Signal

transduction and
Apoptosis
SYBSc Biotechnology
SVU
Content
Mechanism of cell communication

principles and types of cellular communication

Types of Signals

Perception of signals

4 receptor types with examples

Receptor activation.

Adaptor proteins
Hawaiian bobtail squid
Cell Signaling
• All cells have the ability to
respond to their environment
• Cells must perceive and respond to a wide range of signals
GENERAL PRINCIPLES OF CELL COMMUNICATION

A simple intracellular signaling pathway activated by an extracellular signal molecule

Cell Signaling Systems
Four forms of intercellular signaling
Endocrine and neuronal strategies for
long-range signaling.
Extracellular signals can act slowly or rapidly to change the behavior of a target
cell .
Signalling via gap junctions
Extracellular Signal Molecules Bind to
Specific Receptors
• Different types of signals – proteins, peptides, amino acids,
nucleotides, steroids, fatty acid derivatives and even gases like NO and
CO.
• Signals can respond even at very low conc 10-8M and receptors can
bind at high affinity Ka > 108 liters/moles.
cell in a multicellular organism may be
exposed to hundreds of different signal
molecules in its environment
Different Types of Cells Usually Respond
Differently to the Same Extracellular Signal
Molecule
Positions of Developing cell determines
its fate
• Same signal acting on the same cell type can have qualitatively
different effects depending on the signal’s concentration

• Very important in animal development

• Signal is called as morphogen

• it diffuses out from a localized cellular source (a signaling center),

generating a signal concentration gradient
 Important to have a short lifetime signals
• Signal exerts its effects by altering the concentrations of intracellular molecules that are
short-lived (unstable), undergoing continual turnover

• Once the extracellular signal is gone, the replacement of the old molecules by new ones
wipes out all traces of the signal’s action.

• The speed with which a cell responds to signal removal depends on the rate of
destruction, or turnover, of the intracellular molecules that the signal affects

• turnover rate can determine the promptness of the response when an extracellular signal
arrives
Gases as a signaling molecules
• Nitric Oxide Gas acts as a Signals by Directly Regulating the Activity of
Specific Proteins Inside the Target Cell

• Example of hydrophobic signals – have cytosolic receptors

• one of NO’s many functions is to relax smooth muscle- walls of blood

vessels
Receptors
 Nuclear Receptors
•Hydrophobic signals - steroid hormones, thyroid hormones,
retinoids, and vitamin D.

• All acts through similar functions

•They bind to their respective intracellular receptor proteins and

alter the ability of these proteins to control the transcription of
specific genes

•These proteins serve both as intracellular receptors and as

intracellular effectors for the signal.
Nuclear Receptors Are Ligand-Modulated
Gene Regulatory Proteins
• The receptors are all structurally related, being part of the very large
nuclear receptor superfamily

• Orphan Receptors

• Steroid Hormones- Cortisol, Vitamin D, Sex hormones, and Thyroid

hormones
Cell surface receptors
• 3 important classes
• Ion channels
• G protein coupled receptors
• Enzyme coupled receptors

cell-surface receptors act as signal transducers by converting an extracellular

ligand-binding event into intracellular signals that alter the behavior of the
target cell
 ION-CHANNEL-COUPLED RECEPTORS
• Ion-channel-coupled receptors, also known as
transmitter-gated ion channels or ionotropic
receptors.

• involved in rapid synaptic signaling between

nerve cells and other electrically excitable
target cells such as nerve and muscle cells

• Neurotransmitters – signaling molecules

• ion-channel-coupled receptors belong to a

large family of homologous, multipass
transmembrane proteins.
 G-protein-coupled receptors
• Indirectly regulating the activity of a separate plasma-membrane-bound target protein, which is generally either an enzyme or an
ion channel.

• A trimeric GTP-binding protein (G protein) mediates the interaction between the activated receptor and this target protein

• The activation of the target protein can change the concentration of one or more small intracellular mediators (if the target
protein is an enzyme), or it can change the ion permeability of the plasma membrane (if the target protein is an ion channel).

• All of the G-protein-coupled receptors belong to a large family of homologous, multipass transmembrane proteins.
 Enzyme-coupled receptors
• Function directly as enzymes or associate directly with enzymes that they activate
• Single pass transmembrane proteins that have their ligand-binding site outside the cell and their
catalytic or enzyme-binding site inside.
• Majority have protein kinases activity
• Control the specialization of different cell types during development and in tissue renewal and repair
Cell-Surface Receptors Relay Signals Via
Small Molecules and a Network of
Intracellular Signaling Proteins
 Intracellular signalling proteins
• Proteins form a functional network, in which each protein helps to process the signal in one or more of the
following ways as it spreads the signal’s influence through the cell:

• Relay the signal to the next signaling component

• Scaffold to bring two or more signaling proteins together so that they can interact more quickly and
efficiently
• Amplify the signal it receives, either by producing large amounts of a small intracellular mediator or by
activating many copies of a downstream signaling protein
• Transform, or transduce, the signal into a different form, which is suitable for either passing the
signal along or stimulating a cell response
• May receive signals from two or more signaling pathways and integrate them before relaying a signal
onward
• Spread the signal from one signaling pathway to another, creating branches in the signaling stream,
thereby increasing the complexity of the response
• Anchor one or more signaling proteins in a pathway to a particular structure in the cell where the
signaling proteins are needed.
• Modulate the activity of other signaling proteins and thereby regulate the strength of signaling along
a pathway
 Intracellular Signaling Proteins Function as Molecular Switches That Are Activated by
Phosphorylation or GTP Binding

• About 30% of human proteins contain covalently attached phosphate, and the human genome encodes about
520 protein kinases and about 150 protein phosphatases. It is thought that a typical mammalian cell makes
use of hundreds of distinct types of protein kinases at any one time
 Intracellular Signaling Complexes Enhance the Speed,
Efficiency, and Specificity of the Response
3 types of intracellular complexes
• A receptor and some of the intracellular signaling proteins it activates in sequence are
preassembled into a signaling complex on the inactive receptor by a large scaffold
protein. In other cases, a preassembled complex binds to the receptor only after the
receptor is activated.
• A signaling complex assembles on a receptor only after the binding of an extracellular
signal molecule has activated the receptor; here the activated receptor phosphorylates
itself at multiple sites, which then act as docking sites for intracellular signaling proteins
• Activation of a receptor leads to the increased phosphorylation of specific phospholipids
(phosphoinositides) in the adjacent plasma membrane, which then serve as docking sites
for specific intracellular signaling proteins, which can now interact with each other
A specific signaling complex formed using
modular interaction domains
 Cells Can Use Multiple Mechanisms to Respond Abruptly to a Gradually Increasing
Concentration of an Extracellular Signal

Some cell responses to extracellular signal molecules are

smoothly graded according to the concentration of the signal
molecule

In other cases, the relationship between signal and response

can be discontinuous or all-or-none, with an abrupt switch
from one type of outcome to another as the signal
concentration increases beyond a certain value
Examining individual cells to detect all or-none responses
to increasing concentrations of an extracellular signal
 Strength of Signal is important
• Appearance of almost switch like behavior.

• one mechanism, more than one intracellular signaling molecule must bind to its downstream
target protein to induce a response – Eg 4 cAMP require to activate protein kinase A

• sharpening of response is seen when the activation of an intracellular signaling protein requires
phosphorylation at more than one site.

• Responses are also sharpened when an intracellular signaling molecule activates one enzyme and,
at the same time, inhibits another enzyme that catalyzes the opposite reaction

• Glycogen breakdown in skeletal muscle cells induced by the hormone adrenaline- activates an enzyme that
promotes glycogen breakdown and inhibits an enzyme that promotes glycogen synthesis.
To produce true all-or-none responses, one needs
another mechanism, positive feedback
• output of a process acts back to regulate that
same process

• positive feedback, the output stimulates its

own production

• negative feedback, the output inhibits its

own production
 A positive feedback mechanism giving
switch-like behavior
• A positive feedback loop in a signaling pathway can transform
the behavior of the responding cell.

• If the positive feedback is of only moderate strength, its effect

will be simply to steepen and increase the response to the
signal.
•
• if the feedback is strong enough, it can produce a qualitatively
different result: a runaway increase in the quantity of product
when the signal increases above a critical value, leading to a
new steady level of production that is sharply different from
that obtained when the signal was only slightly weaker
 Cells Can Adjust Their Sensitivity to a Signal
• cells and organisms can detect the same percentage of change in a signal over a very wide range of
stimulus strengths. The target cells accomplish this through a reversible process of adaptation, or
desensitization, whereby a prolonged exposure to a stimulus decreases the cells’ response to that
level of stimulus
Apoptosis
Programmed cell death

normal process that is unique to animal cells

Apoptosis occurs through an orchestrated

sequence of events that leads to the death of a cell

Death by apoptosis is a neat, orderly process

characterized by distinct morphological characteristics
and energy-dependent biochemical mechanisms

Inappropriate apoptosis (either too little or too much)

is a factor in many human conditions including
neurodegenerative diseases, ischemic damage,
autoimmune disorders and many types of cancer
 Significance of apoptosis
vital component of various
processes including normal cell
turnover, proper development
occurs normally during and functioning of the immune
development and aging and as system, hormone-dependent
a homeostatic mechanism to atrophy, embryonic
maintain cell populations in development and
tissues chemical-induced cell death

Apoptosis also occurs as a involved in the elimination of

defense mechanism such as in cells that have sustained
immune reactions or when cells irreparable genomic damage
are damaged by disease or
noxious agents
Necrosis
• Irreversible cell injury and eventual cell death due to pathological processes are termed necrosis.

• Follows some type of physical trauma or biochemical insult.

• It is an uncontrolled cell death that results in swelling of the cell organelles, plasma membrane
rupture and eventual lysis of the cell, and spillage of intracellular contents into the surrounding
tissue leading to tissue damage.

• necrosis can also occur as a regulated and programmed process (called necroptosis), although
much less orderly in nature
Causes of Necrosis
• Cell injury can range from external injury to internal abnormalities. The
most common causes of injurious stimulus include:

• Hypoxia: This can occur due to ischemia, shock, or respiratory failure.

• Physical agents: These include external injuries such as trauma, extremes of
temperature, radiation exposure, or electric shock
• Chemical agents: These include poisons, occupational exposure, drug
toxicities, or recreational drugs.
• Biological agents: bacteria, viruses, or fungi
• Immunologic reactions: autoimmune responses
• Necrosis is characterized by the swelling of both the cell and its internal membranous organelles,
membrane breakdown, leakage of cell contents into the medium, and the resulting induction of
inflammation
Why do our bodies have unwanted cells, and where do
we find cells that become targeted for elimination?
Morphology of Apoptosis
Cell shrinkage- the cells are smaller in size; the
cytoplasm is dense, and the organelles are more tightly
packed

Pyknosis- chromatin condensation and this is the most

characteristic feature of apoptosis

The apoptotic cell appears as a round or oval mass

with dark eosinophilic cytoplasm and dense purple
nuclear chromatin fragments
Extensive plasma membrane blebbing occurs followed
by karyorrhexis and separation of cell fragments into
apoptotic bodies during a process called “budding.”
 It has been estimated that 10 -10 – 10 -11 cells in the adult body die every day by apoptosis

apoptosis is involved in the elimination of cells that have sustained irreparable genomic damage.

Apoptosis is also responsible for the death of cells that are no longer required, such as activated T
cells that have responded to an infectious agent that has been eliminated.

apoptosis appears to be involved in neurodegenerative diseases such as Alzheimer ’ s disease,

Parkinson ’ s disease, and Huntington ’ s disease
Inappropriate apoptosis
•Failure

•Overactive
Studies of apoptosis in C.elegans
• Insight into the molecular basis of apoptosis was first revealed in
studies on the nematode worm C. elegans , whose cells can be
followed with absolute precision during embryonic development.

• Of the 1090 cells produced during the development of this worm, 131
cells are normally destined to die by apoptosis.

• Importance of CED-3 gene – Discovery of caspases

Caspases
• Cysteine proteases
• Very important molecules in apoptosis
• There are more than 11 known caspases
 Targets of caspases
Dozen of protein kinases,
including focal adhesion Disrupt cell adhesion, leading to detachment of the apoptotic cell from its
kinase ( FAK ), PKB, PKC, neighbors. Inactivation of certain other kinases, such as PKB, serves to disrupt pro
and Raf1. survival signaling pathways.

Cleavage of Lamins
Disassembly of the nuclear lamina and shrinkage of the nucleus
Cleavage and consequent
inactivation of intermediate
filaments, actin, tubulin,
and gelsolin. Changes in cell shape
Activation of an
endonuclease called
caspase activated DNase
(CAD) Attacks DNA, severing it into fragments.
 Mechanisms of Apoptosis
Highly complex and sophisticated, involving an
energy-dependent cascade of molecular events

Apoptosis can be triggered by both internal stimuli, such as abnormalities in the DNA, and
external stimuli, such as certain cytokines (proteins secreted by cells of the immune system)

Internal signals - epithelial cells of the prostate become apoptotic when

deprived of the male sex hormone testosterone. (Intrinsic pathway)

External signals - extracellular messenger protein

called Tumor necrosis factor (TNF), (Extrinsic pathway)

Cross-talk between these pathways and that extracellular

apoptotic signals can cause activation of the intrinsic pathway.
Additional Pathway – perforin/Granzyme
pathway

involves T-cell mediated cytotoxicity and

perforin-granzyme-dependent killing of the cell

induce apoptosis via either granzyme B or

granzyme A
Biochemical Features of Apoptosis
Biochemical modifications such as protein cleavage, protein cross-linking, DNA breakdown, and phagocytic
recognition that together result in the distinctive structural pathology

Activation of caspases- initiation of a protease cascade.

Extensive protein cross-linking is another characteristic of apoptotic cells and is achieved through the
expression and activation of tissue transglutaminase

DNA breakdown by Ca2+ and Mg2+ dependent endonucleases also occurs, resulting in DNA fragments
of 180 to 200 base pairs

Expression of cell surface markers that result in the early phagocytic recognition of apoptotic cells by
adjacent cells - Annexin I and calreticulin.
 The Extrinsic Pathway of Apoptosis
• Extracellular messenger protein – TNF
• Binds to TNFR1 (death receptors).
• Death domain: protein-protein
interaction - recruitment of several
proteins
• Interaction with pro caspases
• Activation of downstream caspases-
Apoptosis
• Necroptosis- RIPK interacts with RIP3K
that activates MLKL- oligomerize-
cytoplasmic leakage
 Intrinsic Pathway:
• Internal stimuli, such as irreparable genetic
damage, lack of oxygen (hypoxia), extremely high
concentrations of cytosolic Ca 2+ , viral infection,
ER stress, or severe oxidative stress (i.e., the
production of large numbers of destructive free
radicals), trigger apoptosis by the intrinsic pathway.

• Regulated by members of the Bcl-2 family

Perforin/granzyme Pathway:
• T-cell mediated cytotoxicity is a variant of type IV hypersensitivity where sensitized CD8+cells kill
antigen-bearing cells

• These cytotoxic T lymphocytes (CTLs) can kill target cells via the extrinsic pathway and the FasL/FasR
interaction is the predominant method of CTL-induced apoptosis

• involves secretion of the transmembrane pore-forming molecule perforin with a subsequent exophytic
release of cytoplasmic granules through the pore and into the target

• The serine proteases granzyme A and granzyme B are the most important component within the granules

• Granzyme B will cleave proteins at aspartate residues and will therefore activate pro-caspase-10 and can
cleave factors like ICAD (Inhibitor of Caspase Activated DNAse)
• Granzyme A is also important in cytotoxic T cell induced apoptosis and
activates caspase independent pathways. Once in the cell, granzyme A
activates DNA nicking via DNAse NM23-H1, a tumor suppressor gene
product.

• DNAse has an important role in immune surveillance to prevent cancer

through the induction of tumor cell apoptosis.

• The nucleosome assembly protein SET normally inhibits the NM23-H1

gene. Granzyme A protease cleaves the SET complex thus releasing
inhibition of NM23-H1, resulting in apoptotic DNA degradation.
Conclusion
Apoptosis is regarded as a carefully regulated energy dependent process, characterized by
specific morphological and biochemical features in which caspase activation plays a central
role.

Although many of the key apoptotic proteins that are activated or inactivated in the
apoptotic pathways have been identified, the molecular mechanisms of action or activation
of these proteins are not fully understood and are the focus of continued research.

The importance of understanding the mechanistic machinery of apoptosis is vital because

programmed cell death is a component of both health and disease, being initiated by various
physiologic and pathologic stimuli.

Understanding the mechanisms of apoptosis, and other variants of programmed cell death,
at the molecular level provides deeper insight into various disease processes and may thus
influence therapeutic strategy.