Mechanisms of Disease 2 :Cell Damage and Cell Death Flashcards
What is the function of necrosis?
- Removes damaged cells from an organism so that they do not cause chronic inflammation (but necrosis causes acute inflammation)
Why does necrosis cause inflammation?
-Necrosis induces its own acute inflammation to clear cell debris via phagocytosis to avoid more chronic inflammation
What can cause necrosis?
Mainly due to a lack of blood supply This can result because of : -Injury -Infection -Cancer -Infarction -Inflammation
Why does a lack of blood supply cause necrosis ?
Tissue moves away from blood vessel = ⬇ partial pressure of oxygen.
+ ⬇ pH as we move away from blood vessel
What is the step by step process of necrosis ?
- Injury
- ⬇ oxygen = no ATP production
- ATP is required for ion channels on cell membrane, so ATP ion pumps stop working. Osmosis causes influx of water. Cell will swell.
- Osmolality = lysosomes rupture, releasing enzymes which degrade organelles + nuclear material.
- Cell membrane ruptures = cellular debris released = triggers inflammation
What is the difference between apoptosis and necrosis?
Necrosis -Whole group of cells are affected
Apoptosis-Certain cells only
Necrosis-Triggered by external factors
Apoptosis -Cell death generated by normal healthy processes
Necrosis initial events are reversible however apoptosis is not
At what stage is necrosis reversible?
It is reversible where not many organelles have expanded and not too much damage meaning if the cell is re-oxygenated , resupply ATP and the swelling can be decreased.
Outline the necrosis microscopic appearance
1.Chromatin condenses and shrinks
2There is fragmentation of the nucleus, chromosomal DNA is in pieces
3.Dissolution of the chromatin by DNases
Outline the cytoplasmic changes during necrosis
- Opacification-Protein denaturation and aggregation (not clear anymore becomes white)
- Complete digestion of cell by enzymes causing cell to liquify (liquefactive necrosis(Lose cell structures)
Outline the biochemical changes during necrosis
Enzyme release e.g. creatine kinase /lactate dehydrogenase
Release of proteins e.g. myoglobin
This can be used to measure tissue damage extent
Outline Astrocytoma
Tumour of astrocytes that form the supportive tissue of the brain.
Middle of tumour mass is necrotic because as tumour grows, vascularisation becomes further from the middle.
Cancer will try to make its own vascularisation = angiogenesis
Apoptosis =
Apoptosis selectively removes unnecessary/infected/transformed cells (cancerous)
What is apoptosis involved in?
Embryogenesis Metamorphosis Normal tissue turnover (life span) Endocrine-dependent tissue atrophy (extracellular ligands) Pathological conditions
Describe what Apoptosis is
Apoptosis is the programmed cell death of 1/few cells.
Irreversible + requires ATP(energy).
What is the Apoptosis step by step
- Cell shrinks due to cytoskeleton disassembly.
- Cell contents are packaged into membrane-bound vesicles.
- Vesicles express molecules that stimulate phagocytosis without causing an inflammatory response.
Outline the apoptosis microscopic appearance
- Cell shrinks + organelles are packaged into vesicles.
- Cell fragmentation, membrane-bound vesicles will bud off.
- The cell fragments are phagocytosed by macrophages and adjacent cells .
- Limited leakage of cytosolic components = low inflammation.
What is blebbing ?
blebbing = A defined feature of apoptosis. Cell cytoskeleton has broken down, causing membrane to bulge/vesicles outwards.
Tubules and vesicles budding of the membrane
What are the nuclear changes in apoptosis ?
- Nuclear chromatin condenses on nuclear membrane
- DNA cleavage
What are the biochemical changes in apoptosis?
- Expression of charged sugar molecules on outer surface of cell membranes (recognised by macrophages to enhance phagocytosis)
- Protein cleavage by proteases, caspases(family of proteases)
What happens if you run a cell treated by apoptosis through an agarose gel ?
Apoptosis causes characteristic laddering of chromosomal DNA
If you run a normal cell you will get one single high molecular weight band
What are some specific causes of apoptosis?
- There is cell death in embryonic hand and this create fingers.
- Apoptosis induced by growth factor deprivation (neuronal death from lack of NGF).
- DNA damage-mediated apoptosis. If DNA is damaged due to radiation or chemo therapeutic agents, p53 (tumour suppressor gene product) accumulates. This arrests the cell cycle enabling the cell to repair the damage. If repair process fails, p53 triggers apoptosis.
- Death of neutrophils during an acute inflammatory response
- Death of immune cells( both T and B lymphocytes) after depletion of cytokines as well of death of autoreactive T cells in the developing thymus
Give some real life examples of apoptosis uses
Metamorphosis -This is the tadpole tail being lost through apoptosis
Interdigital web loss-This is where webbing is lost through apoptosis between the fingers.
What are the two types of apoptosis?
Intrinsic and extrinsic
What is intrinsic apoptosis?
DNA damage – p53-dependent pathway Interruption of the cell cycle Inhibition of protein synthesis Viral Infection (Once the virus has entered the cell) Change in redox state
What is extrinsic apoptosis?
(Relative to the cell rather than body)
Withdrawal of survival factors e.g. mitogens
Extracellular signals (e.g. TNF)
T cell or NK (Natural Killer) (e.g. Granzyme).
What are caspases?
- These are the point of convergence of apoptosis.
- They are cysteine proteases
- They form an activation cascade as one cleaves and activate next
Outline Caspase activation
Inactive procaspase Y contains three parts:
N terminus
C terminus
It is cleaved by an upstream caspase in two places which release N terminal pro domain and creates a active caspase Y after rearrangement of small and large subunit.
Outline the caspase cascade
There is an imitator caspase (8/9)
These will activate further caspases and then those will activate even more.
Not just caspases , the downstream ones have additional substrates. These are called effector caspases (1,3,6.7)
There is cleavage of cytosolic proteins and cleavage of nuclear proteins (Lamin -Nuclear envelope destruction)
PYRAMID SHAPE _THROUGH SIGNAL AMPLIFICATION
What are some substrates for caspases and what do they effect ?
Lamin A/B -Nuclear envelope PARP- DNA Repair DNA-PK-DNA repair Toposiomerase 2- DNA replication Raf-1-Signalling Akt/PKB-Cell survival STAT1-Signalling eLF4-Translation
What is the effect of caspase activation?
Caspase activation leads to characteristic morphological changes, such as shrinkage, chromatin condensation, DNA fragmentation and plasma membrane blebbing.
Degradation of cytoplasm -Cytoskeleton degradation
How do initiator caspases activate themselves?
They will cleave each other when in close proximity
Describe the structure of the parts of ligand induced multimerization
It activates extrinsic apoptosis
Process:
1.There is an extracellular ligand which will bind to its receptor via the ligand binding domain on the cell membrane
2.There is a shared domain between the receptor and the death adaptor called the death domain.
- There is also a shared domain between the death adaptor and procaspase -8 called the death effector domain.
Finally there is a protease domain
The function of these domains is to allow proteins to bind together to dimerise /multimerise
Outline the process of Ligand induced multimerisation using TNF
Tumour Necrosis Factor
1.TNF will bind to the ligand domain on the TNFR and this brings the death domains to close proximity creating environment where dimerisation can take place.
2.There is a death adaptor protein called FADD (FAS-associated protein with Death domain) with high concentration of death effector domains which will recruit Procaspase-8.
This creates a complex called Death-inducing signalling complex (DISC)
We get proteolysis which activates the caspase 8 which can move from membrane to cytoplasm and move into cytoplasm and activate caspase cascade
How does intrinsic apoptosis become activated ?
This is through the release of cytochrome c release from the mitochondria
What is Cytochrome C ?
It is a mitochondrial matric protein and it becomes released due to oxidative stress which causes it to become more permeable (Permeability transition) .
Outline what molecules are involved in Cytochrome C induced apoptosis
There is cytochrome c
There is a death adaptor protein called APAF-1 (Apoptotic Protease Activating Factor )
There is an initiator caspase -Procaspase 9
The caspase recruiter domain (CARD)
Cytochrome c binding site on top of the APAF1
The whole thing is called APOPTOSOME
What regulates cytochrome C release from mitochondria ?
Family of protein called BCL-2 which form a pore
Two types:
Anti-apoptotic (bcl-2,bcl-XL) -These repress cytochrome release
Pro-apoptotic(Bax,Bad,Bid)-Facilitate cytochrome c release
Not all membrane proteins
All of them contain a BH3 domain which is used for creating dimers
Outline the process of BAX and BCL-2
BAX -Pro apoptotic
Creates a pore which allows cytochrome c to be released
BCL-2 -Anti-apoptotic
It will bind to BAX and cytochrome c will remain inside the mitochondria
BAD - (pro-apoptotic)
BAD will bind to BCL-2 and it will displace it allowing cytochrome c to be released again
How does TP53 regulate BCL-2
TP53-
Transcription factor
DNA Damage triggers TP53 expression
Transcription is driven by TP53 driven by BAX(TP53 target protein) and this causes membrane insertion
This will form new pores which can’t be blocked leading to death as more cytochrome c is released
How does phosphorylation regulate BCL-2 ?
This is through growth factors
BAD
Survival signals will activate kinase (serine -threonine kinase/Protein kinase B)
This kinase will phosphorylate BAD becomes sequestered in cytoplasm can’t displace BCL-2 meaning cytochrome c can’t be released
What happens if survical signals are blocked ?
We do not get activation of PKB meaning we do not get phosphorylation of BAD which can still bind to BCL-2 unblocking the pore and allows cytochrome c to leave and cause cell death .
