Apoptosis

1. Introduction

Apoptosis or Programmed cell death is a normal process that is Unique to

animal cells. Apoptosis occurs through the orchestrated sequence of that leads to

death of cell. Death by apoptosis is a neat orderly process characterised by the

overall shrinkage in volume of the cell and it’s nucleus the loss of adhesion to

neighboring cells the formation of blebs at the cell surface, the dissection of the

chromatin into small fragments and rapid engulfment of ‘corpse’ by phagocytosis.

The term apoptosis was coined in1972 by John Kerr, Andrew Wylie, And

A. R. Currie of the University of Aberdeen, Scotland. They first time described the

coordinated events that occurred during the programmed death of a wide range of

a cells.

Apoptosis plays a crucially important part in animals. In healthy adult

human billions of cells die in a bone marrow and intestine every hour. The normal

cell deaths are suicides, in which the cells activate an intercellular death program

and kill themselves in a controlled way and a process is known as programmed

cell death. Apoptosis is a Greek word meaning falling off, as a leaves from a tree.

Cells dying by apoptosis undergo characteristic morphological changes. They

shrink and condense the cytoskeleton collapse and breaks up into fragments.

The growth development and maintenance of multicellular Organism

depend not only on the production of cells but also on mechanism to destroy them.

The cell surface blebs and if the cell is large breaks up into membrane enclosed

fragments called apoptotic bodies. The surface of the cell or apoptotic bodies

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becomes chemically altered, so that neighbouring cell or macrophage rapidly

engulfs them, before they can spill their contents. In this way, the cell dies neatly

and rapidly cleared away without causing damage inflammatory response.

Animal cells that die in response to an acute insult such as trauma or lack of

blood supply usually do so by a process is called cell necrosis. Necrotic cells swell

and burst spilling their contents over their neighbours and eliciting an

inflammatory response. Necrosis is likely to be caused by energy depletion which

leads to metabolic defects and loss of ionic gradients that normally exist across the

cell membrane.

2. Morphological Changes during Apoptosis

It is a normal physiological response to specific suicide signals or lack of

survival signals. In apoptosis the chromatin condenses and migrates to nuclear

membrane, whereas the cytoplasm undergoes shrinkage without damage to the

plasma membrane.

Figure 1. Morphological Changes during Apoptosis

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The cell contents are packaged in membrane bound bodies, and internal

organelles, though still functioning, are engulfed by neighbours. Normal cell

undergoes shrinkage condensation of chromatin and cell blebbing. The cell is

fragmented to produce apoptotic bodies (Figure 1).

3. Elimination of Unwanted Cells by Apoptosis

Apoptotic cell death occurs in a developing and adult animal tissue. In the

developing vertebrate nervous system more than half of many types of nerve cells

normally die soon after they are formed. Apoptosis helps to sculpt hands and feet

during embryonic development. The individual digits separate only as the cells

between them die. When a tadpole changes to frog at metamorphosis, the cells in

the tail die and the tail which is not needed in the frog disappears. Programmed

cell death also functions as a quality-control process in development, eliminating

cells that are abnormal, misplaced, non-functional or potentially dangerous to the

animal.

Example 1. Sculpting the digits in the developing mouse paw by apoptosis

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Example 2. Apoptosis during the metamophosis of a tadpole into a frog

In vertebrate adaptive immune system, where apoptosis eliminates

developing T and B lymphocytes that fail to produce potentially useful antigen-

specific receptors or produce self-reactive receptors that make the cells potentially

dangerous. It also eliminates most of the lymphocytes activated by an infection,

after they have helped to destroy the responsible microbes.

Apoptosis occurs at a high rate in the adult human bone marrow, where

most blood cells are produced. Neutrophils of white blood cell are produced

continuously in very large numbers. But the majority cells die by apoptosis in

bone marrow within a few days without functioning. The production and

destruction serves to maintain a ready supply of short-lived neutrophils.

Animal cells can recognize damage in their various organelles and they can

kill themselves by undergoing apoptosis. DNA damage which can produce cancer

promoting mutation. If not repaired, cells have various ways of detecting DNA

damage. If they cannot repair it, they kill themselves by undergoing apoptosis.

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4. Apoptosis Depends on an Intracellular Proteolytic Cascade that is

Mediated by Caspases

The intracellular machinery responsible for apoptosis is similar in all

animal cells. It depends on a family of proteases that have a cysteine at their active

site and cleave their target proteins at specific aspartic acids. They are called

caspases (C for cysteine and asp for aspartic acid). Caspases are synthesized in the

cell as inactive precursors or procaspases, which are activated by proteolytic

cleavage. Procaspase cleavage occurs at one or two specific aspartic acids and it is

catalyzed by other active caspases. The procaspase is split into a large and a small

subunit that form a heterodimer and two such dimers assemble to form the active

tetramer. Once activated caspases cleave and activate other procaspases resulting

in an amplifying proteolytic cascade.

The first caspase Identified was a human protein called interleukin-1-

converting enzyme (ICE), which is concerned with inflammatory responses rather

than cell death. ICE cuts out the inflammation-inducing cytokine interleukin-1

form a larger precursor protein. A gene required for apoptosis in C. elegans was

encode a protein that is structurally and functionally similar to ICE, providing the

first evidence that proteolysis and caspases are involved in apoptosis. Not all

caspases mediate apoptosis. Several human caspases are involved in inflammatory

and immune responses, most are involved in apoptosis.

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Figure 2. Procaspase activation during apoptosis

Some of the procaspases that operate in apoptosis act at the start of the

proteolytic cascade and are called initiator procaspases. When activated, they

cleave and activate downstream executioner procaspases, then cleave and activate

other executioner procaspases and also specific target proteins in the cell. Among

the many target proteins cleaved by executioner caspases are the nuclear lamins,

the cleavage of which causes the irreversible breakdown of the nuclear lamina and

the another target is a protein that normally holds the DNA-degrading enzyme in

an inactive form, its cleavage frees the endonuclease to cut up the DNA in the cell

nucleus. Other target proteins include components of the cytoskeleton and cell to

cell adhesion proteins that attach cells to their neighbours, the cleavage of these

proteins helps the apoptotic cell to detach from its neighbour cell, making healthy

neighbouring cells to engulf it.

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The caspases required for apoptosis vary depending on the cell type and

stimulus. Inactivation of the mouse gene encoding caspase-3, an executioner

caspase. Reduces normal apoptosis in the developing brain. As a result, the mouse

dies around birth with a deformed brain that contains many cells. From earlier

stages of an animal development, healthy cells continuously make the procaspases

and other proteins required for apoptosis. Thus, the caspase is an apoptosis

machinery that trigger to activate procaspase (Figure 2).

Initiator procaspases have a long prodomain which contains a caspase

recruitment domain (CARD) that enables them to assemble with adaptor proteins

into activation complexes, when the cell receives a signal to undergo apoptosis.

Once incorporated into such complexes, the initiator procaspases are brought into

close proximity, which is sufficient to activate them, they then cleave each other to

make the process irreversible. The activated initiator caspases then cleave and

activate executioner procaspases thereby initiating a proteolytic caspase cascade,

which amplifies the death signal and spread throughout the cell.

5. Mechanism of Apoptosis

i. Extrinsic or Death receptor pathway

ii. Intrinsic or Mitochondrial pathway

iii. Perforin or Granzyme pathway.

i. Extrinsic or Death receptor pathway

The extrinsic signaling pathways that initiate apoptosis involve

transmembrane receptor-mediated interactions. These involve death receptors that

are members of the tumour necrosis factor (TNF) receptor gene super family.

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Extracellular signal proteins binding to cell surface death receptors triggers

the extrinsic pathway of apoptosis. Death receptor are transmembrane proteins

containing an extracellular ligand-binding domain. The receptors are homotrimers

and belong to the tumour necrosis factor (TNF) receptor family. The ligand that

activate the death receptors are also homotrimers. They are structurally related and

belong to the TNF family of signal proteins.

Figure 3. Extrinsic or Death receptor pathway

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The FAS ligand on the surface of a killer lymphocyte activates FAS death

receptors on the surface of the target cell. Both the ligand and receptors are

homotrimers. The cytosolic tail of FAS then activate the adaptor protein FADD.

Each FADD protein then activates the initiator procaspase (Procaspase-8,

Procaspase-10 or both). The death effector domain on both FADD and the

procaspase, forming a death-inducing signaling complex (DISC). Within the

DISC, the initiator procaspase molecules are brought into close proximity, which

activates them.

The activated procaspases then cleave to one another to stabilize the

activated protease, which is caspase. Activated caspase-8 and caspase-10 then

cleave and activate executioner procaspases, producing a caspase cascade, leading

to apoptosis (Figure 3).

ii. Intrinsic or mitochondrial pathway

The intrinsic signalling pathways that initiate apoptosis involve a diverse

array of non-receptor-mediated stimuli that produce intracellular signals that act

directly on targets within the cell and are mitochondrial-initiated events.

Cells can also activate their apoptosis program from inside the cell, in

response to injury or other stresses such as DNA damage or lack of oxygen,

nutrients or extracellular survival signals. In vertebrate cells, such intracellular

activation of the apoptotic death program occurs. The intrinsic pathway of

apoptosis, which depends on the release into the cytosol of mitochondrial proteins

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that normally reside in the intermediate space of these organelles. Some of the

released proteins activate a caspase proteolytic cascade in the cytoplasm, leading

to apoptosis.

Figure 4. Intrinsic or mitochondrial pathway

A crucial protein released from mitochondria in the intrinsic pathway in

cytochrome C, a water-soluble component of the mitochondrial electron-transport

chain. When released into the cytosol, it binds to the procaspase activating adaptor

protein called Apaf1 (apoptotic protease activating factor-1) causing the Apaf 1 to

oligomerize into a wheel like heptamer called as apoptosome. The Apaf1 proteins

in the apoptosome then activate initiator procaspase proteins, procaspase-9. The

procaspase activate caspase-9. The activated caspase-9 molecules then activate

downstream executioner procaspases to induce apoptosis.

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Mammalian Bcl2 proteins regulate the intrinsic pathway of apoptosis

mainly by controlling the release of cytochrome-C and other intermembrane

mitochondrial proteins into the cytosol. Some Bcl2 proteins are proapoptotic and

promote apoptosis by enhancing the release, whereas others are anti-apoptotic

Bcl2 proteins can bind to each other in various combinations to form

heterodimers, in which the two proteins inhibit each others function. The balance

between the activities of these two functional classes of Bcl2 proteins largely

determines whether a mammalian cell lives or dies by the intrinsic pathway of

apoptosis. The pro-apoptotic Bcl2 proteins consist of two subfamilies- the BH123

proteins and BH 3-only proteins. The main BH123 proteins are Bax and Bak.

When an apoptotic stimulus triggers the intrinsic pathway (Figure 4), the

pro-apoptotic BH123 proteins become activated and aggregate to form oligomers

in the mitochondrial outer membrane inducing the release of cytochrome C. In

mammalian cells, Bax and Bak are the main BH123 proteins. Bak is tightly bound

to the mitochondrial outer membrane even in the absence of an apoptotic signal.

Bax is mainly located in the cytosol and translocates to the mitochondria only after

an apoptotic signal activates it. The anti-apoptotic Bcl2 proteins such as Bcl2 itself

Bcl-XL are also mainly located on the cytosolic surface of the outer mitochondrial

membrane, endoplasmic reticulum and the nuclear envelope.

The BH3-only proteins are the largest subclass of Bcl2 family proteins. The

cell either produces or activates them in response to an apoptotic stimulus. Their

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BH3 domain binds to a long hydrophobic groove on anti-apoptotic Bcl2 proteins,

neutralizing their activity. BH3-only protein provides the crucial link between

apoptotic stimuli and intrinsic pathway of apoptosis.

An Intracellular signalling pathway that depends on the MAP kinase JNK

activates the transcription of the gene encoding the BH3-only protein Bim, which

then triggers the intrinsic pathway. The DNA damage that cannot be repaired, the

tumour suppressor protein p53 accumulates and activates the transcription of

genes that encode the BH3-only proteins Puma and Noxa. These BH3-only

proteins then trigger the intrinsic pathway, thereby eliminating a potentially

dangerous cell that could otherwise become cancerous.

The BH3-only proteins Bid, Bim and Puma can inhibit all of the anti-

apoptotic Bcl2 proteins. Thus, Bid, Bim and Puma are the most potent activators

of apoptosis in the BH3-only subfamily of Bcl2 proteins.

iii. Perforin or Granzyme pathway

Perforin / Granzyme apoptosis pathway is the primary signaling pathway

used by cytotoxic lymphocytes to eliminate virus infected and transformed cells.

In gene disrupted mice indicate that perforin, in combination with Granzyme could

induce apoptosis.

In the Granzyme pathway of apoptosis like cell death, serine proteases such

as Granzyme-A, Granzyme-B are released by cytotoxic T cells / natural killer cells

to pathogen infected cells. The cytotoxic T cells release perforin that makes pores

on membrane of the infected cells through the perforin pores, the Granzyme-A and

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B enters the cell. Granzyme-A forms set complex inducing DNA fragmentation

and apoptosis a caspase independent pathway. Granzyme-B mediates activation of

procaspase-9 and procaspase-3 induction of ROS to promote apoptosis (Figure 5).

Figure 5. Perforin or Granzyme pathway

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6. Summary

Programmed cell death plays a key role in both the maintenance of adult

tissues and embryonic development. The accidental death of cells from an acute

injury most programmed cell death takes place by the active process of apoptosis.

apoptotic cells and cell fragments are then efficiently removed by phagocytosis.

Genes are responsible for the regulation and execution of apoptosis was initially

identified by genetic analysis of C. elegans.

The morphological changes takesplace during apoptosis are normal cell

undergoes shrinkage condensation of chromatin and cell blebbing. The cell is

fragmented to produce apoptotic bodies.

The caspases are a family of proteases that are the effectors of apoptosis.

Caspases are classified as either initiator or effector caspases and both the function

in cascade leading to cell death. Caspases are present in all nucleated animal cells

as inactive precursors called procaspase.

There are three main pathways in apoptosis. They are (i) extrinsic pathway

(ii) intrinsic pathway and (i) perforin/granzyme pathway. The extrinsic pathway is

activated by extracellular ligands binding to cell surface death receptors. The

intrinsic pathway is activated by intracellular signals generated when cells are

stressed.

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Both extracellular signal proteins and intracellular Bcl2 proteins and IAP

proteins regulate the apoptotic program to ensure that cells normally kill

themselves only when it benefits the animals. The high level of Bcl2 protein in the

lymphocytes that promotes the development of cancer by inhibiting apoptosis. In

mammalian cells proapoptotic Bcl2 Family members act as mitochondria where

they promote the release of cytochrome C. Perforin/granzyme-induced apoptosis is

the main pathway used by cytotoxic lymphocytes to eliminate virus-infected or

transformed cells.

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References

1. Albert, B., Johnson, A., Lewis, J., Raff, M., Robert, K. and Walter Petter.
(2008). Molecular Biology of the Cell. V Edition, Garland Science Taylor
and Francis Group Publishers, USA., pp. 1115 -1121.

2. Copper, G.M. and Hausman, R.E. (2016). The Cell: A Molecular Approach.
VII Edition, Library of Congress Cataloguing in Publication, USA, pp. 694-
697.

3. Karp, G. (2013). Cell Biology. VII Edition, John Wiley and Sons Inc.,
Publishers, New Delhi, pp. 656-660.

4. Nirmala J. Grace and Lopus Manu. (2020). Cell death mechanism in

eukaryotes. Cell Biology and Toxicology, 36, 1001-1007.

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CONTENTS

Page No.

1 Introduction 1

2 Morphological Changes during Apoptosis 2

3 Elimination of Unwanted Cells by Apoptosis 3

4 Apoptosis Depends on an Intracellular Proteolytic 5

Cascade that is Mediated by Caspases

5 Mechanism of Apoptosis 7

i. Extrinsic or Death receptor pathway

ii. Intrinsic or mitochondrial pathway

iii. Perforin or Granzyme pathway

6 Summary 14

References 16

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