Mechanisms of Disease II: Cell damage and cell death

Question 1

What are the functions of necrosis?

Answer 1

removes damaged cells from an organism
-failure to do so may lead to chronic inflammation (necrosis induces acute inflammation to clear away cell debris by phagocytosis)

Question 2

Name some of the causes of necrosis

Answer 2

A lack of blood supply to an affected area due to injury, infarction, infection (like gangrene), cancer and inflammation

high distance from a blood vessel causes low PH and low PO2

Question 3

Name some differences between apoptosis and necrosis

Answer 3

Affects a whole group of cells in necrosis but apoptosis is one or a few cells (selective)
Initial events in necrosis are reversible, apoptosis events are irreversible and energy (ATP) dependent

Question 4

What happens in the necrotic cell when there is not enough O2? Think about water balance in the cell

Answer 4

Lack of oxygen prevents ATP production so cells swell due to influx of water this is because ATP is required for ion pumps to work so osmolarity changes

Question 5

Explain what it is in the cell that causes the degradation (not swelling) in necrotic cell

Answer 5

The change in osmolarity (due to no ATP working on ion pumps) causes lysosomes to rupture so enzymes degrade organelles and nuclear material

cell debris then causes inflammation

Question 6

When is the cell irreversibly damaged?

Answer 6

When if it were to be re-oxygenated then it would not be able to produce ATP again

Question 7

Describe how the nucleus of a cell changes under a microscope in a necrotic cell (3)

Answer 7

chromatin condensation/shrinkage
fragmentation of nucleus
dissolution of the chromatin by DNAase enzyme (nuclei vanish!)

Question 8

Describe how the cytoplasm of a cell changes under a microscope in a necrotic cell (2)

Answer 8

becomes more opaque (white) as there is protein denaturation and aggregation like when you fry an egg white
Complete digestion of cells by enzymes causing cells to liquify = liquefactive necrosis

Question 9

Describe some biochemical molecules that are released by the necrotic cell

Answer 9

Release of:

• Enzymes such as creatine kinase or lactate dehydrogenase
• Proteins such as myoglobin (released during muscle damage into bloodstream)

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

Question 10

Define apoptosis

Answer 10

“a selective process for the deletion of superfluous, infected or transformed cells ”

can be normal (e.g. endocrine induced) or pathogenic

Question 10

Is apoptosis reversible?

Answer 10

No, once it happens it is irreversible (necrosis is reversible at early stage). It is also ATP dependant

Question 11

How does the size of the cells change in apoptosis vs necrosis?

Answer 11

Cells shrink in apoptosis due to disassembly of cytoskeleton - this is unlike necrosis where the cells swell

Question 12

Describe the inflammatory response in apoptosis

Answer 12

THERE IS NONE - described below

Question 13

How do the contents of the cells behave in apoptosis?

Answer 13

The contents (organelles) are packaged into membrane bound vesicles - new molecules are expressed on vesicle membranes that stimulate phagocytosis WITHOUT an inflammatory response

Question 14

Describe how the microscopic appearance of a cell changes in apoptosis

Answer 14

Shrinkage of cell, cell fragmentation (membrane bound vesicles bud off), phagocytosis of cell fragments by macrophages and adjacent cells

there is NO leakage (or limited leakage) of cytosolic components as this would trigger inflammation - a lot of apoptosis can cause leakage which causes secondary necrosis due to inflammation
cytoplasm shrinks around the nucleus

Question 15

Describe how the microscopic appearance of a cell nucleus changes in apoptosis

Answer 15

chromatin condenses on nuclear membrane
DNA cleavage

Question 16

What are caspases?

Answer 16

Proteases used in apoptosis

are cysteine proteases
Cysteine Aspartate-Specific Proteases

Question 17

Describe a biochemical change in the cell in apoptosis - what do these do?

Answer 17

Expression of charged sugar molecules on the outer surface of cell membranes are recognised by macrophages to enhance phagocytosis without inflammation
Cleavage of proteins by proteases called caspases

Question 18

How is DNA digestion different in apoptosis vs necrosis on a gel electrophoresis?

Answer 18

DNA is laddered in apoptosis but is a DNA ‘smear’ in necrosis as DNA digestion is non-specific

Question 19

Name the 2 types of apoptosis

Answer 19

Intrinsic and extrinsic

Question 20

Describe some of the causes of intrinsic apoptosis

Answer 20

DNA damage (via p53 dependant pathway)
interruption of the cell cycle
inhibition of protein synthesis
viral infection (once virus is in the cell)
change in redox state

Question 21

Describe some of the causes of extrinsic apoptosis

Answer 21

withdrawal of survival factors
extracellular signals like TNF
T cells

Question 22

How do caspases function?

Answer 22

form an activation cascade where they cleave the next one to activate it - so there is signal amplification. Will cleave various proteins to activate them to promote survival

these are how apoptosis works

Question 23

When there is cleavage of an inactive procaspase Y, what 3 things are released?

Answer 23

cleaved in 2 sites and will release 2 subunits a large and a small as well as a pro domain

the 2 subunits will dimerise to form the active caspase

Question 24

What are lamin proteins?

Answer 24

Are vital for the integrity of the nuclear envelope

Question 25

Name some of the substrates of caspases and their functions

Answer 25

-

Question 26

Explain what is meant by ‘blebbing’

Answer 26

The shape of the apoptotic cell as the cytoplasm shrinks around the nucleus as well as organelles budding off - this is all due to caspase activation

Question 27

Explain how the initiator caspases are activated to begin extrinsic apoptosis

Answer 27

The proteolytic domains cleave (activate) themselves when they are in close proximity to each other - this is explained properly below

Question 28

How does extrinsic apoptosis usually initiate?

Answer 28

By a ligand binding to a receptor causing activation (often multimerisation)

Question 29

What are death adaptors?

Answer 29

Intermediate molecule in ligand-induced multimerisation in the process of extrinsic apoptosis - has death domain + death effector domain?

Question 30

Explain what death domains are. What are death effector domains?

Answer 30

These are domains that are the same in the death adaptor AS WEL AS the receptor death effector domains are the same in the death adaptor and the initiator procaspase

Question 31

So name the 2 domains in procaspase-8

Answer 31

Procaspase-8 is an initial procaspase death effector domain protease domain

Question 31

What is DISC?

Answer 31

This is the multi-protein complex including the ligand, receptor, death adaptors and the procaspase (is shown below) DISC = death-inducing signalling complex

Question 32

Fully explain the process of ligand-induced multimerisation in extrinsic apoptosis leading to the activation of caspases

Answer 32

Ligand (like TNF) binds to its receptor (TNFR)- this allows for similar domains to dimerise/multimerise in the receptor in this case the death domain is called FADD and it dimerises with TNFR death domains the death effector domains (light orange) will then dimerise with death effector domains on procaspase 8 this complex is called DISC the blue domains (proteolytic) are in close proximity and so they are activated!

Question 33

What is the starting event in intrinsic apoptosis?

Answer 33

cytochrome C being released by mitochondria - this is a small protein one form of extrinsic apoptosis (withdrawal from growth factors) also uses cytochrome C

Question 34

Describe what cytochrome C is

Answer 34

mitochondrial matrix protein

Question 35

What is permeability transition?

Answer 35

Cytochrome C is released in intrinsic apoptosis, but it has been known for a long time to be released in response to oxidative stress by a ‘permeability transition’ any inducers of the permeability transition (mitochondria becomes more permeable) eventually induces apoptosis

Question 35

Describe and explain what APAF-1 is

Answer 35

apoptotic protease activating factor this is the equivalent of a death adaptor protein (so is an intermediate)

Question 35

Describe the structure of APAF-1

Answer 35

-

Question 36

Which procaspase is used in this process (cytochrome-C mediated apoptosis)?

Answer 36

Procaspase-9

Question 37

Fully describe the mechanism of how cytochrome C mediates intrinsic apoptossis

Answer 37

When cytochrome C binds, APAF-1 dimerises with procaspase-9 on the CARD domain (this is caspase recruitment domain and is shown in orange) - this complex is an apoptosome the proteasome domains then cleave themselves as normal

Question 37

What is it that actually regulates the release of cytochrome C in intrinsic apoptosis upstream?

Answer 37

Proteins in the BCL-2 family (BCL-2 is also, confusingly, a protein member of the BCL-2 family)

Question 38

Proteins in the BCL-2 family (BCL-2 is also, confusingly, a protein member of the BCL-2 family)

Answer 38

BCL-2 proteins arrange themselves in the mitochondrial membrane to allow for permeability of the membrane to cytochrome C some are not membrane proteins and are cytoplasmic

Question 39

Explain how BCL-2 proteins are classified in 2 ways

Answer 39

Can be pro-apoptotic or anti-apoptotic (either stimulate or repress cytochrome C release to stimulate or inhibit apoptosis)

Question 40

What do all BCL-2 proteins have (domain)?

Answer 40

They all have a BH3 domain. As shown earlier, proteins that all have the same domains often form multimers, dimers, trimers etc.

Question 41

Name one example of a pro-apoptotic BCL-2 protein

Answer 41

BAX

Question 42

Name one example of a anti-apoptotic BCL-2 protein

Answer 42

BCL-2 (the protein and not the family) - it binds to BAX to block the channel so it blocks the efflux of cytochrome C

Question 43

Describe the function of BAD

Answer 43

BAD is a pro-apoptotic protein as it binds strongly to BCL-2 protein so that it can not bind to BAX and therefore the BAX pore is open for efflux of cytochrome C

Question 44

So what regulates BCL-2 proteins since they are what regulate cytochrome C which is essential for intrinsic apoptosis

Answer 44

Gene expression (so TP53 affecting expression of various genes as described below) and post-translational modification (mostly phosphorylation)

Question 45

Explain how TP53 triggers apoptosis in intrinsic apoptosis

Answer 45

In a normal cell, there is a balance between pro and anti-apoptotic factors working on BAX pores. If there is DNA damage, TP53 will be expressed which will upregulate certain genes such as BAX! and BAX will insert into the membrane and cause efflux of cytochrome C as there is not enough BCL-2 to block it

Question 46

Fully explain how survival signals allow for cell survival (NOT activation of apoptosis)

Answer 46

Survival signals = growth factors survival signals are an anti-apoptotic signal that inhibits extrinsic apoptosis - this is by activating protein kinase B which phosphorylates BAD so BAD can’t bind to BCL-2 and displace it so BCL-2 binds to BAX and so there is no efflux of cytochrome C so the cell survives! So a loss of these survival signals/growth factors will be a pro-apoptotic signal by the reverse of the mechanism stated above. This was mentioned that a lack of growth factors will trigger extrinsic apoptosis by the same mechanism as intrinsic apoptosis (with the efflux of cytochrome C)