Week 10 (Apoptosis) Flashcards
What is Apoptosis?
Cell death
–in normal physiology (death can be physiological)
–In disease
What is the function of apoptosis?
- Removes damaged, unwanted or diseased cells
* Programmed cell death = Ordered cell deletion
C. elegans
–powerful model system
–191 predictable deaths in development
–highly tractable system
–study regulation, activation and execution of apoptosis
•Model systems have underpinned current knowledge
What is Apoptosis is crucial for?
–development
–maintenance
–control
Cell Population Control
Proliferation+
Differentiation -/+
Cell Death -
There is no net change in number of cells
Examples of diseases caused by Overactive Apoptosis?
More cells dying than proliferating –Neurodegeneration –Immunodeficiency –Stroke –Coronary Heart Disease –Transplant Rejection
Examples of diseases caused by Underactive Apoptosis
More cells proliferating than dying
–Cancer
–Autoimmune & Inflammatory conditions
–(e.g. SLE and rheumatoid arthritis)
The Importance of Apoptosis
Life-death imbalance can be desirable (physiological)
Why can Underactive Apoptosis be desirable?
–Allows cell accumulation (e.g. accumulation of cells during an immune response)
What why can Overactive Apoptosis be desirable?
–Returns cell populations to normal after challenge
–Neutrophil apoptosis during inflammation
–CTL death
What are the characteristics of apoptosis?
Tightly controlleed/ programmed Cell shrinkage •Organelle integrity •Chromatin condensation •Plasma membrane integrity
- Thermodynamics uphill
- Internal control
- Purposeful
What are the characteristics of necrosis?
Not tightly controlled (accidental/random), caysed by external factors •Cell swelling •Organelle disruption •Nuclear swelling •Plasma membrane rupture
- Thermodynamics downhill
- External “control”
- Accidental
Caspases
- Cysteinyl ASPartate-specific proteASES
- Key cysteine in active site QACxG
- Aspartate at P1 of substrate tetrapeptide
- Tetrapeptide defines caspase specificity
Three broad categories of caspases
- Inflammatory caspases
- Initiator caspase
- Effector caspase
Inflammatory caspases
- Maturation of pro-inflammatory cytokines
* e.g. Caspases 1, 4, 5, 11, 12, 14
Initiator caspase
- Kick off the caspase cascade
* e.g. Caspase 8, 9
Effector caspase
- Cleavage of cellular substrates
- Mediate the killing
- e.g. Caspases 3,6, 7
Describe the activation of Caspases
•Caspases are produced as inactive zymogens
•Pro domain 23 - 220aa
–large pro-domain = initiator caspase (contains recruitment domains)
–Small pro-domain = executioner caspase
•Activation requires a minimum of 2 cleavages
Cleavage at the prodomain and formation of a tetramer
Cleavage of caspase substrates during apoptosis: what is the function of caspases?
- Pro-caspase (inactive) → active caspase
- Break down Structural proteins and DNA replication/repair enzymes
- Inactive nuclease → active nuclease
What is CAD?
caspase-activated DN-ase
What is ICAD?
inhibitor of CAD (caspase substrate)
End goal of apoptosis
detach cell from neighbours
Dismantle cell
destroy DNA
alter the cell surface
Cell suicide occurs in response to
cellular insults
Extrinsic Pathway
–Cell are instructed to die from outside
–Receptors (“Death Receptors”) on cell surface induce death when ligated
- Killer lymphocyte have a protein (fas ligand) on their surface
- Fas death receptor on pre apoptotic cell recognises the fas ligand
- Recruitment of FADD (adaptor protein) which has a death domain and a death effector domain
- Death domain joins the intracellular Fas death receptor
- Death effector domain recognised by caspases (8 or 10)
- FADD recruits procasapases which leads to initiation and activation of procaspases via cleavage
- Caspase cascade takes place
- Resulting in an apoptotic cell
Intrinsic pathway
- cytochrome c released into the cytoplasm from damaged cell which binds to Apaf-1 (adapter protein)
- this recruits inactive procaspases
- activates then
- caspase cascade initiated
Adaptor molecules are important in
recruiting initiator caspases and allowing their activation
Control of cell death - Bcl-2 family
•Family of interactive proteins
- Protect from apoptosis – Pro-survival
- Suppress protection – Pro-death
- Pro- death and pro-survival members
- Bcl-2 - prototypic member of the family
Bcl-2 Pro-survival members
•For example:
–Bcl-2
•Crucial for cell survival
•Essential for life
Bcl-2 Pro-apoptosis members = Pro-death
Divided into 2 categories
Bax-like = BH123 members
–lack BH4 domain (BH123)
–form pores in mitochondria →release of Cyt c
BH3 only
– also called ‘Bad’ proteins
–central to life & death decisions of a cell
–activation during stress will dictate the outcome
Bcl-2 Mechanism of Action
Guard mitochondrial integrity - Regulate Cyt c release
LIFE
Bcl-2 & Bax balance
DEATH
BH3 only members disrupt the balance
Bax will form pores in mitochondria
LIFE
Bcl-2 & Bax balance
Bcl-2 prevents
mitochondrial disruption
BH3 only proteins inhibit
Bcl-2 & tip the balance
Control of Apoptosis: balancing act
•Pro v Anti-apoptosis signals
•Balance can be tipped by cell stress
–e.g. genotoxic damage can stabilise p53 and lead to transcription of pro-death members
IAPs
–Inhibitors of apoptosis
–produced by certain viruses to prevent death
–Inhibit caspases via inhibition of
•activation of pro-caspases
•activity of active caspases
The ultimate step in cell death
- “Burial”
- Cell surface changes act as “eat me” signals (recognised by phagocytes)
- Viable cells eat the dying cell
safe and controlled removal of cells
prevents apoptotic cells falling apart
intracellular contents are detrimental
Cell Removal is the aim of apoptosis
Apoptotic Cell Surface Changes (that are recognised by the phagocyte)
Redistribution of membrane lipids (especially phosphatidylserine)
Receptors on phagocytes
- Recognise apoptosis-induced surface changes
- Apoptotic cells are eaten and degraded
- A silent process
•Safe packaging
–failure leads to inflammation
Biochemical/Cell Biological Features in apoptosis
•Ligation (FAS ligand) of surface death receptors –Generates a death-inducing signalling complex •Mitochondrial Changes –Release of cytochrome c •Cysteine Protease Activity –Protein cleavage after aspartate residues •DNA cleavage –Activation of nucleases (CAD) –Cleavage of DNA into fragments •Surface changes –“eat me signals” –Flip-out of phosphatidylserine •Phagocytosis
How often does cell death occurs
Up to 1million times in some tissues
Phase 1
- Presence of signal
- Intracellular signalling pathways activated or disrupted
- Epopotosis machinery employed (caspase cascade)
- Phagocyte degradation
Phase II
- Extracellular Signals: Cytosine deprivation, gentoxdic (chemical or radiation) DNA damage, death receptor ligation
- Intracellular signals: p53 transcribed, survival signals , caspase
- Both activate the apoptosis engine (caspase cascade)
Which amino acid forms the active site of caspase?
Cysteine 1
Are caspases always activated
No
Only when cells becomes apoptosis
CNS caspases activate other caspases?
Yes
In turn the activated caspases can activate others
What does a small prodomain indicate?
An effector caspase
Are procaspases active or inactive?
Inactive
What are the 4 domains I’d the Bcl-2 family?
BH4
BH3
BH1
bH2
Describe apoptotic cell surface changes
These changes are recognised by phagocytes
- In a healthy cell membrane the phosphadityl serine are facing the cytosol
- When the cell become apoptotic some of the phosphadityl serine face the Extracellular space due to a biochemical change
What type of caspase has a large prodomain?
Initiator casapase
What are the 3 main caspase domains?
Prodomain
Large domain
Small domain
What are the 3 main caspase domains?
Prodomain
Large domain
Small domain
Which caspase has a small prodomain?
Effecror casapa
Where do caspases cleave proteins?
At the P1 aspartase
How does capsase activation occur?
1) Cleave of the prodomain
2) Cleavage and separation of the large/ small domains
3) joining of 2 capspases to form a tetramer
Which end of the protein is the small domain?
Carboxyl end
Which end of the protein is the prodomain?
Amino
Do the survival members of the Bcl-2 family have all 4 domains?
Yes
Which domains do the Bcl-2 pro death members have?
BH123
What would be the effect of an upregulation of BH3?
Induce apoptosis
How does Bcl-2 disrupt mitochondria?
- They have BH123 on their surface (inactive)
- Apoptotic stimulus
- BH123 complex changes in conformation and makes a pore in the mitochondrion
- Mitochondrion releases cytochromes
When is BH123 inactive?
Pro-survival Bcl-2 protein blocks pore formation
An Apoptotic stimulus can replace the Bcl-2 protein thus allowing the BH123 to become a pore
