``` Unregulated cell death associated with • trauma • cellular disruption & • INFLAMMATORY response ```

Apoptosis Flashcards by Rama Jha

Why does programmed cell death occur? •

Harmful cells
• e.g. DNA damaged.

Developmentally defective cells
• e.g. self-antigen B-cells.

Excess cells
• e.g. sculpting of hands during embryonic development (get rid of webbing).

Obsolete cells
• e.g. mammary epithelium at the end of lactation.

Exploitation
• e.g. chemotherapeutic killing of cells.

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Necrosis?

Unregulated cell death associated with
 • trauma
 • cellular disruption
&amp;
 • INFLAMMATORY response

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Apoptosis?

Programmed (regulated) cell death
• controlled disassembly of cellular contents without disruption
&
• NO inflammatory response

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Steps associated with necrosis?

Plasma membrane becomes permeable.
Cell swelling and rupture.
Release of proteases leading to auto-digestion.
Localised inflammation.

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Steps associated with apoptosis?

LATENT PHASE
• death pathways are activated but cell stays morphologically the same

EXECUTION PHASE
1. Loss of microvilli and inter-cellular junctions.
2. Cell shrinkage.
3. Loss of plasma membrane asymmetry.
4. Chromatin and nuclear condensation.
5. DNA fragmentation.
6. Membrane bleb formations.
7. Fragmentation into membrane-enclosed apoptotic bodies.
• So, no inflammation as the plasma membrane remains intact

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DNA modification can also occur during apoptosis - what can this lead to?

Fragmentation of DNA ladders
• seen in agarose gel

AND

Formation of more “ends”
• labelled with an extra fluorescently-tagged base

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Other than necrosis and apoptosis, what are other types of cell death and mechanism?

Apoptosis-like PCD (Programmed Cell Death)
• some, but not all, features of apoptosis
• display of phagocytic recognition molecules BEFORE plasma membrane lyses

Necrosis-like PCD
• variable features of apoptosis BEFORE cell lyses; “Aborted apoptosis”

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The fact that other forms of cell death also exist apart from necrosis and apoptosis, what does this suggest?

The fact that these other forms exist suggests a GRADED response of cell death.

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4 broad mechanisms of apoptotic cell death?

Caspase cascade – the executioners
Death response – death receptors and mitochondria
Bcl-2 family
Stopping the death programme

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What are caspases and their MoA?

Caspases = Cysteine-dependent ASPartate-directed proteASES

a cysteine residue in the active site is required for their activity
they cut proteins after their aspartate residue
activated by a proteolysis cascade

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There are 2 major classes of caspases - what are they and explain it?

Initiator caspases 
 • 2, 9, 8, 10 (TRIGGER APOPTOSIS)
 • 2 subunits – p20 and P10.
 • They have an extra targeting subunit:
  - CARD – Caspase Recruitment Domain
  - DED – Death Effector Domain
  (these direct them to a location)

Effector caspases
• 3, 6, 7 (CARRY OUT APOPTOSIS PROCESS)

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How do caspases mature?

Procaspases are single-chain polypeptides
• to activate, procaspases undergo proteolytic cleavage to form LARGE and SMALL subunits.
• Initiator caspases also cleave the targeting subunits (DED and CARD)

After cleavage, there is a folding of the 2 large and 2 small chains
• to form an active L2S2 HETEROTETRAMER

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Explain the caspases cascade (aka. the main purpose)

Main purpose of the caspase cascade is for:
• amplification
• divergent responses
• regulation

Once apoptosis is triggered, the initiator caspases cleave and activate the effector caspases

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What do the effector capcases do?

Execute the APOPTOTIC PROGRAMME

In 2 ways:

Cleave & INACTIVE proteins/complexes
• e.g. nuclear lamins lead to nuclear breakdown
ACTIVATING enzymes by direct cleavage or cleavage of inhibitors
• e.g. nucleases (CAD)

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What are the 2 broad mechanisms for caspase activation?

Death by design
• Receptor-mediated (extrinsic) pathways

Death by default
• Mitochondrial (intrinsic) death pathway

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What are death receptors?

(extrinsic pathway)

ALL CELLS have death receptors which consist of:
• EC cysteine domains
• Transmembrane domain
• Cytoplasmic tail (death domain)

What can interact with death receptors?

ADAPTOR PROTEINS can interact with these receptors

• FADD (ACTIVATION)

+VE regulator
promotes cell death
DED + DD

• FLIP (INHIBITION)

-VE regulator
inhibits death pathway (allowing for regulation)
DED + DED

Explain and describe the signalling through the death receptors

Fas receptor is upregulated when the cell needs to apoptose
Fas ligand on cytotoxic T-cells binds to the Fas receptor and Fas receptor trimerises
Trimerised DD domains recruit adaptor proteins such as FADD
FADD binding causes recruitment and oligomerisation (links monomers to form dimers/trimers/etc.) of procaspase 8 through DED –> FADD DED
Procaspase 8 + FADD –> DISC (Death-inducing-signalling-complex)
DISC cross-activates other procaspase 8 molecules
Active caspase 8 is released to cleave effector caspases

Explain the oligomerisation of procaspase 8

Occurs to allow procaspase 8 for cleavage and activation (as some initiator procaspases have low catalytic activity)

Initiator procaspases bind to FADD (DED–> DED)
• DED regions bind to DED regions
This brings procaspases into close contact to allow cleavage
Active initiator caspase 8 tetramers release.

Explain how FLIP functions in relation to the death receptors.

Death receptor activation of procaspase 8 is inhibited by FLIP (negative regulator)

*• FLIP incorporates into the trimer
BUT
it has NO PROTEOLYTIC ACTIVITY and so cannot cleave the other procaspases (competes with procaspase 8)

• It can still bind to the DED regions on FADD however*

Why is Caspase 8 important?

It is the INITIATOR CASPASE

Can go on to activate the other EFFECTOR caspases that then carry out the apopotic process

(onenote!!)

Explain and describe the intrinsic (mitochondrial) pathway

Cellular stresses
• e.g. lack of GFs, DNA damage (p53)
Loss of mitochondrial membrane potential
Release of cytochrome C
• and OTHER apoptosis-inducing factors
Stimulation of formation of “apoptosome complex”

What does the Apoptosome (“wheel of death”) consist of?

APAF-1
• Apoptotic Factor 1

Cytochrome C

ATP

Procaspase 9

What do the different parts of the Apoptosome do and how do they relate together?

APAF1 is composed of:
 • CARD
 • ATPase 
and 
 • WD-40 repeats

When cytochrome C binds to WD-40 repeats on APAF-1, it forms a heptamer (the apoptosome) which requires ATP

The CARD domain (also on APAF-1) then binds to CARD on procaspase 9
• so multiple procaspase 9s (x7) can bind to one apoptosome
• proximity of procaspases then allows cross-cleaving

Activated caspase 9 is then released to trigger apoptosis.

What does apoptosis require, determining if this pathway is chosen?

ENERGY - APT Energy levels in the cell may therefore determine whether death is by • necrosis (no ATP) • apoptosis (ATP)

Following on from both the extrinsic & intrinsic pathway, explain the principal mechanisms of apoptosis

Bid links the extrinsic (receptor) and intrinsic (mitochondrial) pathways together • Caspase 8 from the extrinsic pathway can cleave Bid --> enhances activation of the intrinsic pathway • Bid releases Cytochrome C from the mitochondrion which triggers the death pathway Major difference – intrinsic mitochondrial pathway requires energy (ATP) • Apoptosis always uses intrinsic to some degree so will always use ATP • Remember that necrosis uses MUCH LESS ATP.

What are the intrinsic modulators of apoptosis?

Bcl-2 family proteins 3 main groups • ALL contain BH3 domains (required for dimerisation betw. one another) • SOME contain TM domains

What are the 2 main categories of Bcl-2 proteins?

Anti-apoptotic • Bcl-2 • Bcl-xL • Localised in mitochondrial membrane ``` Pro-apoptotic • Bid • Bad • Bax • Bak • Move between cytosol & mitochondrial membrane ```

What are the 2 GF pathways associated with anti-apoptotic effects

1. Ligand binds --> dimerisation --> cross-phosphorylation --> signal transduction and docking of adapter proteins (e.g. Grb2) to adapt pathway direction (e.g. activating Ras --> MAPK/ERK cascade) 2. Another phosphorylation site on tyrosine kinase receptors triggers the PI3-kinase pathway --> cell survival and anti-apoptotic effects.

What does the PI3-kinase pathway lead to?

Cell survival HENCE Anti-apoptotic effects

Explain the PI3-kinase pathway

PI3-K = Phosphatidylinositol 3-kinase = a lipid (not protein) kinase Three main subunits: • Targeting subunit • Adapter subunit • Catalytic subunit PI3-K phosphorylates PIP2 --> PIP3 • which then binds PKB/Akt (protein kinase B) adapter subunit • PKB is then recruited to the cell membrane and it is activated - it has anti-apoptotic effects

Explain how PKB has anti-apoptotic effects

PKB has anti-apoptotic effects by: * Phosphorylating and inactivating Bad (a Bcl-2) * Phosphorylating and inactivating caspase 9 * Inactivating FOXO (promote expression of apoptosis-promoting genes) transcription factors. * Other – stimulates ribosome production.

What are 2 models of regulation of apoptosis by Bcl-2 family proteins via. BH3 heterodimerisation

A - PKB/Akt (PI3-K, GF pathways) B - GF absent

Explain the A model of apoptosis of Bcl-2 family proteins via. BH3 heterodimerisation

Pro-apoptotic proteins (e.g. Bax, Bak) are held INACTIVE in their heterodimers to Bcl-2 and Bcl-xL on the mitochondrial membrane • by the BH3 domain As the pro-apoptotic are held in the inactive heterodimers, cell proliferation and survival is promoted

Explain the B model of apoptosis of Bcl-2 family proteins via. BH3 heterodimerisation

GF is ABSENT!! No PI3-K pathway --> PIP3 not generated --> PKB not recruited --> Bad NOT phosphorylated and so is released from heterodimer with 14-3-3 Bad therefore goes to mitochondrial membrane and binds to Bcl-2 and Bcl-xL • displacing the pro-apoptotic Bcl-2 family proteins Bcl-2 family proteins (Bax, Bak) then form a pore in the mitochondrial membrane to allow cytochrome C to escape to induce apoptosis

What can counteract PI3-K?

PTEN (lipid phosphatase) Counteracts PI3-K signalling and production of PKB SO PROMOTES APOPTOSIS

What are IAPs and what do they do?

Inhibitor of Apoptosis Proteins Regulate programmed cell death by binding to: • Procaspases - preventing activation • Caspases - inhibiting activity ALL extrinsically (as dealing w. caspases)

Summarise the anti-apoptotic pathways

Bcl-2 & Bcl-xL • via the intrinsic pathway FLIP & IAPs • via the extrinsic pathway Growth factor pathways • via PI3-K and PKB/Akt

How can cancer cells avoid apoptosis?

Apoptosis regulators as oncogenes or TSGs Bcl-2 • Oncogene – as overexpression of Bcl-2 results in cancer PKB/Akt • Oncogene – as overexpression of PKB leads to increased cell survival and thus cancer PTEN • TSG – as inactivation raises the PI3-K pathway and thus allows increased survival of cancer.