunctional in
several major cancers. One function of Rb is to prevent excessive cell growth by
inhibiting cell cycle progression until a cell is ready to divide. It plays a pivotal role in
the negative control of the cell cycle and in tumor progression. It has been shown
that Rb protein (pRb) is responsible for a major G1 checkpoint, blocking S-phase entry
and cell growth. When the cell is ready to divide, Rb is phosphorylated to pRb, leading to
the inactivation of Rb. This process allows cells to enter into the cell cycle state. It is also
a recruiter of several chromatin remodeling enzymes such as methylases and
acetylases.

In humans, the protein is encoded by the RB1 gene located on chromosome 13—more
specifically, 13q14.1-q14.2. If both alleles of this gene are mutated early in life, the
protein is inactivated and results in development of retinoblastoma cancer, hence the
name 'Rb'. Retinal cells are not sloughed off or replaced, and are subjected to high levels
of mutagenic UV radiation, and thus most pRb knock-outs occur in retinal tissue, but it
has also been documented in certain skin cancers.

E2F protein
E2F transcription factors regulate the expression of a number of genes important in cell
proliferation, particularly those involved in progression through G1 and into the S-
phase of the cell cycle. The E2 factor (E2F) family of transcription factors are
downstream effectors of the retinoblastoma (RB) protein pathway and are believed to
play a pivotal role in cell division control. Since its discovery, E2F has been viewed as a
positive regulator of genes required for DNA synthesis. As E2F-RB1 complexes are
repressive, loss of E2Fs could also lead to de-repression of genes involved in cell cycle
progression, potentially explaining why loss of E2F can contribute to oncogenesis.

DP protein
Transcription factor DP (Dimerization Partner) is a family of proteins which function as
transcription factors. DP forms a heterodimer with E2F and regulates genes involved in
cell cycle progression. The activities of both DP and E2F proteins are under cell
cycle control, being influenced by the level of phosphorylation imparted through the cell
cycle regulated activity of cyclin-dependent kinases.

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p53
Tumor protein p53, also known as p53 or cellular tumor antigen p53 is the Guardian of
the Genome. It is any isoform of a protein encoded by homologous genes in various
organisms, such as TP53 (humans) and Trp53 (mice). This homolog (originally thought
to be, and often spoken of as, a single protein) is crucial in multicellular vertebrates,
where it prevents cancer formation, and thus functions as a tumor suppressor.

The name p53 was given in 1979 describing the apparent molecular mass; SDS-PAGE
analysis indicates that it is a 53-kilodalton (kDa) protein. However, the actual mass of
the full-length p53 protein (p53α) based on the sum of masses of the amino acid
residues is only 43.7 kDa. This difference is due to the high number of proline residues
in the protein, which slow its migration on SDS-PAGE, thus making it appear heavier
than it actually is.

In humans, the TP53 gene is located on the short arm of chromosome 17 (17p13.1). The
gene spans 20 kb, with a non-coding exon 1 and a very long first intron of 10 kb. The
coding sequence contains five regions showing a high degree of conservation in
vertebrates, predominantly in exons 2, 5, 6, 7 and 8, but the sequences found in
invertebrates show only distant resemblance to mammalian TP53.

p53 plays a role in regulation or progression through the cell cycle, apoptosis, and
genomic stability by means of several mechanisms:

• It can activate DNA repair proteins when DNA has sustained damage. Thus an
important factor in aging

• It can arrest growth 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 if DNA damage proves to be irreparable.

• It is essential for the senescence response to short telomeres.

• Levels of p53 play an important role in the maintenance of stem cells throughout
development and the rest of human life.

In human embryonic stem cells (hESCs)s, p53 is maintained at low inactive levels. This
is because activation of p53 leads to rapid differentiation of hESCs. If the TP53 gene is
damaged, tumor suppression is severely compromised. People who inherit only one
functional copy of the TP53 gene will most likely develop tumors in early adulthood, a
disorder known as Li-Fraumeni syndrome.

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p53 works on multiple levels to ensure that cells do not pass on their damaged DNA
through cell division. First, it stops the cell cycle at the G_1 checkpoint by triggering
production of Cdk inhibitor (CKI) proteins. The CKI proteins bind to Cdk-cyclin
complexes and block their activity (see diagram below), buying time for DNA repair.
p53's second job is to activate DNA repair enzymes. If DNA damage is not fixable, p53
will play its third and final role: triggering programmed cell death so damaged DNA is
not passed on.

Fig. 12 Simplified diagram of how p53 halts the cell cycle at the G1/S checkpoint.

p53 is activated by DNA damage and causes production of a Cdk inhibitor, which binds
to the Cdk-G1/S cyclin complex and inactivates it. This halts the cell in G1 and prevents
it from entering S phase, allowing time for the DNA damage to be fixed.

By ensuring that cells don't divide when their DNA is damaged, p53 prevents mutations
(changes in DNA) from being passed on to daughter cells. When p53 is defective or
missing, mutations can accumulate quickly, potentially leading to cancer. Indeed, out of
all the entire human genome, p53 is the single gene most often mutated in cancers. p53
and cell cycle regulation are key topics of study for researchers working on new
treatments for cancer.

The internal and external cues trigger signaling pathways inside the cell that activate, or
inactivate, a set of core proteins that move the cell cycle forward.

Types of Cyclin dependent kinases

Cyclins drive the events of the cell cycle by partnering with a family of enzymes called
the cyclin-dependent kinases (Cdks). A lone Cdk is inactive, but the binding of a cyclin
activates it, making it a functional enzyme and allowing it to modify target proteins.

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Cyclin-dependent kinases (CDKs) are protein kinases characterized by needing a

separate subunit - a cyclin - that provides domains essential for enzymatic activity.
CDKs play important roles in the control of cell division and modulate transcription in
response to several extra- and intracellular cues.

CDKs are a family of multifunctional enzymes that can modify various protein
substrates involved in cell cycle progression. Specifically, CDKs phosphorylate their
substrates by transferring phosphate groups from ATP to specific stretches of amino
acids in the substrates.

Cyclin-dependent kinases (CDKs) are protein kinases characterized by needing a

separate subunit - a cyclin - that provides domains essential for enzymatic activity.
CDKs play important roles in the control of cell division and modulate transcription in
response to several extra- and intracellular cues. The evolutionary expansion of the CDK
family in mammals led to the division of CDKs into three cell-cycle-related subfamilies
(Cdk1, Cdk4 and Cdk5) and five transcriptional subfamilies (Cdk7, Cdk8, Cdk9, Cdk11
and Cdk20). Unlike the prototypical Cdc28 kinase of budding yeast, most of these CDKs
bind one or a few cyclins, consistent with functional specialization during evolution.

Cyclin-dependent kinases (CDKs) are the families of protein kinases first discovered for
their role in regulating the cell cycle. They are also involved in regulating transcription,
mRNA processing, and the differentiation of nerve cells. They are present in all known
eukaryotes, and their regulatory function in the cell cycle has been evolutionarily
conserved. In fact, yeast cells can proliferate normally when their CDK gene has been
replaced with the homologous human gene. CDKs are relatively small proteins, with
molecular weights ranging from 34 to 40 kDa, and contain little more than the kinase
domain. By definition, a CDK binds a regulatory protein called a cyclin. Without cyclin,
CDK has little kinase activity; only the cyclin-CDK complex is an active kinase. CDKs
phosphorylate their substrates on serines and threonines, so they are serine-threonine
kinases.

CDKs and cyclins in the cell cycle

Most of the known cyclin-CDK complexes regulate the progression through the cell
cycle. Animal cells contain at least nine CDKs, four of which, CDK1, 2, 3, and 4, are
directly involved in cell cycle regulation. In mammalian cells, CDK1, with its partners
cyclin A2 and B1, alone can drive the cell cycle. Another one, CDK7, is involved
indirectly as the CDK-activating kinase. Cyclin-CDK complexes phosphorylate substrates
appropriate for the particular cell cycle phase. Cyclin-CDK complexes of earlier cell-
cycle phase help activate cyclin-CDK complexes in later phase.

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Fig. 13 Cyclin-CDK complexes

Cdc25
Cdc25 is a dual-specificity phosphatase first isolated from the yeast
Schizosaccharomyces pombe as a cell cycle defective mutant. As with other cell cycle
proteins or genes such as Cdc2 and Cdc4, the "cdc" in its name refers to "cell division
cycle". Dual-specificity phosphatases are considered a sub-class of protein tyrosine
phosphatases.

Cdc25 proteins control entry into and progression through various phases of the cell
cycle, including mitosis and S ("Synthesis") phase. The Cdc25s, and in particular Cdc25A
and Cdc25B, are proto-oncogenes in humans and have been shown to be overexpressed
in a number of cancers. The central role of Cdc25s in the cell cycle has garnered them
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considerable attention from the pharmaceutical industry as potential targets for novel
chemotherapeutic (anti-cancer) agents

Function in activating Cdk1

Cdc25 activates cyclin dependent kinases by removing phosphate from residues in the
Cdk active site. In turn, the phosphorylation by M-Cdk (a complex of Cdk1 and cyclin B)
activates Cdc25. Together with Wee1, M-Cdk activation is switch-like. The switch-like
behavior forces entry into mitosis to be quick and irreversible. Cdk activity can be
reactivated after dephosphorylation by Cdc25. The Cdc25 enzymes Cdc25A-C are
known to control the transitions from G1 to S phase and G2 to M phase.

CAK and Wee1

The decreased Wee1 activity alone is not sufficient for mitotic entry: Synthesis of cyclins
and an activating phosphorylation by a Cdk activating kinase (CAK) are also required.
Wee1 plays a role in this checkpoint by coordinating cell size and cell cycle progression.

Wee1 acts as a tumor suppressor in the context of normal cell growth and its functional
loss can be compensated by p53-dependent DNA damage repairing mechanisms,
specific inhibition of Wee1 has deleterious effects on the proliferation and survival of
p53 inactive tumors.

Wee1 inhibits Cdk1 by phosphorylating it on two different sites, Tyr15 and Thr14. Cdk1
is crucial for the cyclin-dependent passage of the various cell cycle checkpoints. At least
three checkpoints exist for which the inhibition of Cdk1 by Wee1 is important:

G2/M checkpoint: Wee1 phosphorylates the amino acids Tyr15 and Thr14 of Cdk1,
which keeps the kinase activity of Cdk1 low and prevents entry into mitosis. During
mitotic entry the activity of Wee1 is decreased by several regulators and thus Cdk1
activity is increased.

Cell size checkpoint: There is evidence for the existence of a cell size checkpoint, which
prevents small cells from entering mitosis. Wee1 plays a role in this checkpoint by
coordinating cell size and cell cycle progression.

DNA damage checkpoint: This checkpoint also controls the G2/M transition. The
lengthening of the G2 phase depends on Wee1; wee1 mutants have no prolonged G2
phase after gamma irradiation.

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nim-proteins
5-nitroimidazole based antibiotics are compounds extensively used for treating
infections in humans and animals caused by several important pathogens. They are
administered as prodrugs and their activation depends upon an anaerobic one electron
reduction of the nitro group by a reduction pathway in the cells. Bacterial resistance
towards these drugs is thought to be caused by decreased drug uptake and/or an
altered reduction efficiency. One class of resistant strains, identified in Bacteroides,
have been shown to carry Nim genes, (NimA, B, C, D and E), which encode for reductases
that convert the nitro group on the antibiotic into a non-bactericidal amine. It is
Nitroimidazole resistance protein (nimE).

SCF complex
Skp, Cullin, F-box containing complex (or SCF complex) is a multi-protein E3 ubiquitin
ligase complex that catalyzes the ubiquitination of proteins destined for 26S
proteasomal degradation. Along with the anaphase-promoting complex, SCF has
important roles in the ubiquitination of proteins involved in the cell cycle. The SCF
complex also marks various other cellular proteins for destruction.

SCF contains a variable F-box protein and three core subunits:

• F-box protein (FBP) – FBP contributes to the substrate specificity of the SCF
complex by first aggregating to target proteins independently of the complex. Each FBP
(e.g. Skp2) may recognize several different substrates in a manner that is dependent on
post-translational modifications such as phosphorylation or glycosylation. FBP then
binds to Skp1 of the SCF complex using an F-box motif, bringing the target protein into
proximity with the functional E2 ubiquitin-conjugating enzyme. FBP is also essential in
regulating SCF activity during the course of the cell cycle. SCF levels are thought to
remain constant throughout the cell-cycle. Instead, FBP affinity for protein substrates is
regulated through cyclin-CDK-mediated phosphorylation of target proteins.

• Skp1 – Skp1 is an adaptor protein that is essential for the recognition and
binding of F-box proteins.

• Cullin (CUL1) – Cullin forms the major structural scaffold of the SCF complex
and links the skp1 domain to the Rbx1 domain. Different combinations of Cullin and
FBPs can generate on the order of a hundred types of E3 ubiquitin ligases that target
different substrates.

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• RBX1 – Rbx1 contains a small, zinc-binding Really Interesting New Gene (RING)
finger domain, to which the E2 ubiquitin-conjugating enzyme binds. This binding event
allows the transferral of ubiquitin from E2 to a lysine residue on the target protein.

Specifically, SCF has been shown to regulate centriole splitting from late telophase to
the G1/S transition. SCF activity is largely regulated by post-translational modifications.
For instance, ubiquitin-mediated autocatalytic degradation of FBPs is a mechanism of
decreasing SCF activity.

Well-characterized cell cycle substrates of SCF complexes include:

• cyclin family proteins: Cyclin D, Cyclin E

• transcriptional regulators: Myc, E2f1, p130

• cyclin-dependent kinase inhibitors (CKIs): p27, p21, Wee1

• centriole proteins: Cep250, Ninein

Anaphase-promoting complex/cyclosome (APC/C)

In addition to driving the events of M phase, MPF also triggers its own destruction by
activating the anaphase-promoting complex/cyclosome (APC/C), a protein complex
that causes M cyclins to be destroyed starting in anaphase. The destruction of M cyclins
pushes the cell out of mitosis, allowing the new daughter cells to enter G1. The APC/C
also causes destruction of the proteins that hold the sister chromatids together,
allowing them to separate in anaphase and move to opposite poles of the cell.

APC/C working

Like a Cdk, the APC/C is an enzyme, but it has different type of function than a Cdk.
Rather than attaching a phosphate group to its targets, it adds a small protein tag
called ubiquitin (Ub). When a target is tagged with ubiquitin, it is sent to
the proteasome, which can be thought of as the recycle bin of the cell, and destroyed.
For example, the APC/C attaches a ubiquitin tag to M cyclins, causing them to be
chopped up by the proteasome and allowing the newly forming daughter cells to enter
G1.

The APC/C also uses ubiquitin tagging to trigger the separation of sister chromatids
during mitosis. If the APC/C gets the right signals at metaphase, it sets off a chain of
events that destroys cohesin, the protein glue that holds sister chromatids together.

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 The APC/C first adds a ubiquitin tag to a protein called securin, sending it for
recycling. Securin normally binds to, and inactivates, a protein called separase.
 When securin is sent for recycling, separase becomes active and can do its
job. Separase chops up the cohesin that holds sister chromatids together, allowing
them to separate.

Fig. 14 APC

Checkpoints and regulators

Cdks, cyclins, and the APC/C are direct regulators of cell cycle transitions, but they
aren’t always in the driver’s seat. Instead, they respond to cues from inside and outside
the cell. These cues influence activity of the core regulators to determine whether the
cell moves forward in the cell cycle. Positive cues, like growth factors, typically increase
activity of Cdks and cyclins, while negative ones, like DNA damage, typically decrease or
block activity.

As an example, let's examine how DNA damage halts the cell cycle in G1. DNA damage
can, and will, happen in many cells of the body during a person’s lifetime (for example,
due to UV rays from the sun). Cells must be able to deal with this damage, fixing it if
possible and preventing cell division if not. Key to the DNA damage response is a
protein called p53, a famous tumor suppressor often described as “the guardian of the
genome.”

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Anaphase-promoting complex (also called the cyclosome or APC/C) is an E3 ubiquitin

ligase that marks target cell cycle proteins for degradation by the 26S proteasome. The
APC/C is a large complex of 11–13 subunit proteins, including a cullin (Apc2) and RING
(Apc11) subunit much like SCF. Other parts of the APC/C still have unknown functions,
but are highly conserved.

It was the discovery of the APC/C (and SCF) and their key role in eukaryotic cell
reproduction that established once and for all the importance of ubiquitin-mediated
proteolysis in eukaryotic cell biology. Once perceived as a system exclusively involved
in removing damaged protein from the cell, ubiquitination and subsequent protein
degradation by the proteasome is now perceived as a universal regulatory mechanism
for signal transduction whose importance approaches that of protein phosphorylation.

The anaphase promoting complex or cyclosome (APC/C) is a multi-subunit cullin-RING

E3 ubiquitin ligase that functions to regulate progression through the mitotic phase of
the cell cycle and to control entry into S phase.

The anaphase-promoting complex (APC) is a large protein complex containing 11–13

subunits, including a RING subunit (Apc11) and a cullin (Apc2). APC activity requires
association with activator subunits (Cdc20 or Cdh1) that contribute to substrate
binding.

Fig. 15 APC- SCF

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Functions of APC

The APC/C's main function is to trigger the transition from metaphase to anaphase by
tagging specific proteins for degradation. The three major targets for degradation by the
APC/C are securin and S and M cyclins. Securin releases separase (a protease) after
being degraded. The separase triggers the cleavage of cohesin, the protein complex that
binds sister chromatids together. During metaphase, sister chromatid is linked by intact
cohesin complexes. When securin undergoes ubiquitination by the APC/C and releases
separase, which degrades cohesin, sister chromatids become free to move to opposite
poles for anaphase. The APC/C also targets the mitotic cyclins for degradation, resulting
in the inactivation of M-CDK (mitotic cyclin-dependent kinase) complexes, promoting
exit from mitosis and cytokinesis.

Licensing factors
A licensing factor is a protein or complex of proteins that allows an origin of replication
to begin DNA replication at that site. They are thought to primarily occur in eukaryotic
cells, since prokaryotes use simpler systems to initiate replication.

Functions-

Origins of replication represent start sites for DNA replication and so their "firing" must
be regulated to maintain the correct karyotype of the cell in question. The origins are
required to fire only once per cell cycle, an observation that led to the postulated
existence of licensing factors by biologists in the first place. If the origins were not
carefully regulated then DNA replication could be restarted at that origin giving rise to
multiple copies of a section of DNA. This could be damaging to cells and could have
detrimental effects on the organism as a whole.

The control that licensing factors exert over the cycle represents a flexible system,
necessary so that different cell types in an organism can control the timing of DNA
replication to their own cell cycles.

Location-

The factors themselves are found in different places in different organisms. For example
in metazoan organisms, they are commonly synthesised in the cytoplasm of the cell to
be imported into the nucleus when required. The situation is different in yeast where
the factors present are degraded and resynthesised throughout the cell cycle but are
found in the nucleus for most of their existence.

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Yamanaka factors
Oct3/4, Sox2, Klf4, c-Myc are highly expressed in embryonic stem (ES) cells, and their
over-expression can induce pluripotency in both mouse and human somatic cells,
indicating that these factors regulate the developmental signaling network necessary
for ES cell pluripotency. iPSC are derived from skin or blood cells that have been
reprogrammed back into an embryonic-like pluripotent state that enables the
development of an unlimited source of any type of human cell needed for therapeutic
purposes.

Oct3/4
 Octamer binding transcription factor (OCT) 3/4 is essential for pluripotency and self
renewal of stem cells
 18 kDa domain transcription factor encoded by POU5F1 gene (POU domain, class 5,
transcription factor 1) at 6p21.3
 Together with SOX2, essential for reprogramming somatic cells into pluripotent stem
cells
 Expressed in germ cells and stem cells, downregulated in all somatic tissues
 Likely to be involved in maintaining the stemness of cancer stem cells
 Marker of germ cells, not expressed in somatic tissues
 Strong diffuse nuclear expression seen in seminoma (dysgerminoma) and embryonal
carcinoma components of germ cell neoplasia, including metastatic disease
 Can be expressed focally in advanced epithelial and lymphatic malignancy

SOX2
SRY (sex determining region Y)-box 2, also known as SOX2, is a transcription factor that
is essential for maintaining self-renewal, or pluripotency, of undifferentiated embryonic
stem cells. Sox2 has a critical role in maintenance of embryonic and neural stem cells. It
play key roles in many stages of mammalian development. This protein family shares
highly conserved DNA binding domains known as HMG (High-mobility group) box
domains containing approximately 80 amino acids.

Varying levels of Sox2 affect embryonic stem cells' fate of differentiation. Sox2 inhibits
differentiation into the mesendoderm germ layer and promotes differentiation into
neural ectoderm germ layer. Npm1/Sox2 complexes are sustained when differentiation
is induced along the ectodermal lineage, emphasizing an important functional role for
Sox2 in ectodermal differentiation

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NANOG
Homeobox protein NANOG is a transcriptional factor that helps embryonic stem cells
maintain pluripotency by suppressing cell determination factors. Therefore NANOG
deletion will trigger differentiation of ESCs. There are many different types of cancer
that are associated with NANOG. The human NANOG protein coded by the NANOG1
gene, consists of 305 amino acids and possesses 3 functional domains: the N-terminal
domain, the C- terminal domain, and the conserved homeodomain motif. The
homeodomain region facilitates DNA binding. The human Nanog 1 gene is located on
chromosome 12, and the mRNA contains a 915 bp open reading frame (ORF) with 4
exons and 3 introns.

NANOG is a transcription factor in embryonic stem cells (ESCs) and is thought to be a

key factor in maintaining pluripotency. NANOG is thought to function in concert with
other factors such as POU5F1 (Oct-4) and SOX2 to establish ESC identity. These cells
offer an important area of study because of their ability to maintain pluripotency. In
other words, these cells have the ability to become virtually any cell of any of the three
germ layers (endoderm, ectoderm, mesoderm). It is for this reason that understanding
the mechanisms that maintain a cell's pluripotency is critical for researchers to
understand how stem cells work, and may lead to future advances in treating
degenerative diseases.

NANOG is highly expressed in cancer stem cells and may thus function as an oncogene
to promote carcinogenesis. High expression of NANOG correlates with poor survival in
cancer patients.

Fig. 16 Yamanaka factors

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Mechanisms of epigenetic regulation

The major epigenetic mechanisms include DNA methylation and hydroxymethylation;
histone protein posttranslational modifications, nucleosome remodeling/repositioning,
and higher-order chromatin reorganization; noncoding RNA regulation; and RNA
editing.

DNA methylation

In the course of life, aging processes, environmental influences and lifestyle factors
such as smoking or diet induce biochemical alterations to the DNA. Frequently, these
lead to DNA methylation, a process in which methyl groups are added to
particular DNA segments, without changing the DNA sequence. Methylation can
change the activity of a DNA segment without changing the sequence. When located in
a gene promoter, DNA methylation typically acts to repress gene transcription.

Two of DNA's four bases, cytosine and adenine, can be methylated. Cytosine
methylation is widespread in both eukaryotes and prokaryotes, even though the rate of
cytosine DNA methylation can differ greatly between species: 14% of cytosines are
methylated in Arabidopsis thaliana, 4% to 8% in Physarum, 7.6% in Mus musculus,
2.3% in Escherichia coli, 0.03% in Drosophila.

DNA methylation is an epigenetic mechanism that occurs by the addition of a methyl

(CH3) group to DNA, thereby often modifying the function of the genes and affecting
gene expression. The most widely characterized DNA methylation process is the
covalent addition of the methyl group at the 5-carbon of the cytosine ring resulting in
5-methylcytosine (5-mC), also informally known as the “fifth base” of DNA. These
methyl groups project into the major groove of DNA and inhibit transcription.

In human DNA, 5-methylcytosine is found in approximately 1.5% of genomic DNA.1 In

somatic cells, 5-mC occurs almost exclusively in the context of paired symmetrical
methylation of a CpG site, in which a cytosine nucleotide is located next to a guanidine
nucleotide. An exception to this is seen in embryonic stem (ES) cells, where a
substantial amount of 5-mC is also observed in non-CpG contexts. In the bulk of
genomic DNA, most CpG sites are heavily methylated while CpG islands (sites of CpG
clusters) in germ-line tissues and located near promoters of normal somatic cells,
remain unmethylated, thus allowing gene expression to occur. When a CpG island in
the promoter region of a gene is methylated, expression of the gene is repressed (it is
turned off).

CpG islands
In mammals, the only exception for this global CpG depletion resides in a specific
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category of GC- and CpG-rich sequences termed CpG islands that are generally
unmethylated and therefore retained the expected CpG content. CpG islands are usually
defined as regions with a length greater than 200bp, a G+C content greater than 50%
and as a region with a ratio of observed to expected CpG greater than 0.6, although
other definitions are sometimes used.

The addition of methyl groups is controlled at several different levels in cells and is
carried out by a family of enzymes called DNA methyltransferases (DNMTs). Three
DNMTs (DNMT1, DNMT3a and DNMT3b) are required for establishment and
maintenance of DNA methylation patterns. Two additional enzymes (DNMT2 and
DNMT3L) may also have more specialized but related functions. DNMT1 appears to be
responsible for the maintenance of established patterns of DNA methylation, while
DNMT3a and 3b seem to mediate establishment of new or de novo DNA methylation
patterns. Diseased cells such as cancer cells may be different in that DNMT1 alone is
not responsible for maintaining normal gene hypermethylation (an increase in global
DNA methylation) and both DNMTs 1 and 3b may cooperate for this function.

Demethylation
DNA demethylation is the removal of a methyl group from DNA. This mechanism is
equally as important and coupled with DNA methylation. The demethylation process is
necessary for epigenetic reprogramming of genes and is also directly involved in many
important disease mechanisms such as tumor progression. Demethylation of DNA can
either be passive or active, or a combination of both. Passive DNA demethylation
usually takes place on newly synthesized DNA strands via DNMT1 during replication
rounds. Active DNA demethylation mainly occurs by the removal of 5-methylcytosine
via the sequential modification of cytosine bases that have been converted by TET
enzyme-mediated oxidation. The ten-eleven translocation (TET) family of 5-mC
hydroxylases includes TET1, TET2 and TET3. These proteins may promote DNA
demethylation by binding to CpG rich regions to prevent unwanted DNA
methyltransferase activity, and by converting 5-mC to 5-hmC, 5-hmC to 5-fC (5-
formylcytosine), and 5-fC to 5-caC (5-carboxylcytosine) through hydroxylase activity.
The TET proteins have been shown to function in transcriptional activation and
repression (TET1), tumor suppression (TET2), and DNA methylation reprogramming
processes (TET3).

DNA methylation as well as many of its contemporary DNA methyltransferases have

been thought to evolve from early world primitive RNA methylation activity and is
supported by several lines of evidence.

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Fig. 17 Methylation Fig. 18 histone deacetylation

Histone acetylation and methylation

Histone acetylation and histone deacetylation involve the addition or removal of an

acetyl group on lysine residues in the N-terminal tail and on the surface of the
nucelosome core of histone proteins. Acetylated and deacetylated histones are
considered epigenetic tags within chromatin by relaxing (euchromatin) or tightening
(heterochromatin) chromatin structure, subsequently increasing or decreasing gene
transcription levels.

Histone acetylation, including histone H3 and histone H4, is involved in the regulation
of chromatin structure and the recruitment of transcription factors to gene promoters.
Quantifying total levels of acetylated histone or levels at specific lysines will help to
elucidate epigenetic regulation of gene activation, and contribute to the development of
HAT or HDAC-targeted drugs.

Histone acetyltransferases (HAT) are enzymes that play a critical role in

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transcriptional regulation of genes, and directly correspond to transcription by

selectively acetylating the epsilon-amino groups of lysines located near the amino
termini of core histone proteins. Abnormal gene silencing by reduced HAT activity has
been linked to the pathogenesis of many diseases, particularly cancer. Determining the
activity of HATs and the potency of their inhibitors contributes to drug discovery and
the development of anti-cancer agents, as well as a better understanding of gene
transcription.

Histone deacetylates
Histone deacetylases (HDACs), also known as lysine deacetylases (KDACs), are a class
of enzymes that remove acetyl groups from core histones (H2A, H2B, H3 and H4),
thereby regulating gene expression. HDACs play a critical role in the suppression of
gene expression by reversing the hyperacetylation of core histones. Eleven HDACs that
deacetylate histones are now recognised in mammalian cells and are classified into two
major classes. Class I includes HDAC1, 2, 3, 8 and which bear significant homology to
the yeast protein RPD3 and are mainly localised to the nucleus. Class II includes
HDAC4, 5, 6, 7, 9 and 10, which are homologous to yeast HAD-1-like enzymes and
shuttle between nucleus and cytoplasm. Class I HDACs are widely expressed and are
found in most cell types, whereas class II HDACs appear to have a more restricted
distribution and may be involved in cellular differentiation. Some HDACs also
deacetylate nonhistone proteins such as α-tubulin, p53, p65 and MyoD.

These epigenetic enzymes influence the structure of chromatin, the protein-DNA

complex that forms into chromosomes residing in the nucleus. By removing an acetyl
group, HDACs enable the tightening of the chromatin structure into what is called
heterochromatin. Specifically, the DNA that wraps around the histones is more compact,
which can reduce the transcription of important genes.

In contrast, histone acetyltransferases (HATs) add acetyl groups to histones and are
associated with euchromatin, a loosened chromatin structure that increases
transcription and gene expression. HDACs are also tightly involved in cell cycle
regulation, cell proliferation, and in the development of human cancer. By studying the
activity and inhibition of HDACs, researchers can gain a better understanding of the
impact histone deacetylation has on diseases and various cellular processes.

Protein Acetylation Modification

Protein acetylation modification, as the name suggests, refers to the grafting of
acetylated groups on the original basis of the protein. In cells, the acetylation
modification reaction is catalyzed by an acetyltransferase, and the acetyl group of
acetyl-CoA is transferred and added to the protein lysine residue. In earlier studies,
acetylation has been considered to be a post-translational modification unique to

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eukaryotic cells, and it has since been discovered that prokaryotic cells also have
protein acetylation modifications in the middle years. Therefore, acetylation
modification is a type of post-translational modification common to prokaryotic and
eukaryotic organisms.

Functions of Protein Acetylation Modification

At present, the research on the function of acetylation modification mainly focuses on
the regulation of cell transcription and the regulation of metabolic pathways. Among
plenty of acetylated proteins, more study has been made on the histones surrounding
the DNA in the nucleus. Histones and DNA entangled to form nucleosomes. The
methylation and acetylation of histones can regulate the tightness of DNA
entanglement and regulate gene expression. Therefore, post-translational modification
of histones is an important part in epigenetic research. In addition, study has also
found that acetylation modifications are ubiquitous in human metabolic enzymes and
is involved in the regulation of metabolic pathways as well as the activity of metabolic
enzymes. Among them, a variety of metabolic enzymes are highly acetylated in
metabolic organs such as the liver. Therefore, the study of new acetylation sites of
proteins and acetylation modification omics can provide important guiding advice for
revealing the causes of more diseases, and carry out new ideas for the development of
new drugs related to diseases.

Histone Acetylation Regulation Process

In the nucleus, histone acetylation is in a dynamic equilibrium with histone
deacetylation, and histone acetylation is jointly regulated by histone acetyltransferase
(HAT) and histone deacetylase (HDAC). HAT transfers the acetyl group of acetyl-CoA to
a specific lysine residue at the amino terminus of histones. The histones, after carrying
a negatively charged acetylation group, repel each other from the same negatively
charged DNA, so that the DNA entanglement becomes slack and can bind to the
transcription factor to initiate expression of the gene. In contrast, HDAC deacetylates
histones and promotes tight binding of negatively charged DNA. The nucleosomes are,
therefore, more tightly packed, making DNA unable to bind to transcription factors,
and gene transcription is inhibited accordingly.

Role of signaling in epigenetic control

Epigenetic changes
Epigenetic changes are defined as inherited modifications that are not present in DNA
sequence. Gene expression is regulated at various levels and not only in response to
DNA modifications. Examples of epigenetic control are DNA methylation, histone
deacetylation and mi-RNA expression.
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Epigenetic changes alter the physical structure of DNA. One example of an epigenetic
change is DNA methylation — the addition of a methyl group, or a "chemical cap," to
part of the DNA molecule, which prevents certain genes from being expressed. Another
example is histone modification.

Fig. 19 Epigenetic control and modifications

Epigenetic modifications are heritable chemical or physical changes in chromatin.

There are two types of epigenetic modifications –

DNA methylation and histone modifications.

DNA methylation is a biological process by which methyl groups are added to the DNA
molecule. Methylation can change the activity of a DNA segment without changing the
sequence. When located in a gene promoter, DNA methylation typically acts to repress
gene transcription.

Histone modification is a covalent post-translational modification (PTM) to histone

proteins which includes methylation, phosphorylation, acetylation, ubiquitylation, and
sumoylation. The PTMs made to histones can impact gene expression by altering
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chromatin structure or recruiting histone modifiers.

Histone modifications

Chromatin architecture, nucleosomal positioning, and ultimately access to DNA for gene
transcription, is largely controlled by histone proteins. Each nucleosome is made of two
identical subunits, each of which contains four histones: H2A, H2B, H3, and H4.
Meanwhile, the H1 protein acts as the linker histone to stabilize internucleosomal DNA
and does not form part of the nucleosome itself. Histone proteins undergo post-
translational modification (PTM) in different ways, which impacts their interactions
with DNA. Some modifications disrupt histone-DNA interactions, causing nucleosomes
to unwind. In this open chromatin conformation, called euchromatin, DNA is accessible
to binding of transcriptional machinery and subsequent gene activation. In contrast,
modifications that strengthen histone-DNA interactions create a tightly packed
chromatin structure called heterochromatin.

Table 4: Major categories of histone modifications

In this compact form, transcriptional machinery cannot access DNA, resulting in gene
silencing. In this way, modification of histones by chromatin remodeling complexes
changes chromatin architecture and gene activation. At least nine different types of
histone modifications have been discovered. Acetylation, methylation, phosphorylation,
and ubiquitylation are the most well-understood, while GlcNAcylation, citrullination,
krotonilation, and isomerization are more recent discoveries that have yet to be
thoroughly investigated. Each of these modifications are added or removed from
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histone amino acid residues by a specific set of enzymes.

Chromatin architecture, nucleosomal positioning, and ultimately access to DNA for

gene transcription, is largely controlled by histone proteins. Each nucleosome is made
of two identical subunits, each of which contains four histones: H2A, H2B, H3, and H4.
Meanwhile, the H1 protein acts as the linker histone to stabilize internucleosomal DNA
and does not form part of the nucleosome itself.

Histone proteins undergo post-translational modification (PTM) in different ways,

which impacts their interactions with DNA. Some modifications disrupt histone-DNA
interactions, causing nucleosomes to unwind. In this open chromatin conformation,
called euchromatin, DNA is accessible to binding of transcriptional machinery and
subsequent gene activation. In contrast, modifications that strengthen histone-DNA
interactions create a tightly packed chromatin structure called heterochromatin. In this
compact form, transcriptional machinery cannot access DNA, resulting in gene
silencing. In this way, modification of histones by chromatin remodeling complexes
changes chromatin architecture and gene activation. Together, these histone
modifications make up what is known as the histone code, which dictates the
transcriptional state of the local genomic region. Examining histone modifications at a
particular region, or across the genome, can reveal gene activation states, locations of
promoters, enhancers, and other gene regulatory elements.

At least nine different types of histone modifications have been discovered. Acetylation,
methylation, phosphorylation, and ubiquitylation are the most well-understood, while
GlcNAcylation, citrullination, krotonilation, and isomerization are more recent
discoveries that have yet to be thoroughly investigated. Each of these modifications are
added or removed from histone amino acid residues by a specific set of enzymes.

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Fig. 20: The most common histone modifications

Types of Histone modifications

Acetylation
Acetylation is one of the most widely studied histone modifications since it was one of
the first discovered to influence transcriptional regulation. Acetylation adds a negative
charge to lysine residues on the N-terminal histone tails that extend out from the
nucleosome. These negative charges repel negatively charged DNA, which results in a
relaxed chromatin structure. The open chromatin conformation allows transcription
factor binding and significantly increases gene expression.

Acetyl groups are added to lysine residues of histones H3 and H4 by histone

acetyltransferases (HAT) and removed by deacetylases (HDAC). Histone acetylation is
largely targeted to promoter regions, known as promoter-localized acetylation.

Methylation
Methylation is added to the lysine or arginine residues of histones H3 and H4, with
different impacts on transcription. Arginine methylation promotes transcriptional
activation while lysine methylation is implicated in both transcriptional activation and
repression depending on the methylation site. This flexibility may be explained by the
fact that that methylation does not alter histone charge or directly impact histone-DNA
interactions, unlike acetylation.

Histone methylation is a stable mark propagated through multiple cell divisions, and for
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many years was thought to be irreversible. However, it was recently discovered to be an

actively regulated and reversible process.

Phosphorylation
Histone phosphorylation is a critical intermediate step in chromosome condensation
during cell division, transcriptional regulation, and DNA damage repair. Unlike
acetylation and methylation, histone phosphorylation establishes interactions between
other histone modifications and serves as a platform for effector proteins, which leads
to a downstream cascade of events. Phosphorylation occurs on all core histones, with
differential effects on each.

Ubiquitylation
All histone core proteins can be ubiquitylated, but H2A and H2B are most commonly
and are two of the most highly ubiquitylated proteins in the nucleus. Histone
ubiquitylation plays a central role in the DNA damage response. Monoubiquitylation of
histones H2A, H2B, and H2AX is found at sites of DNA double-strand breaks.
Monoubiquitylated H2A is also associated with gene silencing, whereas H2B is also
associated with transcription activation.

Histone modifying enzymes: writers and erasers

Histone modifications are dynamically added and removed from histone proteins by
specific enzymes. The balance between these writers and erasers dictates which marks
are present on histones, and at what levels, to ultimately control whether specific
genetic programs and the cellular processes they orchestrate, are turned on or off.

Table 5: The major categories of histone writers and erasers

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Some other mechanisms of epigenetic regulation

Repositioning of nucleosomes
During the remodeling process, the energy of ATP-hydrolysis is used to weaken or
disrupt DNA–histone contacts and facilitate assembly, disassembly or repositioning of
entire nucleosomes or the exchange of certain histones for histone variants.

Such remodeling is principally carried out by :

1) covalent histone modifications by specific enzymes, e.g., histone acetyltransferases
(HATs), deacetylases, methyltransferases, and kinases
2) ATP-dependent chromatin remodeling complexes which either move, eject or
restructure nucleosomes.

Chromosome condensation
Chromosome condensation is one of the major chromatin-remodeling events that
occur during cell division. The changes in chromatin compaction and higher-order
structure organization are essential requisites for ensuring a faithful transmission of
the replicated genome to daughter cells.

RNA editing
Also known as RNA modification, it is a molecular process through which some cells
can make discrete changes to specific nucleotide sequences within an RNA molecule
after it has been generated by RNA polymerase. It occurs in all living organisms, and is
one of the most evolutionarily conserved properties of RNAs. RNA editing is an
important mechanism of genetic regulation that amplifies genetic plasticity by allowing
the production of alternative protein products from a single gene. There are two
generic classes of RNA editing in nuclei, involving enzymatic deamination of either C-
to-U or A-to-I nucleotides.

mTOR pathway

The mechanistic target of rapamycin (mTOR) pathway—is the major nutrient-sensitive

regulator of growth in animals and plays a central role in physiology, metabolism, the
aging process, and common diseases. mTOR is critically important in regulating growth
proliferation and survival. It also called as the PI3K / AKT pathway.

mTOR pathway is an intracellular signaling pathway important in regulating the cell

cycle. Therefore, it is directly related to cellular quiescence, proliferation, cancer, and
longevity. PI3K activation phosphorylates and activates AKT, localizing it in the plasma
membrane.

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The mammalian target of rapamycin, sometimes also referred to as the mechanistic

target of rapamycin and FK506-binding protein 12-rapamycin-associated protein 1, is a
kinase that in humans is encoded by the MTOR gene. mTOR is a member of the
phosphatidylinositol 3-kinase-related kinase family of protein kinases.

Functions of mTOR pathway

Sirolimus, also known as rapamycin, is a macrolide compound that is used to coat

coronary stents, prevent organ transplant rejection and treat a rare lung disease called
lymphangioleiomyomatosis. It has immunosuppressant functions in humans and is
especially useful in preventing the rejection of kidney transplants.

Mechanistic target of rapamycin (mTOR) is a sensor of metabolic stress and integrator

of environmental cues. Activation of mTOR complex 1 (mTORC1) is triggered by
oxidative stress, amino-acid levels and endosomal traffic to the lysosome by small
GTPases such as Rab4A.

The inhibition of mTOR blocks the binding of the accessory protein raptor (regulatory-
associated protein of mTOR) to mTOR, but that is necessary for downstream
phosphorylation of S6K1 and 4EBP1. As a consequence, S6K1 dephosphorylates, which
reduces protein synthesis and decreases cell mortality and size.

mTOR exists in two different complexes :

1) regulation of mTOR occurs through several mechanisms
2) mTOR is a nutrient sensor

So many types of nutrients either nutrient surplus or nutrient deficiency will regulate
mTOR in different ways. Different types of stress mechanisms also regulate mTOR.

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Fig. 21 mTOR pathway

Complexes in pathway

mTOR is the catalytic subunit of two structurally distinct complexes: mTORC1 and
mTORC2. Both complexes localize to different subcellular compartments, thus affecting
their activation and function. Upon activation by Rheb, mTORC1 localizes to the
Ragulator-Rag complex on the lysosome surface where it then becomes active in the
presence of sufficient amino acids

mTORC1 and mTORC2

mTOR is a serine/threonine protein kinase in the PI3K-related kinase (PIKK) family that
forms the catalytic subunit of two distinct protein complexes, known as mTOR Complex
1 (mTORC1) and 2 (mTORC2). mTORC1 is defined by its three core components: mTOR,
Raptor (regulatory protein associated with mTOR), and mLST8 (mammalian lethal with
Sec13 protein 8, also known as GβL). Raptor facilitates substrate recruitment to
mTORC1 through binding to the TOR signaling (TOS) motif found on several canonical
mTORC1 substrates and, as described later, is required for the correct subcellular
localization of mTORC1. mLST8 by contrast associates with the catalytic domain of
mTORC1 and may stabilize the kinase activation loop, though genetic studies suggest it
is dispensible for the essential functions of mTORC1. In addition to these three core
components, mTORC1 also contains the two inhibitory subunits PRAS40 (proline-rich
Akt substrate of 40 kDa) and DEPTOR (DEP domain containing mTOR interacting
protein).

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mTOR Complex 2 (mTORC2) is composed of MTOR, rapamycin-insensitive companion

of MTOR (RICTOR), MLST8, and mammalian stress-activated protein kinase interacting
protein 1 (mSIN1). mTORC2 has been shown to function as an important regulator of
the actin cytoskeleton through its stimulation of F-actin stress fibers, paxillin, RhoA,
Rac1, Cdc42, and protein kinase C α (PKCα). mTORC2 also phosphorylates the
serine/threonine protein kinase Akt/PKB on serine residue Ser473, thus affecting
metabolism and survival.

Inhibition by rapamycin
Rapamycin inhibits mTORC1, and this appears to provide most of the beneficial effects
of the drug (including life-span extension in animal studies). Rapamycin has a more
complex effect on mTORC2, inhibiting it only in certain cell types under prolonged
exposure. Disruption of mTORC2 produces the diabetic-like symptoms of decreased
glucose tolerance and insensitivity to insulin

Apoptosis

Apoptosis can be initiated through one of two pathways. In the intrinsic pathway the
cell kills itself because it senses cell stress, while in the extrinsic pathway the cell kills
itself because of signals from other cells. Weak external signals may also activate the
intrinsic pathway of apoptosis. The two major types of apoptosis pathways are
“intrinsic pathways,” where a cell receives a signal to destroy itself from one of its own
genes or proteins due to detection of DNA damage; and “extrinsic pathways,” where a
cell receives a signal to start apoptosis from other cells in the organism.

Apoptosis is mediated by proteolytic enzymes called caspases, which trigger cell death
by cleaving specific proteins in the cytoplasm and nucleus. Caspases exist in all cells as
inactive precursors, or procaspases, which are usually activated by cleavage by other
caspases, producing a proteolytic caspase cascade. Apoptosis is characterised by a
series of typical morphological features, such as shrinkage of the cell, fragmentation
into membrane-bound apoptotic bodies and rapid phagocytosis by neighbouring cells.

Necrosis

Necrosis is the death of body tissue. It occurs when too little blood flows to the tissue.
This can be from injury, radiation, or chemicals. Necrosis cannot be reversed. Necrosis
can be caused by a number of external sources, including injury, infection, cancer,
infarction, poisons, and inflammation. Black necrotic tissue is formed when healthy
tissue dies and becomes dehydrated, typically as a result of local ischemia.

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Fig. 22 Necrosis v/s Apoptosis

Cell signaling mechanisms

In biology, cell signaling is part of any communication process that governs basic
activities of cells and coordinates multiple-cell actions. A signal is an entity that codes or
conveys information. Biological processes are complex molecular interactions that
involve a lot of signals. Signaling agents could be physical agents like mechanical
pressure, voltage, temperature, light etc. or chemical agents like peptides, steroids,
terpenoids, etc. All cells receive and respond to signals from their surroundings. This is
accomplished by a variety of signal molecules that are secreted or expressed on the
surface of one cell and bind to a receptor expressed by the other cells, thereby
integrating and coordinating the function of the many individual cells that make up
organisms. Each cell is programmed to respond to specific extracellular signal
molecules.

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Three Stages of Cell Signaling:

First, reception, whereby the signal molecule binds the receptor. Then, signal
transduction, which is where the chemical signal results in a series of enzyme
activations. Finally, the response, which is the resulting cellular responses.

Steps involved in Extracellular signaling:

Step 1 : Synthesis and release of the signaling molecule by the signaling cell;

Step 2 : Transport of the signal to the target cell;

Step 3 : Binding of the signal by a specific receptor leading to its activation;

Step 4 : Initiation of signal-transduction pathways.

Four categories of chemical signaling found in multicellular organisms:

paracrine signaling, endocrine signaling, autocrine signaling, and direct signaling across
gap junctions.

Broadly, sensory receptors respond to one of four primary stimuli:

Chemicals (chemoreceptors)

Temperature (thermoreceptors)

Pressure (mechanoreceptors)

Light (photoreceptors)

Fig.23 Overview of cell signalling

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Cells typically communicate using chemical signals. These chemical signals, which are
proteins or other molecules produced by a sending cell, are often secreted from the cell
and released into the extracellular space. There, they can float – like messages in a
bottle – over to neighboring cells.

Fig. 24 Secreting cell , target cell and non-target cell

Sending cell: this cell secretes a ligand

Target cell: this cell has a receptor that can bind the ligand. The ligand binds to the
receptor and triggers a signaling cascade inside the cell, leading to a response.

Nontarget cell: this cell does not have a receptor for the ligand (though it may have
other kinds of receptors). The cell does not perceive the ligand and thus does not
respond to it.

Not all cells can “hear” a particular chemical message. In order to detect a signal (that is,
to be a target cell), a neighbor cell must have the right receptor for that signal. When a
signaling molecule binds to its receptor, it alters the shape or activity of the receptor,
triggering a change inside of the cell. Signaling molecules are often called ligands, a
general term for molecules that bind specifically to other molecules (such as receptors).

The message carried by a ligand is often relayed through a chain of chemical

messengers inside the cell. Ultimately, it leads to a change in the cell, such as alteration
in the activity of a gene or even the induction of a whole process, such as cell division.
Thus, the original intercellular (between-cells) signal is converted into
an intracellular (within-cell) signal that triggers a response.

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Fig.25 Communication via chemical messengers (Source: Pearson Education 2011)

Types of signaling molecules

At least three important classes of signaling molecules are widely recognized, although
non-exhaustive and with imprecise boundaries, as such membership is non-exclusive
and depends on the context:

 Hormones are the major signaling molecules of the endocrine system, though they
often regulate each other's secretion via local signaling (e.g. islet of
Langerhans cells), and most are also expressed in tissues for local purposes
(e.g. angiotensin) or failing that, structurally related molecules are (e.g. PTHrP).
 Neurotransmitters are signaling molecules of the nervous system, also
including neuropeptides and neuromodulators. Neurotransmitters like
the catecholamines are also secreted by the endocrine system into the systemic
circulation.
 Cytokines are signaling molecules of the immune system, with a primary paracrine
or juxtacrine role, though they can during significant immune responses have a
strong presence in the circulation, with systemic effect (altering iron
metabolism or body temperature). Growth factors can be considered as cytokines or
a different class.

Forms of signaling

Cell-cell signaling involves the transmission of a signal from a sending cell to a receiving
cell. However, not all sending and receiving cells are next-door neighbors, nor do all cell
pairs exchange signals in the same way.

There are four basic categories of chemical signaling found in multicellular organisms:
paracrine signaling, autocrine signaling, endocrine signaling, and signaling by direct
contact. The main difference between the different categories of signaling is the distance
that the signal travels through the organism to reach the target cell.
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Paracrine signaling

Often, cells that are near one another communicate through the release of chemical
messengers (ligands that can diffuse through the space between the cells). This type of
signaling, in which cells communicate over relatively short distances, is known
as paracrine signaling.

Paracrine signaling allows cells to locally coordinate activities with their neighbors.
Although they're used in many different tissues and contexts, paracrine signals are
especially important during development, when they allow one group of cells to tell a
neighboring group of cells what cellular identity to take on.

Synaptic signaling

One unique example of paracrine signaling is synaptic signaling, in which nerve cells
transmit signals. This process is named for the synapse, the junction between two nerve
cells where signal transmission occurs.

When the sending neuron fires, an electrical impulse moves rapidly through the cell,
traveling down a long, fiber-like extension called an axon. When the impulse reaches the
synapse, it triggers the release of ligands called neurotransmitters, which quickly cross
the small gap between the nerve cells. When the neurotransmitters arrive at the
receiving cell, they bind to receptors and cause a chemical change inside of the cell
(often, opening ion channels and changing the electrical potential across the
membrane).

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Fig. 26 Signaling and Receiving end

Synaptic signaling. Neurotransmitter is released from vesicles at the end of the axon of
the sending cell. It diffuses across the small gap between sending and target neurons
and binds to receptors on the target neuron.

The neurotransmitters that are released into the chemical synapse are quickly degraded
or taken back up by the sending cell. This "resets" the system so they synapse is
prepared to respond quickly to the next signal.

Fig. 27 Synaptic signalling

Paracrine signaling: a cell targets a nearby cell (one not attached by gap junctions). The
image shows a signaling molecule produced by one cell diffusing a short distance to a
neighboring cell.

Autocrine signaling: a cell targets itself, releasing a signal that can bind to receptors on
its own surface.

Autocrine signaling

In autocrine signaling, a cell signals to itself, releasing a ligand that binds to receptors
on its own surface (or, depending on the type of signal, to receptors inside of the cell).
This may seem like an odd thing for a cell to do, but autocrine signaling plays an
important role in many processes.

For instance, autocrine signaling is important during development, helping cells take on
and reinforce their correct identities. From a medical standpoint, autocrine signaling is
important in cancer and is thought to play a key role in metastasis (the spread of cancer
from its original site to other parts of the body). In many cases, a signal may have both
autocrine and paracrine effects, binding to the sending cell as well as other similar cells
in the area.
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Endocrine signaling

When cells need to transmit signals over long distances, they often use the circulatory
system as a distribution network for the messages they send. In long-distance endocrine
signaling, signals are produced by specialized cells and released into the bloodstream,
which carries them to target cells in distant parts of the body. Signals that are produced
in one part of the body and travel through the circulation to reach far-away targets are
known as hormones.

In humans, endocrine glands that release hormones include the thyroid, the
hypothalamus, and the pituitary, as well as the gonads (testes and ovaries) and the
pancreas. Each endocrine gland releases one or more types of hormones, many of which
are master regulators of development and physiology.

For example, the pituitary releases growth hormone (GH), which promotes growth,
particularly of the skeleton and cartilage. Like most hormones, GH affects many
different types of cells throughout the body. However, cartilage cells provide one
example of how GH functions: it binds to receptors on the surface of these cells and
encourages them to divide^77start superscript, 7, end superscript.

Fig. 28 Endocrine signaling

Endocrine signaling: a cell targets a distant cell through the bloodstream. A signaling
molecule is released by one cell, then travels through the bloodstream to bind to
receptors on a distant target cell elsewhere in the body.

Signaling through cell-cell contact

This is direct signaling across gap junctions. Gap junctions in animals and
plasmodesmata in plants are tiny channels that directly connect neighboring cells.
These water-filled channels allow small signaling molecules, called intracellular
mediators, to diffuse between the two cells. Small molecules and ions are able to move
between cells, but large molecules like proteins and DNA cannot fit through the
channels without special assistance.

The transfer of signaling molecules transmits the current state of one cell to its
neighbor. This allows a group of cells to coordinate their response to a signal that only
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one of them may have received. In plants, there are plasmodesmata between almost all
cells, making the entire plant into one giant network.

Fig. 29 cell-cell contact signaling

Signaling across gap junctions. A cell targets a neighboring cell connected via gap
junctions. Signals travel from one cell to the other by passing through the gap junctions.

In another form of direct signaling, two cells may bind to one another because they
carry complementary proteins on their surfaces. When the proteins bind to one another,
this interaction changes the shape of one or both proteins, transmitting a signal. This
kind of signaling is especially important in the immune system, where immune cells use
cell-surface markers to recognize “self” cells (the body's own cells) and cells infected by
pathogens^{9}9start superscript, 9, end superscript.

Fig. 30 Direct signaling

MAPK/ERK pathway
The MAPK/ERK pathway (also known as the Ras-Raf-MEK-ERK pathway) is a chain of
proteins in the cell that communicates a signal from a receptor on the surface of the cell
to the DNA in the nucleus of the cell.

The signal starts when a signaling molecule binds to the receptor on the cell surface and
ends when the DNA in the nucleus expresses a protein and produces some change in the
cell, such as cell division. The pathway includes many proteins, including MAPK
(mitogen-activated protein kinases, originally called ERK, extracellular signal-regulated

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kinases), which communicate by adding phosphate groups to a neighboring protein

(phosphorylating it), which acts as an "on" or "off" switch.

When one of the proteins in the pathway is mutated, it can become stuck in the "on" or
"off" position, which is a necessary step in the development of many cancers.
Components of the MAPK/ERK pathway were discovered when they were found in
cancer cells. Drugs that reverse the "on" or "off" switch are being investigated as cancer
treatments.

RAF, and ERK (also known as MAPK) are both serine/threonine-selective protein
kinases. One of the first proteins known to be phosphorylated by ERK was a
microtubule-associated protein (MAP). Mitogen-activated protein kinases (MAPKs) are
protein Ser/Thr kinases that convert extracellular stimuli into a wide range of cellular
responses. MAPKs are among the most ancient signal transduction pathways and are
widely used throughout evolution in many physiological processes.

The Ras/Raf/MAPK pathway is probably the best characterized signal transduction

pathway in cell biology. The function of this pathway is to transduce signals from the
extracellular milieu to the cell nucleus where specific genes are activated for cell
growth, division and differentiation.

MAPK is activated by p38 pathway. The mammalian p38 MAPK families are activated by
cellular stress including UV irradiation, heat shock, high osmotic stress,
lipopolysaccharide, protein synthesis inhibitors, proinflammatory cytokines (such as IL-
1 and TNF-α) and certain mitogens.

Ras proteins function as binary molecular switches that control intracellular signaling
networks. Ras-regulated signal pathways control such processes as actin cytoskeletal
integrity, cell proliferation, cell differentiation, cell adhesion, apoptosis, and cell
migration.

Process:

Overall, the extracellular mitogen binds to the membrane receptor. This allows Ras (a
Small GTPase) to swap its GDP for a GTP. It can now activate MAP3K (e.g., Raf), which
activates MAP2K, which activates MAPK. MAPK can now activate a transcription factor,
such as Myc. In more detail:

Ras activation

Receptor-linked tyrosine kinases such as the epidermal growth factor receptor (EGFR)
are activated by extracellular ligands, such as epidermal growth factor (EGF). Binding of
EGF to the EGFR activates the tyrosine kinase activity of the cytoplasmic domain of the
receptor. The EGFR becomes phosphorylated on tyrosine residues.

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Kinase cascade

Activated Ras activates the protein kinase activity of RAF kinase. RAF kinase
phosphorylates and activates MEK (MEK1 and MEK2). MEK phosphorylates and
activates a mitogen-activated protein kinase (MAPK).

Fig. 31 Ras Raf MEK ERK Signaling Pathway

Role in translation and transcription

MAPK regulates the activities of several transcription factors. MAPK can phosphorylate
C-myc. MAPK phosphorylates and activates MNK, which, in turn, phosphorylates CREB.
MAPK also regulates the transcription of the C-Fos gene. By altering the levels and
activities of transcription factors, MAPK leads to altered transcription of genes that are
important for the cell cycle.

The majority of signal transduction pathways involve the binding of signaling

molecules, known as ligands, to receptors that trigger events inside the cell. The binding
of a signaling molecule with a receptor causes a change in the conformation of the
receptor, known as receptor activation.

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