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Cancer > 10. Apoptosis > Flashcards

10. Apoptosis Flashcards

1
Q

What is necrosis?

A
  • Unregulated cell death
  • Associated with trauma, cellular disruption and an inflammatory response
  • Cells burst or membrane permeability increases => loss of contents to environment
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2
Q

What is apoptosis?

A
  • Regulated, controlled disassembly of cellular contents
  • Without disruption - no inflammatory response
  • Plasma membrane remains intact
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3
Q

What happens during necrosis?

A
  • Trauma (mechanical, bacterial etc.)
  • Plasma membrane becomes permeable
  • Cell swelling and rupture
  • Release of proteases leading to auto-digestion and dissolution of the cell
  • Localised inflammation due to attraction of immune (phagocytic) cells
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4
Q

What are the 2 phases of apoptosis and what happens in them?

A
  • Latent phase - death pathways are activated, but cells appear morphologically the same
  • Execution phase - orderly activation of specific proteins and kinases
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5
Q

Outline what happens during apoptosis

A
  • Loss of microvilli and intracellular junctions
  • Dramatic cell shrinkage
  • Loss of plasma asymmetry
  • Chromatin and nuclear condensation
  • DNA fragmentation
  • Formation of membrane blebs
  • Fragmentation into membrane-enclosed apoptotic bodies
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6
Q

What does DNA modification in apoptosis involve and how can this be seen in the lab?

A
  • Fragmentation of DNA ladders - seen on agarose gel

* Formation of more ‘ends’, labelled by adding an extra fluorescently-tagged base in a tunel assay (brighter)

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7
Q

What does apoptosis-like PCD involve?

A
  • Some, but not all, features of apoptosis
  • Display of phagocytic recognition molecules, even before plasma membrane lysis
  • Some inflammatory response
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8
Q

What does necrosis-like PCD involve?

A
  • Variable features of apoptosis before cell lysis

* “Aborted apoptosis”

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9
Q

What are the 4 parts in the mechanism of apoptosis?

A
  • Caspases (“executioners”)
  • Initiating death programme - death receptors (extrinsic) + mitochondria (intrinsic)
  • Bcl-2 family - regulators
  • Stopping death programme
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10
Q

What do caspases have in their active site and what are they activated by?

A
  • Cysteine residue in active site (required for activity)

* Activated by proteolysis - cut proteins just after their aspartate residue

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11
Q

How can caspases be divided into different classes - describe them?

A

Initiator and effector

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12
Q

Describe initiator caspases

A

• Initators are the first to be triggered
• Contain specific motifs:
- CARD - caspase recruitment domain
- DED - death effector domain
• Located at the end of their respective caspases to form homotypic protein-protein interactions (DED with DED)

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13
Q

Describe effector caspases

A
  • Don’t contain specific motifs

* Just proteases

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14
Q

Describe the activation of caspases

A
  • Synthesised as inactive pro-caspases (zymogens) - single chain polypeptides
  • Have a pro-domain to maintain the inactivated stage
  • Proteolysis - cleavage of the pro-domain => formation of the heterodimer
  • Folding of 2 large (L) and 2 small (S) chains to form active L2S2 heterotetramer
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15
Q

What are main purposes of the caspase cascades?

A
  • Amplification
  • Divergent responses
  • Regulation
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16
Q

Once apoptosis is triggered, what do the initiator caspases do to the effector caspases?

A

Initiator caspases cleave and activate the effector caspases, which then carry out the apoptotic programme

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17
Q

What are the 4 different consequences that can occur in the apoptotic programme (from the clipping of different proteins)?

A

Loss of function
• Inactivation - monomeric substrate cleaved by caspase
• Disassembly - multimolecular complex, cleaved at many different points e.g. nuclear lamins

Gain of function
• Activation - direct cleavage resulting in free active component, as it will unfold and change conformation
• Release - caspase cleaves a protein that is holding another protein making it inactive (inhibitory molecule) - results in break down of DNA

18
Q

What do the following caspase mechanisms refer to:
• Death by design
• Death by default

A
  • Design - receptor-mediated (extrinsic) pathways. Particular stimulus outside the cell that tells it to kill itself.
  • Default - mitochondrial (intrinsic) pathway. Cell detects something off inside.
19
Q

What do death receptors on all cells consist of?

A
  • Extracellular cysteine-rich domain
  • Single transcellular domain
  • Cytoplasmic tail (with a death domain)
20
Q

When are death receptors activated?

A

• When they encounter secreted or transmembrane trimeric ligands e.g. TNF-alpha or Fas, (death ligands)

21
Q

What are the adaptor proteins in receptor-mediated apoptosis and what do they do?

A
  • FADD - positive regulator, required for death pathway to be activated and promotes cell death
  • FLIP - negative regulator, inhibits the death pathway and allows it to be regulated
22
Q

What domains do FADD and FLIP have?

A 
DED = death effector domain
DD = death domain
  • FADD = DED + DD
  • FLIP = DED + DED
23
Q

What happens to death receptors when activated (in cytotoxic T lymphocytes) and how does it lead to the execution of the death programme?

A

1) Fas ligand binds to Fas receptor on the cytotoxic T lymphocytes
2) Fas receptor trimerises
3) Receptor brings 3 cytoplasmic DD domains of the positive adapter protein, FADD, together
5) Recruitment of oligomerisation of pro-caspase 8 through its DED, to the FADD DED
6) This binding forms a death-inducing signalling complex (DISC)
7) Disc formation results in the cross-activation of pro-caspase 8 - (at least 3) pro-caspase 8s come into close contact and cleave each other
8) This releases the active initiator caspase 8 tetramer
9) Cleaves effector caspases to execute the death programme

24
Q

What happens once caspase 8 is activated and has carried out its action?

A
  • FLIP adaptor proteins inhibit the caspase system (contain DED domains on their N-terminus)
  • No proteolytic activity
  • Competes with pro-caspase 8 to bind to the DED domains of FADD
  • Prevents trans-cleavage, blocking the formation of active caspase 8
25
Q

When caspase 8 is activated, which effector caspases are activated downstream?

A

Caspase 3 and 7 - these carry out the apoptotic programme

26
Q

Outline the activation of the intrinsic pathway to the formation of the apoptosome?

A
  • Cellular stresses
  • e.g. overstimulation by GFs, DNA damage, reactive oxygen species
  • Causes a loss of mitochondrial membrane potential
  • Release of cytochrome C and other apoptosis-inducing factors
  • Stimulates the formation of an apoptosome complex - equivalent of DISC on death receptors
27
Q

What does the apoptosome consist of and what does it do?

A

• APAF-1, cytochrome C, ATP, pro-caspase 9
• APAF-1 = ATPase
- WD-40 repeats at the C-terminus involved in protein-protein interactions
- caspase recruitment domain (CARD) at the other end
• When cytochrome C binds to the WD-40 repeats, it forms a heptamer
- i.e. monomer associates with 6 other units to make a wheel of 7, bound via the CARD domain
• Process requires ATP

  • The CARD domains of the apoptosome can interact with the CARD domains on pro-caspase 9 (oligomerisation)
  • Close proximity of pro-caspase 9s (that bind to the apoptosome) can cross-cleave and activate each other
  • Pro-caspase 3 comes along, dimerises with caspase 9 and is activated by it
  • Caspase 3 splits into 2 subunits (large and small) - and 2 of each join to produce an active caspase 3 (tetramer)
28
Q

Can ATP levels make a difference between death via necrosis or apoptosis?

A

Yes, as apoptosis is an active process and necrosis doesn’t require energy

29
Q

What does ‘Bid’ do?

A
  • Links the receptor-mediated (extrinsic) and mitochondrial death (intrinsic) pathways
  • Caspase 8 from the receptor-mediate pathway can cleave Bid
  • This enhances the release of mitochondrial proteins
30
Q

What is the difference between the receptor-mediated and mitochondrial death pathway?

A

The mitochondrial pathway requires ATP

31
Q

What family of proteins does Bid belong to and what is it characterised by?

A

Bcl-2 family of apoptosis regulators (Group 3)

32
Q

Describe the Bcl-2 family

A
  • Characterised by domains in their protein sequence = Bcl-2 homology (BH) domains (1-4)
  • Some of them have a transmembrane domain
  • Bcl-2 is the found member - group 1
  • Group 3 only contains BH3
  • BH3 (Bcl-2 homology domain 3) is a dimerisation motif
  • Pro-apoptotic - Bax, Bid, Bad, Bak (move between cytosol and mitochondria)
  • Anti-apoptotic - Bcl-2, Bcl-xL (localised to mitochondrial membrane)
33
Q

Describe how growth factors create anti-apoptotic effects?

A
  • Ligand binding causes dimerisation and cross-phosphorylation of tyrosine kinase receptors
  • This initiates signal transduction pathways
  • Creates docks for adaptor proteins, which can mediated interactions e.g. activating Ras, activation of MAPK/ERK => growth

• Another phosphorylation site on the TK receptors triggers the PI3-kinase pathway
- site on cytoplasmic tail attracts and adaptor p85 (which is with p110, forming PI3 kinase)
• This (p110 part) phosphorylates a lipid (so must be close to the membrane)
• PIP2 => PIP3 phosphorylation [by PI3-K]
• Recognised by adapter subunit pf PKB/Akt (protein kinase B)
• PKB is recruited to the cell membrane and activated
• Results in mitogenic and anti-apoptotic signals

34
Q

What are the 3 main subunits of PI3-K?

A
  • Targeting subunit
  • Adapter subunit
  • Catalytic subunit
35
Q

Why is the activation of PKB/Akt anti-apoptotic?

A
  • Phosphorylates and inactivates Bad
  • Phosphorylates and inactivates caspase 9
  • Inactivates FOXO transcription factors (FOXOs promote expression of apoptosis-promoting genes)
  • Other e.g. stimulates ribosome production and beneficial protein synthesis
36
Q

How do the pro-apoptotic Bcl-2 family proteins play a part in the apoptosis mechanism?

A
  • When GFs are absent, the PI3-kinase pathway is not activated
  • PKB is not activated, so Bad is not phosphorylated/held in an inactive heterodimer with PKB
  • Bad is free to go to the mitochondrial membrane and binds through its BH3 domain to the BH3 domains of the anti-apoptotic Bcl-2 family
  • This displaces the pro-apoptotic members from inhibition by the anti-apoptotic members
  • The pro-apoptotic members can form a pore in the mitochondrial membrane, allowing cytochrome C to escape into the cytosol
  • Apoptosis is induced
37
Q

Where do Bcl-2 and bax dimerise?

A

They are transmembrane domains that dimerise on the mitochondrial surface

38
Q

What Bcl-2 members are phosphorylated (inactive) in the cytosol

A

Bad and Bax

39
Q

What does PTEN (lipid phosphatase) do?

A
  • Counteracts the production of PKB

* This reduces regulation of cell survival, therefore promotes apoptosis

40
Q

What do IAPs do?

A
  • Bind to pro-caspases and prevent activation
  • Also bind to active caspases and inhibit activity
  • This regulates programmed cell death
41
Q

Which proteins regulate the extrinsic, and intrinsic pathway?

A
  • Extrinsic - FLIP, IAPs

* Intrinsic - Bcl-2, Bcl-xL

42
Q

Which proto-oncogenes/TSGs are associated with apoptosis?

A
  • Bcl-2 (oncogene - over-expression promotes cancer)
  • PKB/Akt (oncogene - over-expression promotes cancer)
  • PTEN (tumour suppressor - inactivation promotes cancer)

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