3. MECHANISMS OF DISEASE II - CELL DEATH & DAMAGE Flashcards
What are the two types of cell death?
- Necrosis
2. Apoptosis
What is necrosis?
- Necrosis is the process by which cells die when they are damaged due to harm such as toxins
- The function of necrosis is to remove damaged cells
- If necrosis doesn’t occur, there’s chronic inflammation. But necrosis itself does also cause inflammation to allow the cell debris to be cleared by phagocytosis
What are the causes of necrosis?
- Normally a lack of blood supply to an area results in necrosis such as:
1. Injury
2. Cancer
3. Inflammation
4. Infarction
What are the steps of necrosis?
- Necrosis occurs due to an injurious agent affecting whole groups of cells. Initial/early events of necrosis are reversible but the later stages aren’t
- Necrosis results in a loss of oxygen or hypoxia
- ATP formation is reduced due to the loss of oxygen as oxygen is needed to produce ATP via metabollic pathways such as gylcolysis
- Reduced ATP means that the ATP dependent ion pumps aren’t as effective and are unable to maintain osmolality. There’s an influx of sodium ions followed by water
- The influx of water results in swelling of the cell. The cytoplasm osmolality changes putting pressure on the membrane
- Lysosomal enzymes rupture & degrade organelles & nuclear material rapidly when released into the cytoplasm
- Cellular debris are released into the extracellular environment triggering infllamation
- Inflammation allows cell debris to be phagocytosed
What are the nuclear changes in changes in necrotic cells?
- Chromatin condenses/shrinks
- Fragmentation of nucleus
- Degradation of chromatin DNAase
What are cytoplasmic changes in necrotic cells?
- OPACIFICATION - Cytoplasm becomes opaque rather than transparent due to protein denaturation & aggregation
- LIQUEFACTIVE NECROSIS - Lysosomal enzymes cause complete digestion of cells resulting in the cell liquefying
What are the biochemical changes in necrotic cells?
- Release of intracelular enzymes such as creatinine kinase or lactate dehydrogenase
- Release of intracellular proteins
What is apoptosis?
- Apoptosis is a type of programmed cell death
- It is a selective process for the deletion of damaged infected or transformed cells
- Apoptosis is involved in embryogenesis, metamorphosis, nromal tissue turnover & pathogical conditions
What are the steps of apoptosis?
- Programmed cell death of one or few cells
- Events in apoptosis are IRREVERSIBLE & are ATP dependent
- Cells shrink due to disassembly of the cytoskeleton
- Organelles & nuclear fragments are packaged into the membrane bound vesicles
- New molecules (phsophatidyserine) are expressed on the membrane of vesicles to cause phagocytosis without an inflammatory response
What are the differences between necrosis & apoptosis?
- Necrosis affects groups of cells whereas apoptosis only affects a few cells
- Early stages of necrosis are reversible but all stages of apoptosis are irreversible
- The loss of ATP triggers necrosis, but the changes in Apoptosis are ATP dependent
- Cells undergoing necrosis swell but cells undergoing apoptosis shrink
- Cells undergoing necrosis release debri, but in apoptosis organelles are packed into vesicles
- Release of debris triggers inflammation in necrosis, but there’s minimal inflammation in apoptosis
What are the 4 cytoplasmic changes in apoptotoc cells?
- Cells shrink & organelles packed into vesicles
- Cell fragmentation, vesicles bud off via BLEBBING
- Phagocytosis of cell fragemnts by macrophages or adjacent cells
- No leakage of debris or minimal debris
What are the nuclear changes in apoptotic cells?
- Nucelar chromosme condenses on nuclear membrane
2. DNA cleavage
What are the biochemical changes in apoptotic cells?
- Expression of new molecules on vesicle membrane (e.g phosphatidyl serine) to enhance phagocytosis
- Protein cleavage by proteases (caspases)
Give some examples of apoptosis
- Cell death in embryonic handto form digits
- DNA damage/P53 mediated apoptosis
- Apoptosis mediated by removal of growth factors
- Death of neutrophils during acute infection
- Death of immune cells after depletion of cytokines
What are the two types of apoptosis?
- Extrinsic apoptosis - Pro-Caspase 8
2. Intrinsic apoptosis - Procaspase-9
What are examples of intrinsic apoptosis?
- DNA damage - P53 dependent pathway
- Interruption of the cell cycle
- Inhibition of protein synthesis
- Viral infection (virus is extrinisc, but it causes apoptosis once it’s inside the cell)
What are examples of extrinsic apoptosis?
- Removal of growth factors
- Extracellular signals (e.g TNF)
- T cell or Natural Killer
What are caspases & what do they do?
- Casapases are a type of protease - cysteine aspartate specifc proteases
- Involved in both extrinsic & intrinsic apoptosis & therefore a converegnce point
What are the two groups of caspases?
- Initiator caspases - activate effector caspases via cleavage
- Effector caspases - cause the effects of apoptosis
Describe the process of caspase activation
- Caspase cascade involves signal amplification with the activation of multiple effector caspases
- Effector caspases are activated by cleavage which also produces substrates
- Substrates responsibel for effects
Give some substrates that caspases cleave & their functions
- Lamin A& B = nuclear envelope
- PARP, DNA-PK = DNA Repair
- Top III = DNA replication
- Raf 1, STAT1 = Signalling
- Akt/PKB = Cell survival
- elF4 = Translation
What is the effect of caspase activation?
- Caspases are responsible for the biochemical changes of apoptosis through cleavage
- Includes blebbing, DNA fragmentation, chromatin condensation
How are initiator caspases activated?
- Intiator caspases are also known as pro-caspases.
- They are activated by AUTOPROTEOLYSIS
- When they come into cose proximity with each other, they cleave themselves
What is extrinsic apoptosis?
- Extrinsic apoptosis occurs when a ligand binds to a receptor resulting in receptor dimerisation or multimerisation
What are the three molecules involved in apoptosis?
- RECEPTOR - has a ligand binding domain & death domain
- DEATH ADAPTOR - has a death domain & death effector domain
- PRO-CASPASE 8 - has a death effector domain & protease domain
Describe the mechanism of extrinsic apoptosis using TNF as an example
- TNF binds to ligand binding domain of TNF receptor
- Results in dimerisation fo TNF receptor via death domain
- Dimerised death domain recruits death domain (FADD) of Death adaptor protein
- Death effector domain recruits Pro-caspase 8 via detah effector dimerisation
- Pro-caspase 8 brought into close proximity with other resulting in activation to Caspase 8 via autoproteolysis
- Casapse 8 triggers cascade signalling cascade & forms a DEATH-INDUCING SIGNALLING COMPLEX (DISC)
What is intrinsic apoptosis?
- The intrinsic apoptosis pathway is triggred by the release of Cytochrome C
- However, the removal of growth factors also involves the release of Cytochrome C which is an exception
What is Cytochrome C?
- Cytochrome C is a mitochondrial matrix protein which is released in response to oxidative stress, resulting in a change in permeability
- Anything that causes a change in permeability can cause apoptosis
What are the three molecules involved in intrinsuc apoptosis?
- Cytochrome C
- APAF 1 (Apoptotoc protease activating factor)
- has a cytochrome C binding site, APAF domain & Caspase recruitment domain (CARD) - Pro-caspase 9
Describe the mechanism for intrinsic apoptosis
- Cytochrome C binds to cytochrome C bidning site on APAF 1
- CARD on many APAF1 molecules brought into close proximity causing dimerisation
- Dimerised CARD recruits Pro-caspase 9
- Forms apoptosome
- Pro-caspase 9 activated to Caspase 9 through autoproteolysis of protease domain
- Caspase 9 triggers signalling cascade resulting the release of Cytochrome C
What are the initiator caspases for intrinsic & extrinsic apoptosis?
- Intrinsic apoptosis = Pro-caspase 9
- Extrinsic apoptosis = Pro-caspase 8
What are BCL2 family proteins & their properties?
- The BCL2 family proteins can be anti-apoptotoc or pro-apoptotic
- These proteins from a pore through which Cytochrome C is released
What are the pro-apoptotic proteins?
- BAX
- BAD
- BID
- promote apoptosis/ cell death
What are the anti-apoptotic proteins?
- BLC2
- BCL2-X
- Promite cell survival, inhibiting apoptosis
What is the effect of the Bax protein on Cytochrome C release?
- Bax protein is a pro-apoptotic protein
- Bax forms a pore, allowing the release of Cytochrome C causing apoptosis
What is the effect of the BCL2 protein on Cytochrome C release?
- BCL2 protein is anti-apoptotoc, can block the Bax pore to prevent the release of Cytochrome C
- Prevents apoptosis & prevents cell survival
What is the effect of the BAD protein on Cytochrome C release?
- Bad is a pro-apoptotic protein
- Bad can bidn to BCL2 & displace it form the Bax pore
- Cytochrome C can then be released from the Bax pore causing apoptosis
What two things regulate BCL2 protein activity?
- Transcription
2. Phopshorylation
How does TP53 regulate BCL2 activity?
- P53 activated in response to DNA damage & P53 target genes are transcribed including Bax
- Bax inserts itself into the membrane froming new pores
- Not enough BCL2 to block the now increased number of Bax proteins so they won’t all be blocked
- Cytochrome C is released
How is BCL2 activity regulated through Bad phopshorylation?
- Growth factors trigger activation of serine threonine kinase Akt/PKB
- Akt/PKB phosphorylates BAD
- Phosphorylated BAD can no longer displace BCL2 from the pore, so BCL2 continues to block pre
- No cytochrome C release therefore no apoptosis
Describe the extrinsic pathway (removal of growth factors) which involves Cytochrome C
- Removal of growth factors means no activation of serine threonine kinase Akt/PKB
- No phosphorylation of BAD -> BAD can displace BCL2 from BAX pore
- BAX pore is unblocked & cytochrome C is released leading to apoptosis
