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Y2 - CANCER > Apoptosis > Flashcards

Apoptosis Flashcards

1
Q

Define Necrosis

A
  • Unregulated cell death associated with trauma, cellular disruption and an inflammatory response
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2
Q

Define Apoptosis

A
  • Regulated cell death; controlled disassembly of cellular contents without disruption – no inflammatory response
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3
Q

Describe the process of necrosis

A
  • The plasma membrane becomes more permeable – the cell swells and the membrane ruptures
  • Proteases are released leading to dissolution and autodigestion of the cell
  • There is localised inflammation as immune cells are attracted
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4
Q

What are the two phases of apoptosis? Describe them

A

Latent phase:

  • Death pathways are activated, but cells appear morphologically the same

Execution phase:

  • Loss of microvilli and intercellular junctions
  • Cell shrinkage
  • Loss of plasma membrane asymmetry
  • Chromatin and nuclear condensation
  • DNA fragmentation
  • Formation of membrane blebs
  • Fragmentation into membrane enclose apoptotic bodies (these are then taken up by macrophages)
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5
Q

What is an important feature of apoptosis that distinguishes it from necrosis?

A
  • Plasma membrane remains intact – no inflammation
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6
Q

What DNA modification is seen during apoptosis and by what techniques can you observe these changes?

A
  • Fragmentation of DNA ladders (seen in agarose gel)
  • Formation of more ends, which are labelled by adding an extra fluorescently-labelled tag in a TUNEL assay
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7
Q

What other types of cell death are there other than necrosis and apoptosis?

A
  • Apoptosis-like cell death - some but not all features of apoptosis. There may be a display of phagocytic recognition molecules, even before cell surface membrane lysis
  • Necrosis-like cell death (sort of like an aborted apoptosis that ends up being necrosis)
  • NOTE: cell death is GRADED
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8
Q

What are caspases?

A
  • Cysteine-dependent aspartate-directed proteases
  • They are the executioners of apoptosis
  • They are activated by cleavage
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9
Q

Which caspases are effector caspases?

A
  • 3, 6 and 7
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10
Q

Which caspases are initiator caspases?

A

2, 8, 9 and 10

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11
Q

Describe the structure of effector caspases

A
  • They are single chain polypeptides consisting of a small and large subunit
  • The subunits are released by proteolytic cleavage
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12
Q

Describe the structure of initiator caspases

A
  • They have the same two (one large and one small) subunits found in effector caspases
  • … but they also have a targeting subunit (protein-protein interacting domain)
  • They have CARD and DED motifs that effector caspases do not have
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13
Q

How do effector caspases actually carry out the apoptotic programme - give 2 example mechanisms?

A
  1. Cleave or inactivate various proteins and complexes - e.g. nuclear lamins leading to nuclear breakdown
  2. Activating enzymes by cleavage or indirect cleavage of inhibitor molecules - e.g. Caspase Activated DNAases which break down the DNA
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14
Q

What are the two types of targeting subunit that initiator caspases can have?

A
  1. CARD – caspase recruitment domain
  2. DED – death effector domain
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15
Q

How are active caspases formed?

A
  • Cleavage of inactive procaspases is followed by the folding of 2 large and 2 small chains to form an active L2S2 heterotetramer
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16
Q

What are the two mechanisms of apoptosis?

A
  1. Extrinsic - Death by design (receptor-mediated)
  2. Intrinsic - Death by default (mitochondrial death pathway)
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17
Q

Describe the structure of death receptors

A
  • Cysteine-rich extracellular domain
  • Transmembrane domain
  • Intracellular tail with a death domain (DD)
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18
Q

What are the two important adaptor proteins in the death by design pathway and how are they different?

A
  1. FADD – positive regulator that promotes cell death – DED + DD
  2. FLIP – negative regulator – DED + DED
19
Q

Describe signalling of apoptosis through Fas

A
  • Fas ligand (FasL) binds to Fas receptor (on cytotoxic T lymohocytes) and the Fas receptors undergo trimerisation, which brings the three DDs in the intracellular domain together
  • The trimerised DDs recruit FADD, which binds via its own DD
  • FADD then recruits and oligomerises procaspase 8 through the DED of procaspase 8 and its own DED
  • Binding of procaspase 8 to FADD forms DISC (death-induced signalling complex)
  • DISC formation results in cross-activation of procaspase 8 by allowing cleavage
  • Active caspase 8 is released and forms an active caspase 8 tetramer, which then activates effector caspases
20
Q

Describe the importance of oligomerisation in the Fas / FasL pathway

A
  • Some initiator caspases have intrinsic low catalytic activity
  • Oligomerisation brings them close enough together to allow transcleavage which allows activation
  • Also, at least 2 procaspases are required to form an active caspase tetramer
21
Q

Describe how FLIP acts as an inhibitor of apoptosis

A
  • FLIP is evolutionarily related to caspases but has lost its catalytic activity
  • It has two DED domains and can compete with procaspase 8 to bind to the DED domains of FADD
  • It can therefore incorporate into receptor-procaspase complexes or in other words in between the procaspases that have bound with FADD and therefore prevent cross-activation of the pro-caspases by interfering with the trans-cleavage
  • So you don’t form the active caspase-8 tetramers that would otherwise go on to activate the effector caspases involved in apoptosis
22
Q

As an overview, describe death by default (intrinsic pathway)

A
  • Cellular stress causes a change in mitochondrial membrane potential
  • This leads to release of cytochrome C from the mitochondrion
  • This stimulates formation of the apoptosome complex
23
Q

What does the apoptosome consist of?

A
  • APAF-1 (apoptotic activating factor 1)
  • Cytochrome C
  • ATP
  • Procaspase 9
24
Q

Describe the domains found within APAF-1 and briefly state their roles

A
  • CARD domain - caspase recruitment domain - attracts procaspase 9
  • ATPase domain - cleaves ATP required to provide energy for binding
  • WD-40 repeats (protein-protein interactions) - binding site for cytochrome C
25
Q

Explain fully, how the intrinsic (death by default) pathway leads to caspase activation and therefore apoptosis

A
  • The cytochrome C released from the mitochondria bind to the WD-40 repeats of APAF-1 and make it form a heptamer structure (apoptosome)
  • This requires ATP, and APAF-1 uses its ATPase domain for this
  • It has 7 CARD domains in the middle, which can interact with CARD domains of procaspase 9
  • Seven procaspase 9 bind via their CARD domains to the APAF-1 molecules and their close contact allows them to cross-cleave each other to generate activate caspase 9
  • Active caspase 9 can go on to activate effector caspases which ultimately carry out the apoptotic programme
26
Q

1) What type of caspases are caspase 8 and 9?
2) How do caspase 8 and 9, activated by the death of design and death by default apoptotic pathways result in the actual carrying out of the apoptotic programme?

A

1)

  • Initiator caspases

2)

  • They both converge on activating caspase 3 - an effector caspase which is the thing that actually carries out the apoptotic pathway
27
Q

What pro-apoptotic protein links the death by default and death by design pathways? Explain how it works

A
  • Bid
  • Caspase 8 (generated by the death by design pathway) cleaves Bid, which travels to the mitochondrion and promotes the release of cytochrome C – thus triggering the mitochondrial death pathway
28
Q

How can energy levels of a cell show whether a cell is going through apoptosis or necrosis?

A
  • Apoptosis requires energy whereas necrosis does not
29
Q

What is an important family of proteins that act as intrinsic modulators of apoptosis?

A
  • Bcl-2 family
30
Q

There are three main groups of Bcl-2 proteins. What is common to all three groups?

A
  • BH3 domain – this is a dimerisation motif, which allows members of the family to form dimers with each other
31
Q

What are the anti-apoptotic Bcl-2 proteins and where are they found?

A
  • Bcl-2
  • Bcl-xL
  • They are found localised on the mitochondrial membrane
32
Q

What are the pro-apoptotic Bcl-2 proteins and where are they found?

A
  1. Bid
  2. Bad
  3. Bax
  4. Bak
  • These are found in the cytoplasm and in the mitochondrial membrane
33
Q

Other than Ras signalling, what other pathway does growth factor binding to growth factor receptors activate?

A
  • PI3-kinase promoting cell survival
34
Q

What type of molecule is PI3-K?

A
  • Lipid kinase
35
Q

What are the main subunits of PI3-K?

A
  • Adaptor subunit
  • Targeting subunit
  • Catalytic subunit
36
Q

What is the main action of PI3-K?

A
  • PI3-K converts PIP2 to PIP3
37
Q

Outline the whole pathway that goes from growth factor binding then via PI3-K and how it has its effects in regards cellular survival and apoptosis

A
  • Growth factors bind receptors
  • This causes dimerisation of these receptors
  • This dimerisation results in cross-phosphorylation of tyrosine kinase domains within the intracellular parts of the receptor
  • This phosphorylation causes morphological change allowing binding of adaptor proteins
  • An adaptor binds which then recruits PI3-K
  • PIP3-K (kinase) carries out PIP2 → PIP3
  • PIP3 is recognised by the adaptor subunit of Protein Kinase B (PKB/Akt)
  • This allows PKB to move to the cell membrane where it becomes activated
  • PKB phosphorylates and inactivates Bad (a member of the BCL-2 anti-apoptotic family) and has many other effects
  • Therefore it promotes cell survival and proliferation
38
Q

Describe the arrangement of the anti-apoptotic and pro-apoptotic proteins when growth factor signalling and the PI3-K pathway is active

A
  • This means PI3-K can produce PIP3
  • So PKB/Akt is activated meaning that Bad is phosphorylated and inactivated Bad is held in an inactive heterodimer with 14-3-3 On the mitochondrial membrane, Bak and Bax are held in inactive heterodimers with Bcl-2 and Bcl-xL
39
Q

Describe how loss of growth factor signalling can lead to apoptosis

A
  • This means loss of activation of the PI3K pathway
  • So less PIP3 produced
  • So less activation of PKB/Akt
  • Bad is allowed to get dephosphorylated and dissociated from its inactive heterodimer Bad then moves to the mitochondrial membrane and binds to the anti-apoptotic proteins (Bcl-2 and Bcl-xL) via its BH3 domain
  • This displaces Bax and Bak from their inactive heterodimers
  • So Bax and Bak then form a pore in the mitochondrial membrane allowing the release of cytochrome C from the mitochondrion – this leads to apoptosis
40
Q

Summarise the effects of PKB/Akt in promoting cell survival

A
  • Phosphorylates and inactivates Bad
  • Phosphorylates and inactivates caspase 9
  • Inactivates FOXO transcription factors (FOXOs promotes the expression of apoptosis-promoting genes)
41
Q

Outline regulation of apoptosis by BCL-2 family proteins via BH3 heterodimerisation, and thus go over again (there’s another flashcard in here about it) how absence of growth factors and the PI3-K pathway leads to apoptosis

A
  • Pro-apoptotic proteins belonging to the BCl-2 family including Bax and Bak are held in their inactive heterodimers by their BH3 domains by the anti-apoptotic proteins belonging to the BCl-2 family including BCl-2 and BCl-xl
  • When growth factors are absent, the PIK-3 pathway is inactive
  • So PIP3 is not formed
  • So PKB is not activated
  • This means that Bad cannot be phosphorylated and held in an inactive heterotrimer
  • Therefore it is dephosphorylated in the absence of active PKB
  • Thus it is released from its heterotrimer
  • Now it can form pores in the mitochondrial membrane, which allows cytochrome C to escape into the cytosol and induce apoptosis - cytochrome C apoptosome thing
42
Q

1) Name two extrinsic regulators of apoptosis (anti-apoptotic) and describe their actions
2) Then mention an intrinsic anti-apoptotic regulator - no need to describe their actions

A

1)

PTEN

  • Lipid phosphatase
  • Counteracts the activation of PKB
  • Reduces cell survival and promotes apoptosis

IAPs (Inhibitor of Apoptosis proteins)

  • Binds to procaspases and prevents their activation
  • Can bind to activate caspases and inhibit their activity

2)

Anything in the BCL-2 family

43
Q

Are the following tumour suppressor genes or oncogenes? a. Bcl-2 b. PTEN c. PKB/Akt

A

a. Bcl-2 Oncogene – increased activation would mean reduced likelihood of apoptosis (cancers are anti-apoptotic)
b. PTEN Tumour suppressor gene –inactivation will mean reduced likelihood of apoptosis
c. PKB/Akt Oncogene

Y2 - CANCER (14 decks)

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