Lecture 11 - Apoptosis Flashcards
Why is apoptosis necessary?
- essential for the development of a multi-cellular organism
- must destroy cells when they become superfluous
What is apoptosis required for in embryogenesis?>
- required for normal tissue development
- ew.g. removal of interdigital tissues
What are some of the roles of apoptosis?
- Development (e.g. metamorphasis in insects)
- Tissue homeostasis (e.g. intestinal microvilli generation/death)
- Elimination of premalignant cells (p53 activation)
- removal of damaged cells (e.g in infection, cytoxic T lymphocytes and gransymes induce apoptosis)
What are the differences between necrosis and apoptosis?
Apoptosis
- Physiological
- Cellular condensation
- Nuclear fragmentation
- Rapid phagocytosis
- lack of inflammation
- Blebbing
- Clustereed nuclear pores
Necrosis
- Pathological
- Organelles swell
- Membranes ruptures
- Leakage of cell contents
- Marked inflammation
- Lesions
- Even nuclear pores
What is the series of morphological changes that occur in apoptosis?
Mild convolution
Chromosome compaction and segregation
Condensation of cytoplasm
Nuclear fragmentation
Blebbing
Cell fragmentation
Phagocytosis of the apoptotic bodies
How are trophic factors involved in apoptosis?
- most cells require trophic signals to stay alive
- removal of trophic factors can activate apoptosis
What are the two main types of Bcl-2 proteins that are involved in apoptisus?
Anti-apoptotic proteins(e.g. Bcl-2, Bcl-XL)
Pro-apoptotic proteins (e.g. Bax, Bid, Bad)
What are the three subgroups of Bcl-2 proteins?
Group I proteins (Bcl-2, Bcl-XL) [AA]
Group II proteins (Bax) [PA]
Group III proteins (Bid, Bad) [PA]
Determined by their various different domains
What is the process of blebbing?
small pockets of membrane containing cellular contents are formed, and phagocytosed by white blood cells, phagocytes, lymphocytes and delivered to the lysosome
What are features of the Bcl-2 family?
- Intracellular regulators of apoptosis
- control the release of cytochrome C and other apoptotic regulators from mitochondria
- have either pro- or anti-apoptotic functions (
What are the domain similarities and differences between different Bcl-2 proteins?
Bim, Bax, Bcl-2, Bid
- Bim, Bax and Bcl-2 have transmembrane domains
- All contain BH3 domains (Bcl homology domain 3)
- Bcl-2 has a BH4 domain (excerts anti-apoptotic activity)
- Bcl-2 and Bax have BH1 domains
- Bcl-2 and Bax have BH2 domains
What are the interactions between the Bcl-2 family of proteins and Cytochrome C?
- Bcl-2/Bcl-2 homodimer or Bcl-2/Bcl-XL heterodimers inhibit cytochrome C release from the mitochrondia
- The function of Bad is normally inhibited by being bound to 14-3-3, but if phosphorylated Bad is released from 14-3-3 and recruited to the mitochrondia
- Once activated, Bad can bind to Bcl-2 (or Bcl-XL) dimers and inhibit their function
- This recruits Bax to the mitchondrial membrane, increasing mitchondrial membrane permeability allowing cytochrome C to be released
- cytochrome C initiates an intracellular apoptosis signalling cascade, mediated by caspases
How can the spectrum of Bcl-2 proteins expressed by a cell determine it fate?
The balance between anti-/pro- Bcl-2 proteins decides whether cell lives/dies
if the amount of Bcl-2 > Bax the cell is protected from apoptosis
if the amount of Bcl-2 < Bax, the cell is susceptible to apoptosis
What are the features of caspases?
- 11 known caspases in humans
- Two groups of caspases (Initiators/activators, cleave other caspases [2,8,9,10,12] and Effectors/excecutioners [3, 6, 7])
- Cysteine proteases that cleave their substrates on the C-terminal of aspartate residues
- synthesised as inactive zymogens (proenzymes) and require proteolytic modification to be activated
- have homodimeric structure, chains from each monomer required for two active sites
How are procaspases activated by cleavage?
- two inactive procaspase molecues have their pro domains cleaved off to allow caspases to dimerise and form an active caspase molecule
- with one large subunit and one small subunit
How are initiated caspases and effector caspases activated?
- Initiator caspases are initiated by dimerisation
- Effector caspases are activated cleavage then dimerisation to become active. When 2 are cleaved they form a homodimeric active enzyme with 2 catalytic sites
What is the amplifying caspase cascade?
- one molecule of an initator caspase cleaves and activates effector caspases
- these go on to cleave subsequent caspases
- amplifying the signal
What is the sequence of events following the release of cytochrome C that leads to cell death?
- Cytochrome C binds Apoptotic activating factor 1 (Apaf1)
- This recruits procaspase 9 to the apoptosome
- Dimerisation anf cleavage of procaspase 9 by Apaf1 results in active caspase 9 (inititator caspase )
- Active caspase 9 activates excecutioner caspases (procaspase 3 to active caspase 3)
- leads to degradation od intracellular substrates and cell death
How is procaspase 9 recruited to the apoptosome?
- Apaf1 and procaspase 9 contain CARD domains
- When Apaf1 is bound to cytochrome C it allows binding to the CARD domain of procaspase 9, forming a large oligomeric apoptosome complex
How do caspases acheive nuclear fragmentation, disruption of the cytoskeleton, membrane blebbing and cell fragmentation ?
- Initiator caspases (caspase 9) target effector caspases (caspase 3,6,7)
- more than 40 target protein substrates
- caspases cleave nuclear (laminins) and cytoskeletal (actin) proteins
- > cleave and activate gelsolin, induces actin severing
- another key target = Initiator of Caspase Activated DNAase (ICAD)
- > ICAD is cleaved resulting in the release of CAD (DNAase) which fragments DNA
- leads to nuclear fragmentation, disruption of the cytoskeleton, membrane blebbing and cell fragmentation
What are trophic factors?
Factors that induce differentiation/survival e.g.
NGF (nerve growth factor)
EDF (Epidermal growth factor)
PDGF (platelet derived growth factor and others)
-binding of growth factors can activate downstream signals e.g.
pI3-kinase/Akt signalling
Ras/Raf/MAPK signalling pathways
What is the prcess of PI3-kinase/Akt signalling?
- In the presence of a trophic factor, pI-3 kinase is stimulated to activate Akt (PKB) which phosphorylates Bad
- Phospho-Bad forms a complex with the 14-3-3 protein preventing Bad from interacting with Bcl-2
What can engagement of the death receptors lead to and what inhibits this pathway?
growth factor independent mechanism of apoptosis
- the activation of proapoptopic proteins (Bid, Bik) and cause apoptosome assembly and the initiation of the caspase cascade
- inhibited by Z-VAD
What growth factor deprivation lead to and what inhibits this pathway?
the activation of proapoptopic proteins (Bad) and cause apoptosome assembly and the initiation of the caspase cascade
-inhibited by Akt
What can nuclear damage lead to?
the activation of proapoptopic proteins (Rad-9), or stimulation of p53 to activate Bax/Noxa and cause apoptosome assembly and the initiation of the caspase cascade
What are death receptors?
growth factor independent mechanism of apoptosis
cell surface receptors that initiate apoptosis following ligand binding e.g.
-Fas (CD95/APO-1) with FasL (CD95L) ligans
-TNF-R1 (tumour necrosis factor 1) with TNFα or TRAIL (TNF-related apoptosis-inducing ligand)
What is the process of Fas/FasL mediated apoptosis?
- FasL binds to the Fas death receptor
- Ras interacts with an adaptor protein FADD (Fas-associated death domain)
- FADD complexes with procaspase 8 (a DED-caspase (death effector domain) to activate it via dimerisation
- caspase 8 cleaves Bid to tBid,activating it. tBid can then inhibit Bcl-2 in the mitochondrial membrane and the release of cytochrome C, leading to activation of caspase 9 via apoptosome formation
- Active caspae 8 also activates caspase 3/7 (excecutioner caspases) forming a death inducing signal complex (DISC - FADD and caspase and ligand and receptor)) which also leads to the inhibition of Bcl-2 in the mitochondiral membrane and release of cytochrome C
how do death receptors interact with adaptor proteins?
through mutual death domains
What is the role of Fas in AIDS?
- normally, all CD4+ T cells express Fas, but HIV infected cells also express high levels of FasL
- this induces non infected cells to commit suicide resulting in failure of the immune response
- infected cells survive as high FasL levels prevent interaction with their own Fas
What is the role of Fas in influenza defence?
- Target cell is infected with influenza. MHCI presents the viral peptide on the surface of the infected cell
- MHCI interacts with the cytotoxic T cells via the T cell receptor and FasL on the T cells interacts with Fas on target cells inducing apoptosis
