6: Apoptosis (30.01.2020) Flashcards
1
Q
Why does apoptosis occur?
A
- Harmful cells (e.g. cells with viral infection, DNA damage).
- Developmentally defective cells (e.g. B lymphocytes expressing antibodies against self antigens).
- Excess / unnecessary cells:
(embryonic development: brain to eliminate excess neurons; liver regeneration; sculpting of digits and organs). - Obsolete cells (e.g. mammary epithelium at the end of lactation).
- Exploitation - Chemotherapeutic killing of cells.
2
Q
Apoptosis vs necrosis
A
- Necrosis - unregulated cell death associated with trauma, cellular disruption and an inflammatory response (does not require energy)
- Apoptosis (programmed cell death) - regulated cell death; controlled disassembly of cellular contents without disruption; no inflammatory response; requires energy (ATP)
3
Q
What happens in necrosis?
A
- Plasma membrane becomes permeable
- Cell swelling and membranes are compromised, rupture of cellular membranes
- chromatin condenses
- Release of proteases leading to autodigestion and dissolution of the cell -> phagocytic cells invade
- Localised inflammation
4
Q
What happens in apoptosis?
A
Latent phase – death pathways are activated, but cells
appear morphologically the same
Execution phase – (here the cells change morphologically)
Loss of microvilli and intercellular junctions
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
Phagocytosis by neighbouring cells or by phagocytes. Macrophages engulf the apoptotic bodies.
Plasma membrane remains intact – no inflammation
5
Q
4 types of cell death
A
- Necrosis - Unregulated cell death associated with cellular disruption and an inflammatory response
- Apoptosis (programmed cell death, PCD) - Regulated cell death; controlled disassembly of cellular contents; no inflammatory response
- Apoptosis-like PCD - some, but not all, features of apoptosis. Display of phagocytic recognition molecules before plasma membrane lysis
- Necrosis-like PCD - Variable features of apoptosis before cell lysis; “Aborted apoptosis”
=> there are actually even more (~10) types. There is a graded response, so cell death types can have features of both apoptosis and necrosis and don’t have to fit in strictly into one or the other category.
6
Q
Mechanisms of apoptotic cell death
A
- The executioners – Caspases
- Initiating the death programme
- Death receptors (extrinsic)
- Mitochondria (intrinsic)
- The Bcl-2 family
- Stopping the death programme
7
Q
Caspases
A
- Cysteine-dependent aspartate-directed proteases
- Executioners of apoptosis
- Activated by proteolysis
- Cascade of activation
8
Q
Caspase maturation
A
- procaspases (zymogens) -> active enzymes (by the cleavage of Prodomain)
- Cleavage of the inactive procaspase precursor is followed by..
- folding of 2 large and 2 small chains to form an active L2S2 heterotetramer (LS and SS from both caspase 3 and 8 via proteolytic cleavage, 2 pairs of the 2 Paris will form the heterodimer)
- heterodimerisation of caspase 8 and caspase 3
9
Q
Caspase cascades
A
- amplification
- divergent responses
- regulation
- Initiator caspases – trigger apoptosis by cleaving and activating effector caspases (directly caspase 3 and 7 which carry out the effecter programme and activate further caspases)
- Effector caspases – carry out the apoptotic programme
10
Q
How do effector caspases execute the apoptotic programme?
A
- recognise specific sites and clip them
- Cleave and inactivate proteins or complexes (e.g. nuclear lamins leading to nuclear breakdown) -> can activate or inactivate monomoreic and multi protein complexes by clipping them
- Activate enzymes
- incl. protein kinases
- nucleases- Caspase-Activated DNase, CAD)
=> by direct cleavage, or cleavage of inhibitory molecules
11
Q
Mechanisms of caspase activation
A
- Death by design – Receptor-mediated (extrinsic) pathways
- Death by default – Mitochondrial (intrinsic) death pathway
12
Q
Death Receptors
A
- ligand binds and they become trimeric (3 molecules form 1 unit)
- ec: death receptor
- ic: death domain
13
Q
Adapter proteins in receptor-mediated apoptosis
A
- FADD: has DED and DD; activation
- FLIP: has 2 DED domains: inhibition
DED= death effecter domain; DD= death domain.
14
Q
Signalling through Death Receptors
A
e.g. Fas/Fas-ligand
- Receptor (Fas) trimerisation due to ligand binding(Fas-L on lymphocyte) -> signal for the cell to commit suicide
- Recruitment of adapter protein (FADD) through its DD to DD of Fas
- Recruitment and oligomerisation of procaspase 8 through its DED to FADD DED –> this produces the Death-Inducing Signalling Complex (DISC)
(e.g. Fas, upregulated on infected cells) -> you want these cells to die!
15
Q
DNA modification in apoptosis
A
- in apoptosis there is fragmentisation of the DNA
- you can run a gel
- first the DNA is quite large and does not want to move in the gel (15 mins)
- there is “ladder formation” at 45 and 70 minutes -> fragmentation
16
Q
TUNEL assay
A
- method for detecting DNA fragmentation
- by labeling the 3′- hydroxyl termini in the double-strand DNA breaks generated during apoptosis
- yellow spots on the assay suggest DNA fragmentation and apoptosis
17
Q
The BCL-2 family
A
= modulates apoptosis
- The Bcl-2 Family consists of a number of evolutionarily-conserved proteins that share Bcl-2 homology (BH) domains.
- The Bcl-2 family is most notable for their regulation of apoptosis, a form of programmed cell death, at the mitochondrion.
- The Bcl-2 family proteins consists of members that either promote or inhibit apoptosis, and control apoptosis by governing mitochondrial outer membrane permeabilization (MOMP), which is a key step in the intrinsic pathway of apoptosis.
18
Q
What are the different types of caspases?
A
Initiator
- 2,9,8,10
- have very defined domains
- 2 and 9 have CARD (caspase recruitment domains) and p10 and p20
- 8 and 10 have 2 DED (death effector domain) each and p10 and p20
- homotypic protein-protein interactions (cf. adaptors) -> same type i.e. caspase 8 dimerises with caspase 8
Effector
- 3,6,7
-> All have p10 and p20 domains
19
Q
CARD and DED
A
- caspase recruitment domain
- death effector domain
20
Q
Initiator procaspase 8 oligomerisation
A
- Initiator procaspase 8 binds to adapter protein (FADD) via DED-DED
- 3 procaspases are recruited to a trimeric structure
- there is cross cleavage of the procaspases (transcleavage, they cleave each other, they are in close proximity to one another)
- active initiator caspase 8 is formed (tetramer of the fragments)
- need at least to procaspases to form and active tetramer because they have to cleave one another
- Some initiator procaspases have intrinsic low catalytic activity – oligomerisation allows transcleavage
- others are activated by conformational change on oligomerisation
=> caspase 8 activates downstream effecter caspases
21
Q
Oligomerisation
A
The process of converting a monomer or a mixture of monomers into an oligomer.
22
Q
FLIP
A
- Death Receptor activation of caspase 8 is inhibited by FLIP
- FLIP - caspase homology in DED domain, but no proteolytic activity therefore COMPETES with procaspase for binding to receptor tails / FADD via DED domains
- Incorporates into receptor-procaspase complexes and interferes with transcleavage
- there is a short and long form of FLIP (they have different p10 and p20 domains)
23
Q
Mitochondrial regulation of apoptosis
A
- Cellular stresses e.g. lack of or overstimulation by growth factors, DNA damage (p53), reactive oxygen species
- Loss of mitochondrial membrane potential (ΔΨ)
- release of cytochrome c
- Release of other apoptosis-inducing factors
- Formation of the apoptosome complex
24
Q
Apoptosome complex
A
- apoptosome = wheel of death
- important: Apaf 1, cytochrome c, ATP, procaspase 9
- heptameter: CARD domains of apaf1 in the center, WD40 repeats facing outwards and interact with cytC
- the center of the wheel will attract to pc-9 dimer via binding to CARD domain (CARD-CARD)
- caspase 3 will be activated -> initiation of CASPASE CASCADE
- apoptosome includes the heptameter, cyt c and procaspases
- this process requires cytochrome c and ATP to occur
25
Apaf-1
apoptotic activating fator 1
- CARD
- ATPase
- WD40 repeats at C terminal (modulate structure of protein protein interactions)
- > forms heptameter (CARD domains in the center, WD40 repeats facing outwards and interacts with cytC)
26
What may determine if necrosis or apoptosis occurs?
- Energy levels in the cell may determine whether
death is by necrosis or apoptosis
- the apoptosome requires ATP
27
Connection between the intrinsic and extrinsic activation of apoptosis
- Caspase 8 cleaves Bid which enhances release of mitochondrial proteins, thus engaging the intrinsic pathway
- extrinsic activation enhance the intrinsic activation
28
Bcl-2 family proteins in apoptosis
- modulate apoptosis
- some are anti-apoptotic; some are pro-apoptotic.
- divided into 3 groups (1,2 and 3)
- BH3 present in all of them, dimerisation motif.
29
Which Bcl-2 proteins are pro/anti-apoptotic?
Pro: move between cytosol and mitochondria
- Bid
- Bad
- Bax
- Bak
Anti: mitochondrial
- Bcl-2
- Bcl-xL
30
PI3’-Kinase Signalling Pathway in Cell Cycle and Apoptosis Regulation
- GF e.g. EGF binds to GFR e.g. EGFR
- > 1) Ras -> ERK -> growth (mitogenic)
- > 2) PI3'K -> PDK-1 -> PKB/Akt -> survival, proliferation
- PI3 Kinase regulates both the cell cycle and apoptosis
- PI3K is made up of a p85 domain (adapter part) and p110 domain (kinase domain)
- PI3K phosphorylates a lipid: PIP2 -> PIP3
- Phosphatidylinositol 3’-kinase (PI3’-K) – a lipid kinase (not a protein kinase) involved in growth control and cell survival
- PIP3 recruits PKB/Akt to membrane (protein kinase)
- PKB/Akt has an anti-apoptotic and mitogenic effect
31
How does PKB/Akt induce cell survival?
=> by blocking apoptosis
1. Phosphorylates and inactivates Bad (Bcl family, Bad is pro-apoptotic)
2. Phosphorylates and inactivates caspase 9 (main executor of extrinsic pathway)
3. Inactivates FOXO transcription factors (FOXOs promote expression of apoptosis-promoting genes)
4. Other, e.g. stimulates ribosome production and protein synthesis
=> phosphorylation of many molecules, control of many different aspects that are necessary for cell survival and function
32
A model for the regulation of apoptosis by Bcl-2 family proteins via BH3 heterodimerisation
In the presence of GF:
- Bad is phosphorylated by PKB/Akt sequestered in the cytosol by protein 1433
- Bax is also in the cytosol
- in the mitochondrial matrix there are heterodimers Bcl-2 and Bax (heterodimered via BH3) -> inactivated
=> cell survival
In the absence of GF:
- Bad is dephosphorylated, will be released and comes into the mitochondria
- displaces Bcl-2/-xL from -> Bax/Bak;
- Pore -> allows cytochrome c to be released into the cytoplasm -> this starts apoptosis
33
PTEN
- "inactivates" the PKB/Akt pathway
- =lipid phosphatase
- counteracts PI3K signalling
- dephosphorylates PIP3 -> PIP2 (reverses the PIP2->PIP3 reaction)
34
IAPs
- Inhibitor of Apoptosis Proteins (IAPs)
- regulate PCD
- extrinsic pathway:
- Bind to procaspases and prevent activation
- Bind to active caspases and inhibit their activity
35
Cytoprotective / anti-apoptotic pathways
- Bcl-2, Bcl-xL: intrinsic pathway (at the mitochondri exclusively)
- FLIP, IAPs: extrinsic pathway
- growth factor pathways via PI3’-K and PKB/Akt
36
How can cancer cells avoid apoptosis?
Apoptosis regulators as oncogenes or tumour suppressors
37
Apoptosis and cancer:Programmed cell death – therapeutic uses
- Harmful (oncogenic) cells (e.g. cells with viral infection, DNA damage)
- Chemotherapeutic killing of tumour cells, e.g. Dexamethasone stimulates DNA cleavage
