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Year 2 MCD - Cancer > Apoptosis > Flashcards

Apoptosis Flashcards

1
Q

What cells do we need to remove?

A
  • harmful cells (viral infection/DNA damage)
  • developmentally defective cells
  • excess/unnecessary cells
  • obsolete organs (e.g. mammary epithelium at the end of lactation)
  • exploitation (chemotherapeutic killing of cells)
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2
Q

What is necrosis?

A

Unregulated cell death associated with trauma, cellular disruption and an INFLAMMATORY RESPONSE

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3
Q

What is apoptosis?

A

programmed cell death

Regulated, controlled disassembly of cellular contents without disruption – NO INFLAMMATORY RESPONSE

active process (requires energy)

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4
Q

What happens during necrosis?

A
  • plasma membrane becomes permeable
  • cell swelling and rupture of cellular membranes
  • release of proteases leading to auto-digestion and dissolution of the cell (unregulated action
  • localised inflammation occur due to the attraction of immune cells
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5
Q

What are the phases of apoptosis?

A

Latent phase – death pathways are activated, but cells appear morphologically the same.

Execution phase – an orderly activation of specific proteins and kinases:

  • Loss of microvilli and intercellular junctions
  • Dramatic cell shrinkage
  • Loss of plasma membrane asymmetry
  • (Phosphatidylserine lipid appears in outer leaflet)
  • Chromatin and nuclear condensation
  • DNA fragmentation
  • Formation of membrane blebs
  • Fragmentation into membrane-enclosed apoptotic bodies
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6
Q

Why is the no inflammation in apoptosis?

A

Up to the blebbing, the plasma membrane remains intact.

Because the contents aren’t released, there is no inflammation

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7
Q

How is DNA modifies in apoptosis?

A

Because the nucleus gets condensed and destroyed, whatever is inside the nucleus must be degraded

If you extract DNA from the cell undergoing apoptosis, and run it on agarose gel, there is fragmentation of the DNA.

  • Fragmentation of DNA ladders (in agarose gel)
  • Formation of more ‘ends’, which are labelled by adding an extra fluorescently-tagged base in a TUNEL assay
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8
Q

What is apoptosis-like PCD?

A

some, but not all, features of apoptosis. There may be a display of phagocytic recognition molecules, even before plasma membrane lysis

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9
Q

What is necrosis-like PCD?

A

Variable features of apoptosis before cell lysis – “Aborted apoptosis” (can occur up to a certain point down the process)

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10
Q

Whatare the mechanisms of apoptotic cell death?

A
  1. The executioners – Caspases (key enzymes)
  2. Initiating the death programme: via death receptors (extrinsic) and mitochondria (intrinsic)
  3. The Bcl-2 family
  4. Stopping the death programme
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11
Q

How are caspases activated?

A
  • Executioners of apoptosis that have a cysteine residue in their active site (required for activity)
  • Activated by proteolysis, and cut proteins just after their aspartate residue
  • Cascade of activation – have a recognition motif, which will cleave when interacting with these motifs
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12
Q

What are the classes of caspases?

A

initiators and effectors

  • Initiator caspases are the first to be triggered (2, 9, 10 and 8) - contain specific motifs (e.g. CARD for 2 and 9, DED for 10 and 8)
  • Effector caspases (3, 6 and 7) don’t contain these motifs
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13
Q

Describe the maturation of caspases

A
  • synthesised as pro-caspases (zymogens)
  • have a pro-domain to maintain the inactivated stage
  • Proteolysis results in cleavage of the pro-domain -> formation of the heterodimer.
  • Cleavage of the inactive pro-caspase precursor is followed by folding of 2 large and 2 small chains to form an active L2S2 heterotetramer.
  • These cleavages are done by the caspases themselves
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14
Q

What is the purpose of the caspase cascade?

A

Amplification, Divergent responses, Regulation

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15
Q

Describe the caspase cascade

A

apoptosis is triggered -> the initiator caspases cleave and activate the effector caspases -> allows the commencement of cascades

Initiator caspases trigger apoptosis by cleaving and activating

Once effector caspases are activated, they carry out the apoptotic programme (activate further caspases downstream)

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16
Q

How do effector caspases carry out apoptosis?

A
  • Cleaving and inactivating various proteins and complexes (e.g. nuclear lamins leading nuclear breakdown)
  • Activating enzymes by direct cleavage, or cleavage of inhibitor molecules (e.g. protein kinases, nucleases such as Caspase-activated DNAse (CAD))
17
Q

How are caspases activated?

A
  • Death by design – Receptor-mediated (extrinsic) pathways

- Death by default – Mitochondrial (intrinsic) death pathway

18
Q

What do death receptors consist of?

A
  • Extracellular cysteine-rich domain
  • Single transcellular domain
  • Cytoplasmic tail (with a death domain)
19
Q

What activated death receptors?

A

only activated when they encounter secreted or transmembrane trimeric ligands (e.g. TNF-alpha or Fas) – these are called death ligands

20
Q

What do death receptors do?

A

When activated, receptors activate the caspases

Once activated, they will dimerise/trimerise -> attract adaptor protein

  1. Receptor (Fas) trimerisation by ligand (Fas-L on lymphocyte)
  2. Recruitment of adapter protein (FADD) through its DD to DD of Fas
  3. Recruitment and oligomerisation of pro-caspase 8 through its DED to FADD DED -> DISC (Death-Inducing Signalling Complex)
  4. active caspase 8 is then released, and it cleaves effector caspases to execute the death programme
21
Q

What are the adaptor proteins in receptor-mediated apoptosis?

A

FADD: POSITIVE regulator (required for the death pathway to become activated) and promotes cell death.

FLIP: negative regulator (inhibits the death pathway and allows it to be regulated)

FADD = DED + DD
FLIP = DED + DED
  • DED = Death Effector Domain
  • DD = Death Domain
22
Q

How is initiator caspase?

A

Once recruited, the trimerised receptors FADD contain DED domains. The pro-caspase 8 (initiator) will interact with the DED domain. There will be 3 pro-caspase 8 molecules

They will be cleaved (trans-cleavage) by one another -> the tetramer can be assembled together to form an ACTIVE CASPASE 8. The trimerised receptor enables close proximity of the pro-caspase, so trans-cleavage can take place -> active initiator caspase.

Initiator pro-caspases bind, via their DED domains, to the DED domains of FADD - three initiator pro-caspase 8s come into close contact, which allows cleavage -> releases the active initiator caspase 8 tetramer

23
Q

How does FLIP inhibit activation of caspase 8?

A

FLIP – caspase homology in DED domain, but has no proteolytic activity therefore it competes with pro-caspase

Because of the DED domain of the FLIP inhibitor protein, it will insert itself in-between the pro-caspase, and PREVENT the efficient cleavage -> it blocks the formation of new, active caspase 8

24
Q

What happens in mitochondrial regulation of apoptosis?

A

intrinsic pathway whereby cellular stresses (e.g. lack of/overstimulation by growth factors, DNA damage etc.) cause a loss of mitochondrial membrane potential -> release of cytochrome C and other apopotosis-inducing factors -> stimulate the formation of an apoptosome complex

25
Q

What is the apoptosome complex?

A

The equivalent of the DISC on death receptors

The apoptosome consists of: APAF-1 (apoptotic activating factor 1), Cytochrome C, ATP, Pro-caspase 9

APAF-1 is an ATPase protein – at the C-terminus, it has WD-40 repeats

  • At one end, APAF-1 contains a number of repeats that are involved in protein-protein interactions
  • There is also an ATPase domain within APAF-1
  • At the other end of APAF-1 there’s a caspase recruitment domain (CARD) – caspase recruitment domain

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

26
Q

How does the apoptosome activate caspase 9?

A

The CARD domains at the centre of the apoptosome can interact with the CARD domains on procaspase-9 (so seven pro-caspase 9s can bind to the apoptosome)

The close proximity of the pro-caspase 9s that bind to the CARD domains of the apoptosome can cross-cleave and activate each other to produce caspase

27
Q

What links the receptor-mediated and mitochondrial death pathways?

A

Bid

Caspase 8 from the receptor-mediated pathway can cleave Bid, which enhances release of mitochondrial proteins, thus engaging the intrinsic pathway

Bid promotes the release of cytochrome C from the mitochondrion, which triggers the mitochondrial death pathway

28
Q

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

A

mitochondrial pathway requires energy

29
Q

What are Bcl-2 family proteins?

A
  • intrinsic modulators of apoptosis
  • There are THREE main groups of Bcl-2 proteins, all of which contain BH3 domains
  • Some of the proteins contain other domains including a transmembrane domain
  • BH3 is a dimerisation motif (for protein-protein interaction) that allows proteins in the Bcl-2 family to associate and dimerise with each other
30
Q

What are some pro-apoptotic and anti-apoptotic Bcl-2 family proteins?

A

PRO-APOPTOTIC: Bid, Bad, Bax, Bak (move between cytosol and mitochondria)

ANTI-APOPTOTIC: Bcl-2, Bcl-xL (localised to the mitochondrial membrane)

31
Q

What pathways do GFs activate?

A

*anti-apoptotic effects

  1. Ras -> ERK ->growth
  2. PI3 kinase pathway -> cell survival
32
Q

What happens in the PI3 kinase pathway?

A

GF receptors that dimerise.

There is phosphorylation of a tyrosine site on the cytoplasmic tail -> attracts an adaptor -> recruits PI3 kinase, which contains 2 subunits: p85 and p110.

This doesn’t phosphorylate another protein, but phosphorylates a lipid. So it is important that it is recruited close to the membrane.

PIP2 -> PIP3 phosphorylation takes place (PIP3 kinase is a lipid kinase involved in cell growth survival) -> recognised by the adapter subunit of PKB/Akt (protein kinase B) [an anti-apoptotic enzyme]

PKB is then recruited to the cell membrane and it is activated – it has anti-apoptotic effects

  • PKB phosphorylates and INACTIVATES Bad (part of the Bcl-2 family)mitogenic and anti-apoptotic signals
33
Q

What are the subunits of phosphatylinositol3-kinase?

A
  • lipid kinase involved in growth control and cell survival
  • Targeting subunit
  • Adapter subunit
  • Catalytic subunit
34
Q

How does PKB/Akt induce cell survival?

A
  1. Phosphorylates and inactivates Bad (key regulator of apoptosis)
  2. Phosphorylates and inactivates caspase 9
  3. Inactivates FOXO transcription factors (FOXOs promote expression of apoptosis-promoting genes)
  4. Other, e.g. stimulates ribosome production and protein synthesis
35
Q

How does Bad lead to apoptosis?

A

Bad is dephosphorylated and released from the heterodimer -> can then go to the mitochondrial membrane, where it can bind through its BH3 domain to the BH3 domains of the anti-apoptotic Bcl-2 family members thus DISPLACING the pro-apoptotic Bcl-2 family members (e.g. Bax and Bak) -> form a pore in the mitochondrial membrane, which allows cytochrome C to escape into the cytosol and induce apoptosis.

36
Q

What is PTEN?

A

lipid phosphatase that counteracts the production of PKB, therefore reducing the regulation of cell survival and promoting apoptosis

37
Q

What do IAPs do?

A

Inhibitor of Apoptosis Proteins
bind to pro-caspases and prevent activation.
IAPs also bind to active caspases and inhibit their activity

38
Q

What regulates the anti-apoptotic pathways?

A

Bcl-2, Bcl-xL: regulate the intrinsic pathway

FLIP, IAPs: regulate the extrinsic pathway

Growth factor pathways via PI3’-K and PKB/Akt – these COUNTERACT APOPTOSIS

39
Q

Which proto-oncogens/ tumour suppressor genes are associated with apoptosis?

A
  • Bcl-2 (oncogene because over-expression of Bcl-2 will promote cancer)
  • PKB/Akt (oncogene because over-expression of PKB/Akt will promote cancer)
  • PTEN (tumour suppressor because inactivation of this gene promotes cancer)

Year 2 MCD - Cancer (15 decks)

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