Cell death and damage

Question 1

What is the function of necrosis?

Answer 1

-Removes damaged cells from an organism

Failure to do so may lead to chronic inflammation, more damage subsequently done
- Necrosis causes its own acute inflammation to clear cell debris via phagocytosis

Question 2

What are the causes of necrosis?

Answer 2

Usually lack of blood supply e.g

Injury

Infection e.g gangrene

Cancer

Infarction

Inflammation

Question 3

What are the steps of necrosis?

6 steps, start: injurious agent/event.

End” cell membrane ruptured

Answer 3

1. Result of an injurious agent or event (whole groups of cells are affected).
2. Initial events are reversible, later ones are not. So if conditions are right the cell can undo the damage and survive, only to a certain point before the events are irreversible.
3. Lack of oxygen prevents ATP production from glycolysis or TCA cycle
4. Cells swell due to the influx of water (ATP is required for ion pump to work). This changes the osmolarity of cytoplasm and puts more pressure on membranes within cell due to expanded volume
5. Lysosomes rupture; enzymes degrade other organelles and nuclear material hapzardly random manner
6. Cell membrane ruptured, cell debris released in extracellular environment, triggering inflammation

Question 4

What nuclear changes occur during necrosis?

Answer 4

1. Chromatin condensation/shrinkage at beginning of necrosis
2. Fragmentation of nucleus, can see chromosomal DNA
3. Dissolution of the chromatin by DNAse

Question 5

What cytoplasmic changes occur during necrosis?

Answer 5

1. Opacification: protein denaturation and aggregation to become whiter and more solid mass
2. Complete digestion of cells by enzymes causing cell to liquify (liquefactive necrosis)

Question 6

What biochemical changes occur during necrosis?

Answer 6

1. Release of enzymes such as creatine kinase or lactate dehydrogenase
2. Release of other proteins such as myoglobin into blood stream and urine

These biochemical changes are useful in the clinic to measure the extent of tissue damage

Question 7

What is the function of apoptosis? And what is it involved in?

Answer 7

Selective process for the deletion of superfluous, infected or transformed cells

Involved in:

• Embryogenesis
• Metamorphosis
• Normal tissue turnover to be replaced by newer cells
• Endocrine-dependent tissue atrophy
• A variety in pathological conditions

Question 8

What is the step by step process of apoptosis

Answer 8

1. Programmed cell death of one or a few cells – distinction from necrosis which is multiple cells at once, apoptosis is selective
2. Events are irreversible and energy (ATP) dependent
3. Cells shrink as the cytoskeleton is disassembled
4. Orderly packaging of organelles and nuclear fragments into membrane bound vesicles
5. New molecules are expressed on vesicle membranes that stimulate phagocytosis without an inflammatory response

Question 9

What cytoplasmic changes occur during apoptosis?

Answer 9

1. Shrinkage of cell. Organelles packaged into membrane vesicles
2. Cell fragmentation. Membrane bound vesicles bud off
3. Phagocytosis of cell fragments by macrophages and adjacent cells
4. No leakage of cytosolic components – leakage into extracellular environment minimised compared to necrosis where it leaks and triggers inflammation

Question 10

What nuclear changes occur during apoptosis?

Answer 10

1. Nuclear chromatin condenses on nuclear membrane
2. DNA cleavage

Question 11

What biochemical changes occur in apoptosis?

Answer 11

1. Expression of charged sugar molecules on outer surface of cell membranes (recognised by macrophages to enhance phagocytosis)
2. Protein cleavage by proteases, caspases

Question 12

What are the two types of apoptosis?

Answer 12

Intrinsic

• DNA damage – p53 dependent pathway
• Interruption of the cell cycle
• Inhibition of protein synthesis
• Viral infection ie once virus is in the cell
• Change in redox state

Extrinsic – relative to the cell not body

• Withdrawal of survival factors e.g mitogens
• Extracellular signals e.g TNF
• T cell or NK (natural killer) e.g Granzyme

Question 13

What are caspases?

Answer 13

Involved in apoptosis.

The point of convergence for causes of apoptosis. Extrinsic and intrinsic causes feed into casases, causing apoptosis.

Caspases are cytesine proteases (cysteine aspartate-specific proteases) - cysteine is the type of protease caspase is, aspartate is the amino acid motif

Question 14

Describe activation of caspase

Answer 14

Inactive procaspase is cleaved by upsteam caspase in two places, releasing n terminal prodomain. Turns inactive procaspase into active caspase.

Question 15

Describe the caspase cascade

Answer 15

Top caspase in cascade is initiator caspase – in humans this is typically 8 and 9

Activates downstream caspases which typically activates many more further downstream

Not just activation of caspases, downstream caspases have additional substrates that are not caspases. Get cleavage of cytosolic proteins and nuclear lamin proteins (nuclear envelope proteins)

Caspases with additional substrates are effector caspases (in humans 1,3,6,7) more and more molecules with each step

Not a linear cascade but like a pyramid

There is signal amplification

Question 16

What does caspase activation cause?

Answer 16

Caspase activation leads to characteristic morphological changes such as shrinkage, chromatin condensation, DNA fragmentation and plasma membrane blebbing

Question 17

How is the intitiator caspase activated?

Answer 17

Initiator caspases activate themselves when in close proximity – cleaves itself

Activation therefore means bringing initiator caspases together. - lots of them in same point in space and time

Question 18

What induces extrinsic apoptosis?

Answer 18

Ligand binding to its receptors, causing receptor dimer (or multimer)-isation of that receptor.

Multimerisation of the receptor leads to activation of the caspase.

Question 19

In ligand induced multimerisation, what do the death adaptor and receptor share?

What do the death adaptor and procaspase-8 share?

What domain does procaspase-8 contain?

What do shared domains between proteins allow?

Answer 19

1. The death domain
2. Death effector domain
3. Death effector doman and protease domain, catalytic part.
4. Allow proteins to bind together because similar domains will dimerise/multimerise.

Question 20

Explain ligand induced multimerisation in Tumour necrosis factor (TNF)

Answer 20

• TNF bound to ligand binding domain of TNFR, bringing death domains into close proximity
• Creates environment where dimerisation with other proteins with death domains is favoured
• Death adaptor protein, FADD creates environment with high conc. of death effector domain proteins
• Death effector proteins recruit procaspases-8. Multi-protein complex has assempled - called DISC, death-inducing signaling complex
• Catalytic domains of procaspase-8 in close proximity, activate themselves
• Get proteolysis, caspases become active.
• Caspase-8 production from procaspase-8
• Caspase-8 departs from membrane into cytoplasm, activates caspase cascade

Question 21

What is intrinsic apoptosis induced by?

Answer 21

Induced by cytochrome c released from mitochondria.

Growth factor withdrawal (extrinsic apoptosis) is an exception that uses cytochrome c.

Question 22

What is cytochrome c?

Answer 22

• a mitochondrial matrix protein
• Known for many years to be released in response to oxidative stress by a “permeability transition”
• Any induces of the permeability transition also eventually induce apoptosis

Question 23

Cytochrome C-induced apoptosis

What domain is shared between APAF-1 (apoptotic protease activating factor 1) and procaspase-9?

What domain is on procaspase-9?

Where is the cytochrome c binding site?

Answer 23

1. Caspase recruitment domain (CARD)
2. Protease domain.
3. On APAF-1.

Question 24

How does cytochrome C trigger the caspase cascade?

Answer 24

• Cytochrome C is released from mitochondria, binds to its binding domain in APAF-1, bringing the molecules together
• There’s dimerisation and multimeriation and recruitment of procaspase-9
• This whole molecule is called an apoptosome
• There is the proteolytic, catalytic domains of procaspase-9 in close proximity which can target itself and cleave itself - autoproteolysis
• Results in active caspase-9 which triggers caspase cascade

Question 25

How is the release of cytochrome c from the mitochodnria regulated?

Answer 25

A pore made of BCL-2 family protein, through which cytochrome c can be released. Anti-apoptotic repress release of cytochrome c, cells survive e.g BCL-2, BCL-XL Pro-apoptotic facilitates release if cytochrome c leading to cell death e.g BAX, BAD

Question 26

Are all BCL-2 proteins membrane proteins? How does BAD work if there is also BAX and BCL2?

Answer 26

No, instead are cytoplasmic predominantly. However they all have BH3 domain used to form dimers. BH3 domain facilitates the formation of oligomers, dimers, trimers. is pro-apoptotic, binds strongly to BCL-2 (more strongly than BAX), displacing it from binding to BAX, allows cytochrome c to be released.

Question 27

What regulates BCL-2 proteins?

Answer 27

TP53. For example, DNA damage triggers activity of tP53 and expression of TP53 target genes e.g BAX, pro-apoptotic molecule. BAX insets into membrane, get formation of new pores that aren't blocked by anti-apoptotic BCL2. Release of cytochrome c and death.

Question 28

What type of apoptosis is removal of growth factors?

Answer 28

Extrinsic apoptosis, works through cytochrome C. GF are pro-survival signalling mechanism. No growth factors, survival signals blocked, no activation of AKT or PKB, BAD no longer phosphorlated. BAD binds to BCL-2, displacing it from the pore, allows release of cytochrome c, causes cell death.

Question 29

What do growth factors normally do to the cell?

Answer 29

Normally cell is enriched with growth factors that act as survival signals and activate serine/threonine kinase (AKT/PKB). Kinase phosphorylates BAD, phosphorylated BAD is sequestered in the cytoplasm. Therefore is no longer available to bind to BCL-2, can't dispalce it from pore