Apoptosis Flashcards
What is apoptosis?
- programmed cell death – cells go through sequence of changes leading to death and engulfment
What is necrosis?
cells burst, releasing intracellular contents
Is apoptosis irreversible once started?
Yes
Apoptosis process (5 steps)
- Chromosomal DNA begins to fragment due to cleavage by nucleosomes
- Chromatin condenses and nucleus breaks up into small pieces
- Cytoskeleton breaks up
- Cell shrinks and breaks up into membrane-enclosed fragments
- Known as blebbing
- Apoptotic cell is recognized and phagocytized by macrophages.
What is the signal for phagocytization in apoptotic cells?
- Phosphatidylserine translocated from inner leaflet to outer leaflet of plasma membrane.
Function of Caspases?
- They are cysteine proteases that cleave after aspartate residues
- Normally present as inactive precursors.
- Activated by cleavage by other caspases
- Cleave proteins at only a few residues
- Initiators and executioners.
- Examples of Cleaved:
- Inhibitor of Dnase that cleaves genomic DNA between nucleosomes
- Nuclear lamins
- Cytoskeletal proteins
- Inhibitors of apoptosis
What 4 genes play a role in apoptosis in C. elegans?
- Required for apoptosis to occur:
- ced-3
- ced-4
- egl-1
- Negative regulator of apoptosis
- ced-9
Apoptotic pathway of C. elegans?
- ced-9 binds to ced-4, preventing it from activating ced-3
- When ced-9 is removed by egl-1, ced-4 now able to bind to ced-3
- ced-3 cleaves itself to form active caspase
- ced-3 now cleave proteins in cell destined to die.
Role of ced-3?
Cleaves itself to form active caspase then cleaves proteins in cell destined to die.
Role of ced-4?
Activates ced-3
Role of egl-1?
Removes ced-9 from ced-4.
Role of ced-9?
Binds to ced-4, thus preventing the activation of ced-3 and starting apoptosis.
What family of proteins determines if apoptosis occurs in invertebrates?
- Bcl-2 family of proteins
- Play the role of both activators and inhibitors of apoptosis.
What are the two pathways of vertebrate apoptosis?
Intrinsic/stress
Extrinsic/death receptors
Bcl-2 family proteins:
Name three members that activate apoptosis and how they do so.
- Bad, Bax, Bid
- Activate apoptosis by translocating to mitochondria, where they are thought to disrupt integrity allowing cytochrome C to release.
Bcl-2 family proteins:
Name three members that activate apoptosis and how they do so.
- Bcl-2, Bcl-xL, Bcl-w
- Inhibit apoptosis by sequestering pro-apoptotic proteins.
Function of Bcl-2 proteins with 1 BH3 domain
- Pro-apoptotic
- Bid, Bad, Bim, Noxa, Puma
- Have both activator and enabler/derepressor activities
- Activated by multiple stimuli to initiate apoptosis
Function of Bcl-2 proteins with 3 BH3 domains
- Pro-apoptotic
- Bax, Bak
Function of Bcl-2 proteins with 4 BH3 domains
- Anti-apoptotic
- Bcl-2, Bcl-w, Bcl-xL
Mitochondrial pathway for apoptosis?
- Predominantly release of proteins that trigger activation of apoptosis
- Could also involve disruption of Electron transport or reactive oxygen species
- Formation of apoptosome
- Damage to DNA or mitochondria occurs
- Apaf-1 and cytochrome c complex with caspase-9 and ATP/dATP in cytoplasm
- (Apoptosome)
- Apaf-1 and cytochrome c complex with caspase-9 and ATP/dATP in cytoplasm
- Apaf-1 ans caspase-9 interact through N-terminal CARDs
- Caspase-9 is activated by autocleavage
- activates downstream caspases
- Damage to DNA or mitochondria occurs
Mitochondrial Apoptosis
Downstream caspases?
- Directly Activated:
- Caspase 3
- Indirectly activated:
- Caspases -2,-6,-8,-10
How is caspase-9 activated?
Autocleavage
Components of apoptosome?
Apaf-1, cytochrome c, caspase-9 and ATP/dATP
Death receptor pathway?
In immune system, Fas receptor controls apoptosis
Ligand for Fas is FasL, a trimer of indentical polypeptides.
Fas/FasL pathway mechanism
- Membrane-bound FasL binds to Fas on adjacent cell
- Induces trimerization of Fas
- Trimerized Fas binds to adaptor (FADD protein) via death domains at C-termini of both proteins
- TRADD - required by some death receptors but not all
- FADD binds to inactive caspase-8
- Complex becomes “death-inducing signaling complex” (DISC)
- Autocleavage of caspase-8
- Caspase-8 translocates from cytoplasmic tail of Fas-FADD complex to cytoplasm
- Activates downstream caspases
- Like, caspase-1
- Also cleaves Bid, Bcl2 protein that then disrupts the mitochondria.
Comparison of apoptotic pathways
- C. elegans
- Egl-1 inhibits CED-9, which was inhibiting CED-4, CED-4 now activates CED-3. CED-3 induces apoptosis.
- Mammals
- Bcl-2 pathway
- Bik and Bax inhibit Bcl-2 which was inhibiting Apaf-1.
- Apaf-1 now activates caspase-9 and thus apoptosis
- Fas/CD95 pathway
- CD95 activates FADD which activates Caspase-8 leading to apoptosis.
- Bcl-2 pathway
Where does FADD bind to Caspase-8
FADD has a death effector domain (DED) at it’s N-terminus. It binds inactive caspase-8 here to activate it.
Two pathways of inhibiting apoptosis
PI3-kinase pathway
PKB pathway
Describe PI3-Kinase pathway
- Activated by tyrosine kinases or G-protein
- PI3-kinase phosphorylates PIP2 to form PIP3
- PIP3 recruits Akt, serine-threonine kinase, to the plasma membrane
- Akt is phosphrylated by PDK
- Active Akt phosphorylates proteins: Bad and Caspase-9 which regulate apoptosis.
- Bad when phosphorylated binds to 14-3-3 which inactivate it in the cytosol.
Describe the PKB pathway?
- Survival factor binds to receptor
- Activates protein kinase B
- PKB phosphorylates and inactivates Bad
- Also, phosphorylates and inactivates regulatory proteins that stimulate transcription of apoptosis promoting proteins.
Positive feedback and amplification loops in apoptosis regulation?
- Caspases activate downstream caspases
- Cleave anti-apoptotic proteins
- Fragments then promote apoptosis.
Regulation of apoptosis by buffers/dampeners?
- Bcl-2 family of anit-apoptotic proteins
- Inhibitors of apoptosis proteins (IAPs)
- Bind to procaspases to prevent activation
- Bind to caspases and either inhibit activation or cause ubiquitination
- FLIPs
- Similar to caspase-8 but lacks catalytic residues
- Competes with caspase-8 for binding to FADD
- Decoy receptors
- Bind to apoptosis-inducing ligand, but cannot transduce apoptotic signal.
What proteins inhibit IAPs?
Smac/DIABLO and Omni
Sequesters IAP in mitrochondrial intermembrane space.
