APOPTOSIS

BIOCHEMICAL FEATURES &

MECHANISMS
Name: Dr KACHINDA
LAMU-PATHOLOGY
2016
INTRODUCTION
‘Apo’ from ancient Greek means ‘away from’ & ‘ptosis’ means
‘falling’
Cell death plays a crucially important role in animal and plant
development
We now know that ‘normal’ cell death are suicides, in which cells
activate intracellular death program and kill themselves in a
controlled way – process known as programmed cell death
In contrast to necrosis which results from traumatic cell death and
illicit an inflammatory response, apoptosis does not illicit any
inflammatory response.
CAUSES OF APOPTOSIS
Apoptosis in Physiologic Situations
 The programmed destruction of cells during embryogenesis, including
implantation, organogenesis, developmental involution, and
metamorphosis
Involution of hormone-dependent tissues upon hormone withdrawal,
such as endometrial cell breakdown during the menstrual cycle, ovarian
follicular atresia in menopause, the regression of the lactating breast
after weaning, and prostatic atrophy after castration
Cell loss in proliferating cell populations, such as immature lymphocytes
in the bone marrow and thymus that fail to express useful antigen
receptors, B lymphocytes in germinal centres, and epithelial cells in
intestinal crypts, so as to maintain a constant number
Elimination of potentially harmful self-reactive lymphocytes,
either before or after they have completed their maturation,
so as to prevent reactions against one’s own tissue
Death of host cells that have served their useful purpose,
such as neutrophils in an acute inflammatory response, and
lymphocytes at the end of an immune response. In these
situations cells undergo apoptosis because they are deprived
of necessary survival signals, such as growth factors
Apoptosis in Pathologic Conditions
 DNA DAMAGE. Radiation, cytotoxic anticancer drugs, and
hypoxia can damage DNA, either directly or via production of
free radicals. If repair mechanisms cannot cope with the injury,
the cell triggers intrinsic mechanisms that induce apoptosis
Accumulation of mis-folded proteins. Improperly folded
proteins may arise because of mutations in the genes encoding
these proteins or because of extrinsic factors, such as damage
caused by free radicals. Excessive accumulation of these proteins
in the ER leads to a condition called ER stress, which culminates
in apoptotic cell death
Cell death in certain infections, particularly viral infections,
in which loss of infected cells is largely due to apoptosis that
may be induced by the virus (as in adeno- virus and HIV
infections) or by the host immune response (as in viral
hepatitis).
Pathologic atrophy in parenchymal organs after duct
obstruction, such as occurs in the pancreas, parotid gland,
and kidney.
MORPHOLOGICAL FEATURES OF
APOPTOTIC CELLS
Cell shrinkage – cell reduces in size due to cytoskeleton collapse
Nuclear fragmentation
Chromatin condensation - most characteristic feature of apoptosis
where the chromatin aggregates peripherally, under the nuclear
membrane, into dense masses of various shapes and sizes
Formation of cytoplasmic blebs and apoptotic bodies - the
apoptotic cell first shows extensive surface blebbing, then
undergoes fragmentation into membrane-bound apoptotic bodies
composed of cytoplasm and tightly packed organelles, with or
without nuclear fragments
APOPTOTIC CELLS ARE BIOCHEMICALLY
RECOGNIZABLE
• Cell undergoing apoptosis not only have characteristic morphology
but also display characteristic biochemical changes which is used to
identify apoptotic cells
• Important changes occurs in the plasma membrane of apoptotic cells
• Negatively charged phospholipid phosphatidylserine is exclusively
located in the inner leaflet of the lipid bilayer of plasma membrane
• But it flips to the outer leaflet in apoptotic cells serving as a cell
marker for phagocytosis
• Visualisation of this marker on the cell surface is achieved by Annexin
V protein which specifically binds this phospholipid
• Phosphatidylserine also blocks the inflammation often associated with
phagocytosis: the phosphatidylserine – dependent engulfment of
apoptotic cells inhibits the production of inflammation – inducing
signal proteins cytokines by the phagocytic cells
• Cells undergoing apoptosis often lose the electrical potential that
normally exists across the inner membrane of the mitochondria
• The membrane potential is measured by the use of positively charged
fluorescent dyes that accumulate in the mitochondria driven by the
negative charge of inner mitochondria
• The relocation of cytochrome C and other proteins from the
mitochondria to the cytosol is used as another marker of apoptosis
INTRACELLULAR MECHANISM OF
APOPTOSIS
• Intracellular machinery responsible for apoptosis depends
on a family of proteases that have a cysteine at their active
site and cleave their target proteins at specific aspartic acids
and these are called caspases
• These procaspases are split in to a large and small subunits
that form a heterodimer and two such dimers assemble to
form the active tetramer. Once activated the caspases cleave
and there by activate, other procaspases resulting in
amplifying proteolytic cascade
• Procaspases that start apoptosis in the proteolytic cascade
are called initiator procaspases; when activated will cleave
downstream executioner proteins procaspases as well as
target proteins in the cell
HOW IS THE FIRST PROCASPASES IN THE
CASCADE ACTIVATED?

• Initiator procaspases have along prodomain, containing the

caspases recruitment domain (CARD) that enables them to
assemble with the adapter molecules into activation
complexes when the cell receives a signal to undergo
apoptosis
• Once incorporated into the complex initiator procaspases are
brought into close proximity, that is sufficient to activate
them cleaving each other irreversibly
• Initiator procaspases activate executioner procaspases,
initiating a proteolytic caspases cascade amplifying the death
Extrinsic (Death Receptor–Initiated)
pathway
• Extrinsic pathway of apoptosis is triggered by the binding of extracellular signal
proteins to the cell-surface death receptors
• It contains the extracellular ligand-binding domain, a single transmembrane
domain and an intracellular death domain
• These receptors are homotrimers belonging to the tumour necrosis factor (TNF)
receptor family and includes the TNF itself and the Fas death receptor
• Activation of the Fas on the surface of the target cell by fas ligand on the surface
of the killer lymphocyte (cytotoxic), the death domains on the cytosolic tails of
the Fas death receptor recruit intracellular adaptor proteins, which in turn recruit
initiator procaspases( procaspases-8 and or procaspases-10 forming a death-
inducing signalling complex (DISC) which activate downstream the executioner
procaspases then induce apoptosis
The Extrinsic (Death Receptor–Initiated) Pathway
of Apoptosis
CONTROL MECHNISM OF EXTRINSIC
PATHWAY
• Cell surface Decoy receptors, which have a ligand-binding
domain but no death domain
• Cell can produce Intracellular blocking proteins such FLIP
resembling an initiator procaspases but lacks the proteolytic
domain
• Such inhibitory mechanisms help prevent the inappropriate
activation of the extrinsic pathway of apoptosis
Intrinsic (mitochondrial)pathway
• Apoptosis can be activated from inside the cell in response to injury or
stress such as DNA damage or lack of oxygen, nutrients or extracellular
survival signals
• This mechanism involves the release into the cytosol of mitochondrial
proteins that reside in the intermembrane space of these organelles
activating the caspases proteolytic cascade in the cytoplasm
• Cytochrome c is a crucial protein released from the mitochondria; a
water-soluble component of the electron transport chain
• When released into the cytosol, it binds a procaspases-activating
adaptor protein called the Apaf1 (apoptotic protease activating- factor-
1)
• The Apaf1 oligomerize into wheel like heptamer called the
apoptosome
• The apoptosome then recruit initiator procaspases protein
(procaspases-9) which are then activated in the proximity
• The activated caspases-9 then activate down- stream
executioner procaspases to induce apoptosis
Bcl2 proteins regulate the intrinsic
pathway
• They are a major class of intracellular regulators of apoptosis
• They tightly regulate to ensure that cells kill them selves when
appropriate
• Some Bcl2 proteins are pro-apoptotic and promote apoptosis by
enhancing the release of mitochondrial proteins while others anti-
apoptotic inhibiting or blocking the release of mitochondrial proteins
• Bcl2 and bcl-XL are pro-apoptotic with two subfamilies BH123
proteins and the BH3-only protein
• Bax and Bak are from BH123 family and when apoptotic stimulus is
available, they aggregate to form oligomers leading to the release of
cytochrome c to the cytosol
• Bax and Bak operate on the surface of the endoplasmic reticulum and nuclear
membrane when activated in response to ER stress they are thought to release
the release ca2+ into the cytosol activating the mitochondrial-dependent intrinsic
pathway
• Anti-apoptotic Bcl2 proteins include the Bcl2 itself and the Bcl-XL are located on
the cytosolic surface of the outer mitochondrial membrane, ER and nuclear
envelope
• Preserve the integrity of membrane by preventing inappropriate release of
proteins from the mitochondria and ca2+ from the ER
• These proteins inhibit apoptosis mainly by binding to and inhibiting pro-apoptotic
Bcl2 proteins either on these membranes or in the cytosol
• BH3 only proteins are usually produced or activated in response to an apoptotic
stimulus, promote apoptosis mainly by inhibiting anti-apoptotic Bcl2 proteins
• BH-3 only protein domain binds to a long hydrophobic
groove on the anti apoptotic Bcl2 proteins neutralizing
their activity
References
• 1. Bruce alberts eta’l 5th Edition,(2008) Molecular Biology of the cell,
Talyor and Francis
• 2. Kumar and Abas 7th Edition, (2005) Robbins Pathological basis of
Disease. Elsevier Saunders