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Lecture 1
Introduction to Apoptosis
 Apoptosis, concept of which was established in 1972, is associated with fragmentation of genomic DNA
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Characteristics of apoptosis and necrosis
Apoptosis (Death by design)
Affects single cells

Initiated by direct cell damage (pathological)

An active process: requires macromolecule synthesis

Passive: does not require macromolecule synthesis

Cells shrink

Cells swell

Membrane blebs (buds off) but integrity is maintained

Membrane integrity lost

Increased mitochondrial membrane permeability

Organelle swelling and lysosomal leakage

Chromatin condenses and genomic DNA fragments into a 200 bp
ladder

No condensation of chromatin and random degradation of
genomic DNA

Formation of apoptotic body

No formation of apoptotic body

Apoptotic bodies ingested by neighbouring cells

Lysed cells ingested by macrophages

Does not cause inflammation

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Affects groups of neighbouring cells

Initiated by a signal transduction process (physiological)

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Necrosis (Death by accident)

Causes significant inflammation

Caenorhabditis elegans (C
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Why do we use them? Because they are small, fast growing, culture them on agar plates, they are tough as
they can even survive space shuttle disaster
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elegans programmed cell death machinery:-

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Order remains the same whatever kind of system it may be
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elegans called
ced-3
To make the proteases activated, we need an adaptor protein in C
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Because we don’t want the proteases to be activated too quickly, we need a regulatory process
wherein, ced-9 inhibits the activity of ced-4 else, ced-4 would activate ced-3 all the time giving
unwanted cell death
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The block also needs to be able to be released
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So, egl-1 inhibits
ced-9 activity
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Mammalian apoptosis follows the same pathway but with many more components (Inhibitors,
Regulator, Adaptor, Protease, Death)
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elegans and humans are separated by years of evolution, the rules and proteins
of how they work are very similar
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 95% of such animals do not survive postnatally
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e
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C=Cranioschisis i
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failure of the skull to close
Defects in formation of eyes (D-E)

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 Thicker retina, smaller lens
Persistence of interdigital webs (I-K)

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Sculpting mechanisms of apoptosis during development

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Of those mice that survive adulthood, males are infertile because of degeneration of the
spermatogonia germ cells
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Because the death signals are stronger, they get into the middle of the ball of cells
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In the lumen, there is more death that survival at this point
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Normally, same amount of cells produced are turned over
If there is too much apoptosis, the systems may fail
Example: If pancreatic islets are killed there is the occurrence of diabetes
If there is defective apoptosis like a tumour for example, may persist and carry on dividing undesirable
cells
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 Too much apoptosis of the trophoblast cells can lead to complication such as pre-eclampsia
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 Cancer
Consequences of ineffective apoptosis in cancer
 Mutations that cause cancer, can lead to excessive proliferation
 Cells with badly damaged DNA such as UV exposure, normally undergo apoptosis preventing
propagation of genetic damage
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 Most cancer treatments (chemo- and radio-) rely on being able to induce DNA damage in cells to cause
them to undergo apoptosis
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Apoptosis and Lifestyle
 Sex
 Measure of apoptosis in animals given alcohol
 Animals given a lot of ethanol will always have a small amount of apoptosis in their sperm
production
 More ethanol, more apoptosis
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 Drugs
 Alcohol can cause apoptosis in muscles in liver
 Heroin: Pure heroin does not cause apoptosis in comparison to heroin mixed morphine and 6monoacetylmorphine
 Cannabinoids: Low doses enhances cell proliferation (Good), too much causes cell cycle arrest
and cell death (Bad)
 Cocaine: Apoptosis in myocardial cells in fetuses and adults
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 Excessive noise
 Exposure to intensive noise causes apoptosis of the hair cells in the ear within 5 minutes of
exposure to 75 seconds of sound > 155 dB

Lecture 2
Pathways of Apoptosis in Mammals
 Recap:
o Apoptosis is controlled compared to necrosis
o Important in development, maintenance of an appropriate cell number
o Mutations in crucial apoptosis genes designated ced - cell death abnormality
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o Early in apoptosis, there is expression of PS in the outer layers of the cell membrane, which has been
flipped out from the inner layers
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o Followed by characteristic breakdown of DNA into large 50-300 kb pieces
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o Activated caspase cleaves many vital cellular proteins and break up the nuclear scaffold and
cytoskeleton
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o Active caspase is a homodimer with each monomer consisting of a large and a small subunit
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o Several Alpha-helices and short Beta-strands are located on either side of the central Beta-sheet which gives
rise to globular fold
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o The four active-site loops determine the sequence specificity of caspase substrates
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o The S4 subsites of caspase-8 and -9 occupy intermediate positions and show preference for small
hydrophobic Val or Leu residue
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Initiator and effector caspases
o First mammalian caspase (caspase-1 or ICE) was identified as an important regulator of the
inflammatory response
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 CED-3 is the only apoptotic caspase in nematodes and functions as both, an initiator and
effector caspase
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o Initiator caspase is characterized by an extended Asp residue that separate the large (p20) and small
(p10) subunits
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o As the activation of an initiator caspase in cells occurs, it inevitably triggers a cascade of downstream
caspase activation, it is tightly regulated and often requires the assembly of a multi-component complex
under apoptotic conditions
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o Once activated, the effector caspases are responsible for the proteolytic cleavage of a broad spectrum
of cellular targets, which ultimately leads to cell death
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Ced-9

Effector caspases with short prodomains perform downstream execution steps of apoptosis by
cleaving multiple cellular substrates and are typically processed and activated by upstream caspases
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Important role of downstream caspases in the execution of apoptosis was demonstrated by
caspase-3 and caspase-7 double-mutant mice which exhibit strong apoptotic deficiency, including
immediate death after birth, exencephaly, resistance of double-mutant mouse embryonic
fibroblasts (MEFs) to both mitochondrial and death receptor-mediated apoptosis
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Ced-9 mutant worm can be given Bcl-2, the first human homolog, first discovered in humans to be disrupted in Bcell leukemia
In this leukemia, there is a translocation
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This causes overexpression of Bcl-2 in B-cells therefore causing inhibition of apoptosis is therefore an oncogene
(stops cells from dying)
There are subfamilies of Bcl-2 proteins that have opposing apoptotic activities
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o Pro-apoptotic proteins BAX, BAK promote apoptosis has loss of a BH4 domain
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Some have TM
domains and some do not
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o Thus, different combinations, sizes of proteins exist but all of the same class due to Bcl-2 homology domain
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o Anti-apoptotic family members such as BCL-2 and BCL-XL inhibit BAX and BAK
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o Anti-apoptotic bind to pro-apoptotic and BH3-only to prevent their oligomerisation
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Activation of the zymogens by proteolytic cleavages separates the large and small subunits and removes the
prodomain
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On the basis of their known major functions, caspases are grouped into two subfamilies, pro-apoptotic and proinflammatory subfamilies
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Pro-inflammatory caspases (caspase-1-4,-5,-11,-12) regulate cytokine maturation during inflammation
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On the basis of this classification, caspases are divided into initiator caspases (caspase-2,-4,-5,-8,-9,-10,-11,-12)
and effector caspases (caspase-3,-6,-7)
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Intrinsic Pathways
 Intracellular problems - DNA damage, chemicals, radiation
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2
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 Contacts between cells: Ligand: Receptor Interactions
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1
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1
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 This pathway leads to the activation of caspase-9
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 Cytochrome C
 Normally found within mitochondria in the intermembrane space
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 Pro-apoptosis is not favoured and at a certain point, the pro-apoptotic proteins begin to
dimerise with each other
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 These monomers then undergo conformational change that induces oligomerisation
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 Instead, this is believed to cause a load of Ca2+ release from the mitochondria which causes the
opening of the permeability transition pore complex (PTPC) which opens in response to the
Ca2+ leak
 Once open, the pore is big enough for Cytochrome C to come out into the cytoplasm along with
other things that are normally sequestered in the mitochondria which is the first signal for
things going wrong
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 Formation of the Apoptosome
 Apoptosome is a multimeric protein complex involving Apaf-1, cytochrome c and the
cofactor dATP/ATP
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 CARD is responsible for the interaction with the prodomain (CARD) of caspase-9 which is
essential to the recruitment and activation of caspase-9
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 Caspase-9 which is activated through an apoptosome-induced conformational change
further processes the downstream caspases, such as caspase-3 and caspase-7 to execute
apoptosis
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 In response to a trigger like DNA damage, Cytochrome C comes out of the mitochondria
so,
 WD-40 prefers Cytochrome C to itself when begins to open up the molecule

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When this happens ATP hydrolysis occurs which,
Causes another conformational change allowing the CARD domains at the N-terminus to
interact with each other which are also locked up by auto-inhibition
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In the absence of apoptotic signal, Apaf-1 is present in monomeric form
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Cytochrome C-Apaf-1-dependent activation of caspase-9 is absolutely required for
neuronal and fibroblast cell-death processes
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Going from a monomeric locked up form to a multimeric joined up form that is activated
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NLRs include NODs and three subfamilies of proteins involved in the formation of caspase1 activating inflammasome complexes, namely NALPs and NAIPs
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NALP1 can be regulated by BCL-2 and BCL-XL in a manner that is reminiscent of caspase
activation in C
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Procaspase-9 (Initiator caspase, when inactive - a caspase is in pro-version)
 Procaspase-9 monomers are inactive, dimers are active
 Procaspase-9 has another CARD domain recruits itself onto Apaf-1
 The CARDs on procaspase-9 have opposite charges
 Procaspase-9 is activated by induced proximity and induced conformational change of
apoptosome
 How does this work?

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Prodomain with two parts of caspase-9

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When caspases are cleaved, there is a small and a large subunit which dimerise and form an
active caspase
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Procaspase-9 cleaves itself to form a dimer which then goes on to cleave procaspase-3
The fact that caspase-9 exhibits a much higher level of catalytic activity in the apoptosome
suggests that a conformational change may occur in the active site of the apoptosome-bound
caspase-9
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Once procaspase-3 is activated, it is the key effector of the caspase
Caspase-3 cleaves other caspases that amplifies the cascade further and these go on to cleave
many intracellular components
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Extrinsic Pathway (Death receptor-mediated pathway)
 Stimulus occurs from outside the cell
 Requires formation of a complex containing:
 Ligand, receptor, adaptor protein, procaspase-8 or -10
 Receptors: Death Receptors
 Death receptors are a family of Type I transmembrane proteins characterized by the presence of
multiple cysteine-rich repeats in the extracellular domain and the protein-protein interaction
module known as the death domain (DD) in the cytoplasmic tails
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 Main ones are: CD95/Fas - activated by CD95L/Fas/L ligand
 TRAIL-R1 - activated by TRAIL (TNF-related apoptosis inducing ligand)
 TNF-R1 (Tumour Necrosis Factor Receptor) activated by TNF-alpha
 DR6, Ectodysplasin A receptor (EDAR) and nerve growth factor receptor
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 DISC formation results in the activation of caspase-8, which plays the central role in transduction
of the apoptotic signal
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 Triggering members of the DR family by death ligands results in the transduction of either
apoptotic or survival signals
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Interactions between the molecules at the DISC are based on homotypic contacts
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This is then released into the cytosol to propagate the apoptotic signal
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FLIPL and FLIPS inhibit activation of procaspase-8 at the DISC by blocking its processing
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Two types of CD95 signaling have been established
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Activated caspase-8 directly leads to the activation of downstream effector caspases
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In this case, signalling requires an additional amplification loop that involves the cleavage
by caspase-8 of the Bcl-2 family protein Bid to generated truncated Bid and subsequent
tBid-mediated release of cytochrome C from the mitochondria
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In these situations, loss of BID or overexpression of BCL-XL inhibits cell death
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Controlling signalling via the CD95/Fas receptor
 There are several levels of modulation of DR signaling
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If there is some ligand floating around, it can bind to
a DcR if present
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There are other DcR that do
not float around and are on the membrane so lack internal death domains and
cannot transduce signals (DcR1 and DcR2 for TRAIL)

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Intracellular proteins that can control this such as cFLIP (cellular FLICE-like inhibitory
protein) competes with procaspases for binding to the DISC as it has a death domain
but does not have an enzyme part to it
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IAPs (inhibitors of apoptosis) inhibit effector caspase activation
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Cellular Inhibitor of Apoptosis Proteins (IAPs)
 IAP gene family regulates the cell’s decision to live or die in response to daily stresses and
insults
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 Human IAP family has rapidly expanded to include 7 others members: XIAP, cIAP1, cIAP2
etc
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 Some of them have the RING domain shown to possess E3 ubiquitin ligase activity directly
regulating auto- or trans-ubiquitination and protein degradation
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 Presence of a CARD domain is unique to cIAP1 and cIAP2 which function in proteinprotein interactions
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 Survivin, the smallest IAP, contains a single BIR domain possesses a coiled-coil domain
required for its interactions with chromosomal passenger proteins and for maintenance of
residency in the nucleus and does not inhibit caspase activity in vitro
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 Second level of regulation involves specific inhibition of active caspases by naturally
occurring cellular inhibitors
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 XIAP suggested to inhibit the extrinsic apoptotic signaling by blocking the
activity of downstream effector caspases as opposed to interfering directly
with caspase-8 activation
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 BIR2 of XIAP binds to the active site of caspases-3 or -7 but not to caspase-1, 6, -8 or -10 so can inhibit their activity even after they have been switched on
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 Three residues have been revealed by mutational analysis namely,
Trp310, Glu314, His343 which are indispensable to XIAP-mediated
inhibition of caspase-9
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 This interaction is essential to XIAP-mediated inhibition of caspase-

9
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 So, binding by XIAP is necessary but not sufficient for the inhibition
of caspase-9
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 Also changes the shape of the procaspase-9 active site to an inactive form
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On receiving an apoptotic signal through the intrinsic mitochondrial stress
pathway, both Smac and cytochrome c are released with similar, but not
necessarily identical kinetics
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Other
studies however, suggest that Smac and cytochrome c release is mediated
through different mechanisms
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During apoptosis, Direct IAp binding protein with LOw pl is released from
mitochondria after outer membrane permeabilization into the cytoplasm
where it uses a similar IAP-binding tetrapeptide motif to bind the BIR3 domain
of XIAP thereby, competitively removing caspase-9
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Although SMAC/DIABLO tetrapeptide in isolation can remove XIAP-mediated
caspase-9 inhibition, it is not capable of removing IAP-mediated inhibition of
effector caspases like caspase-3,-7
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Fragment responsible for inhibiting caspase-3,-7 is located between the BIR1
and BIR2 domain of XIAP rather than the surface groove of BIR2/BIR3 domain
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Regulatory mechanisms for caspases
 Phosphorylation
 Protein phosphorylation is the most widely used cellular regulatory mechanism
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 In response to growth factor stimulation, ERK/MAPK have been shown to
phosphorylate caspase-9 on Ser196 and Thr125 respectively
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 Example: Cell survival mediated by the oncogene Akt, is dependent on glucose
metabolism to inhibit Bax activation and cytochrome C release
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Therapeutic possibilities
 One common chemotherapy drug - Cisplatin
 Platinum from this molecule binds to the DNA forming irreversible crosslinks
between bases
 Lots of side-effects such as kidney, nerve damage, hearing loss, nausea, vomiting,
electrolyte imbalance
 Alternative strategy 1 - Inhibition of anti-apoptotic Bcl-2 family members
 One good example of these agents is the drug oblimersen sodium, which is a Bcl-2
antisense oblimer
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 Using small molecule inhibitors such as first generation Gossypol from cotton plants
which would inhibit Bcl-2 but also leads to hypokalemia (low K+ levels) but second
generation (Apogossypol) seems to be better
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Shown to exhibit cytotoxicity in lymphoma, small cell lung carcinoma cell line and
primary patient-derive cells and caused regression of established tumours in animal
models with a high percentage of cure
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This wasn’t perfect, has
side-effects
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Navitoclax was refined to make ABT-199 (Venetoclax)
Venetoclax has been given a breakthrough therapy status due to efficacy in cancers
such as Chronic Lymphocytic Leukemia (CLL)
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 Example: use of Bcl-2 specific siRNA been shown to specifically inhibit the expression
of target gene in vitro and in vivo with anti-proliferative and pro-apoptotic effects
observed in pancreatic carcinoma cells
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 Small molecules like Phikan083 and CP-31398 have been found to intercalate with
DNA and alter and destabilize the DNA p-53 core domain complex, resulting in the
restoration of unstable p53 mutants
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Alternative strategy 5 - Targeting the IAPs for cancer therapy
 In vitro experiments demonstrate that upregulation of IAP expression increases
resistance to chemotherapeutic and radiation resistance
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 Antisense oligonucleotides wherein, ssODN hybridization to the mRNA Watson-Crick
base pairing, prevents the target gene from being translated into protein, thereby
blocking the action of the gene, and resulting in the degradation of the target mRNA
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SMCs were
intended to de-repress XIAP-mediated inhibition of apoptosis in cancer cells
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 Survivin small molecule antagonists which are under development, YM155 is
undergoing clinical trials of suppressing survivin gene expression
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Alternative strategy 6- Targeting caspases
 Apoptin is a caspase-inducing agent designed synthetically to activate caspases
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 They are pro-apoptotic and have the ability to induce auto-activation of procaspase 3
directly
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Alternative strategy 7 - Activate death receptors
 Using agonistic antibodies against a receptor can cause oligomerization and
activation
 First tried with CD95/Fas using local administration of the anti-Fas antibody to
arthritic rats which showed improvements but other studies of whole body
administration showed severe hepatic injury
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o Pattern of fragmentation is the result of endonuclease-mediated chromatin cleavage at internucleosomal
sites, and is typically accompanied by condensation of the chromatin
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o Evidence of DNA fragmentation being important for avoiding unwanted immune activation has come from
studies of mice that are deficient in nucleases involved in DNA fragmentation within the dying cell (caspaseactivated DNase (CAD))
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o This suggests that apoptosis-associated DNA degradation helps to prevent the accumulation of DNA that if
released into the extracellular space, could provoke autoimmune responses
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o Smaller fragments move faster
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o Nothing seen in the first couple hours, then lower molecular weight bands seen in a characteristic ordered
process
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o What is causing DNA fragmentation?
 DNA fragmentation factors (DFF) have been found, major one being CAD (caspase activated DNAse)
which is a nuclease that cleaves DNA
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 During apoptosis, ICAD is cleaved by caspases, which results in the liberation of CAD and
fragmentation of chromatin
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This is sequestered as an inactive monomer by a chaperone/inhibitor (ICAD)
Both are made and bind to each other preventing inhibitor activity
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ICAD loses its ability to inhibit, allows CAD to dimerise and become an active dimer of DNA
ICAD loses its function, CAD gains its function
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If there is a protein that binds DNA it protects the DNA from cleavage
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Histones are positively charged and bind DNA quite strongly
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Only those bits of the DNA that are not wrapped by histone are the bits in between
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Some of the DNA is still intact as it is still wrapped around by protein
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 Apoptotic trigger then given such as a drug or ligand
 Lyse cells
 DNA starts to be cleaved at this point
 Then apply an electric current, if the cell does not go through apoptosis, DNA is still intact
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 The comets formed can be measured as a % of DNA in head vs
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TdT-mediated dUTP nick end labelling (TUNEL) assay
 Cleavage of phosphodiester backbone by CAD leaves hydroxyl groups at 3' end
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Cytoskeleton Alterations
o On activation, caspases cleave many of the major constituents of the cell cytoskeleton
o These substrates include components of actin microfilaments such as actin itself, and actin-associated
proteins including myosin, filamin, Alpha-actinin and intermediate filaments (vimentin, keratin, laminus)
o Proteolysis of these cytoskeleton constituents probably contributes to the rounding and retraction of the
cell that is seen in the early stages of apoptosis; thus, a nice spread out cell is now weakened
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Pharmacological inhibitors of caspases efficiently block apoptosis-associated plasma-membrane blebbing
Caspase substrate strongly implicated in this phase of apoptosis is the Rho effector ROCK1 a regulator of
actin cytoskeleton dynamics
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Extensive membrane blebbing that is seen during apoptosis might result from myosin-dependent
contraction of cortical bundles of actin, pushing the cytosol against other areas of the cell cortex and causing
blebs in areas where the cytoskeleton has been weakened
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In the early stages of apoptosis, dying epithelial cells stimulate their neighbours to form a network of actinmyosin cables and these effectively extrude the apoptotic cell from the epithelium as the cables contract
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Other nuclear events
o In order to prevent cells from wasting energy to repair themselves which are already marked out to die,
need to stop enzymes that try to repair this
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o Caspase-mediated cleavage of nuclear lamins (caspase-6) weakens the nuclear lamina, allowing nuclear
fragmentation and nuclear envelope proteins are proteolysed
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o Acinus (apoptotic chromatin condensation inducer in the nucleus) also cleaved resulting in condensation of
DNA
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o Ribosomal proteins and some ribosomal RNA are also degraded
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Inhibition of translation correlates with apoptosis induction
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Add either of the apoptosis inducers, some limited translation still occurs
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All of these come together to form the eIF4G complex
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During apoptosis, this gets cleaved
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The very first tRNA for messengers is Methionine tRNA which has to be bound by a factor and used in
initiation
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To do another translation round, it needs to be recycled which uses a GEF called eIF2
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Cleavage of the dsRNA protein kinase (PKR)
During apoptosis, PKR's inhibitory domain at N-terminus gets cleaved off
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However, eIF2Alpha also gets cleaved
Even though this is phosphorylated, it can cleave itself and can bypass eIF2B
eIF2 can still recycle through the system
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o PS becomes externalized to the outer surface by enzymes known as Flippases, Scramblases, Floppases,
Translocases to initiate cell death
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 Fluorescent tags like FITC (green fluorophore binding to the outside of the cell) or PE (red protein) can
be added to Annexin V
 How do we know chemotherapy is working when treating a patient?
 When a patient has an organ transplant, how do we know that the organ is not being attacked by the
internal systems?
 Annexin V can bind to apoptotic cells
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 This allows cells to be eliminated with their plasma membranes intact and precludes the potentially
damaging release of cellular constituents into the surrounding milieu
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i
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“eat-me” signals
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 Exposure of PS by apoptotic cells is of particular interest because it induces membrane ruffling and
efferocytosis and it has been implicated in many of the immunomodulatory effects associated with
efferocytosis
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 During apoptosis, Calreticulin increases on the cell surface and redistribution into PS-rich patches
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 Tethering and Tickling
 Some efferocytosis receptors appear to perform primarily a tethering function by holding the
apoptotic cell and phagocyte close together while other receptors are primarily involved with
transducing the engulfment signal
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 Example: milk-fat-globule-factor (MFG)-E8 and growth-arrest-specific-factor 6 (Gas6) are soluble
bridging molecules that directly link PS on the apoptotic cell surface with their corresponding
phagocyte receptors
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Signalling activates remodelling of engulfing cell via Rho-GTPases, meaning ROCK1 signalling is
probably involved

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