Mechanisms of Disease II: Cell Damage and cell death

Question 1

What is the function of necrosis?

Answer 1

1. Removes damaged cells from an organism
2. Failure to do so may lead to chronic inflammation
3. Necrosis causes acute inflammation (to prevent further bigger inflammation) to clear cell debris via phagocytosis

Question 2

What are the causes of necrosis?

Answer 2

1. Usually lack of blood supply
   e.g. injury, infection, cancer, infarction, inflammation
2. As the distance away from the blood vessel increases, the pH drops and partial pressure of oxygen drops significantly

Question 3

What are the steps of necrosis?

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.
3. Lack of oxygen prevents ATP production.
4. Cells swell due to influx of water (ATP is required for ion pumps to work).(due to osmosis here)
5. Lysosomes rupture; enzymes degrade other organelles and nuclear material haphazardly
6. Cellular debris released, triggering inflammation

Question 4

What are some nuclear changes that occur in a cell during necrosis? (from a microscope view)

Answer 4

Nuclear Changes:

1. Chromatin condensation/shrinkage.
2. Fragmentation of nucleus.
3. Dissolution of the chromatin by DNAse.

Question 5

What are some cytoplasmic changes that occur in necrosis of a cell? (microscopic view)

Answer 5

1. Opacification: (cytoplasm becomes more white instead of a see-through watery colour) protein denaturation & aggregation.
2. Complete digestion of cells by enzymes causing cell to liquify (liquefactive necrosis).

Question 6

What are some biochemical changes that occur in necrosis of a cell?

Answer 6

1. Release of enzymes such as creatine kinase or lactate dehydrogenase
2. Release of other proteins such as myoglobin. These biochemical changes are useful in the clinic to measure the extent of tissue damage

Question 7

What is 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
- Endocrine-dependent tissue atrophy
- A variety of pathological conditions

Question 8

What happens in apoptosis?

Answer 8

1. Programmed cell death of one or a few cells.
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 (so a v clean way of disposing cellular content)

Question 9

What is a distinct feature in regards to number of cells involved in necrosis and apoptosis?

Answer 9

Necrosis - multiple cells at once
Apoptosis - very selective, usually one or so cells at a time

Question 10

What is a distinct feature in regards to reversibility in necrosis and apoptosis?

Answer 10

Necrosis - not every part/stage is irreversible Apoptosis - ALL events are irreversible and steps require ATP

Question 11

What cytoplasmic changes can be seen when cell apoptosis happens?

Answer 11

Cytoplasmic Changes:

1. Shrinkage of cell. Organelles packaged into membrane vesicles.
2. Cell fragmentation. Membrane bound vesicles bud off.
3. Phagocytosis of cell fragments by macrophage and adjacent cell.
4. No leakage of cytosolic components.

Question 12

What nuclear changes can be seen when cell apoptosis happens?

Answer 12

Nuclear Changes:

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

Question 13

What biochemical changes can be seen when cell apoptosis happens?

Answer 13

Biochemical changes:

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 14

What are some examples of things that cause apoptosis?

Answer 14

1. Cell death in embryonic hand to form individual fingers.
2. Apoptosis induced by growth factor deprivation (neuronal death from lack of NGF).
3. 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.
4. Cell death in tumours causing regression.
5. Cell death in viral diseases (ie viral hepatitis).
6. Cell death induced by cytotoxic T cells (ie. Cellular immune rejection or vs. host disease).
7. Death of neutrophils during an acute inflammatory response.
8. 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.

Question 15

What are caspases?

Answer 15

→ A family of proteases whose activation is central to all types of apoptosis

→ Caspases are the point of convergence for causes of apoptosis:
both intrinsic and extrinsic → caspases → apoptosis

→ Caspases are cysteine proteases (cysteine aspartate-specific proteases)

→ Caspases form an activation cascade, where one cleaves and activates the next (analogous to kinase cascades)

→ Types of caspases include: initiator and effector

Question 16

What are the 2 types of apoptosis?

Answer 16

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 the body)

→ Withdrawal of survival factors e.g. mitogens
→ Extracellular signals (e.g. TNF)
→ T cell or NK (Natural Killer) (e.g. Granzyme).

Question 17

What does caspase activation lead to?

Answer 17

Caspase activation leads to characteristic morphological changes, such as:

→ Shrinkage
→ Chromatin condensation
→ DNA fragmentation and plasma membrane blebbing.

Initiator caspases activate themselves when in close proximity

Activation, therefore, means bringing initiator caspases together

Question 18

How do we activate the initiator caspases?

Answer 18

Initiator caspases activate themselves when in close proximity by cleaving themselves

Activation, therefore, means bringing initiator caspases together

Question 19

What is extrinsic apoptosis induced by?

Answer 19

Induced by ligand binding to receptors, causing receptor dimerisation or multimerisation - which ultimately leads to activation of the caspases

Ligand-induced multimerization: Domains shared between proteins allow them to bind together

Question 20

How does ligand-induced multimerization work (use an example)?

Answer 20

You have 4 parts to the process:

a. the ligand itself
e.g. TNF

b. the receptor with two domains - ligand binding and death
e.g. TNFR

c. death adaptor (intermediate protein group) with two domains - death and death effector
e.g. FADD

d. procaspase-8 with two domains - death effector and protease

Procaspase-8 autoproteolysis to become caspase-8 and be released into cytoplasm from membrane to initiate the caspase cascade

These shared domains allow for binding because similar domains dimerise or multimerise. Formation of DISC.

Question 21

What is intrinsic apoptosis induced by? Are there any exceptions?

Answer 21

Induced by cytochrome c released from mitochondria

Note: growth factor withdrawal (extrinsic apoptosis) an exception that uses cytochrome C

Cytochrome C: Mitochondrial matrix protein Known for many years to be released in response to oxidative stress by a “permeability transition” Any inducers of the permeability transition also eventually induce apoptosis.

Question 22

What is cytochrome C?

Answer 22

→ Mitochondrial matrix protein

→ Known for many years to be released in response to oxidative stress by a “permeability transition”

→ Any inducers of the permeability transition also eventually induce apoptosis

Question 23

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

Answer 23

A PORE MADE OF BCL-2 FAMILY PROTEINS

Can be pro- or anti-APOPTOTIC

→ Anti-apoptotic: Repress cytochrome c release, leading cells to survive- cytochrome c therefore remains in the mitochondria

→ Pro-Apoptotic: Facilitate cytochrome c release
a. Some are not membrane proteins, therefore cytoplasmic
b. All have a BH3 domain used to form dimers

Question 24

BCL-2 family proteins examples

Answer 24

→ Anti-apoptotic:
BCL-2, BCL-XL

→ Pro-Apoptotic:
Bax, Bad, Bid

Question 25

IF BCL-2 FAMILY PROTEINS REGULATE CYTOCHROME C RELEASE FROM mitochondria, WHAT regulates BCL-2 PROTEINS? (not the only mechanism but the main one)

Answer 25

Gene expression (transcription) and post-transcriptional modifications (e.g. phosphorylation)

Example 1 - TP53:

1. DNA damage leads to transcription driven by TP53
2. The membrane of the cell begins to express more BAX (a pro-apoptotic protein) that creates a new pore for cytochrome c to enter the cell without the BCL-2 block leading to cell death

Example 2 - phosphorylation:

1. Loads of growth factor act as survival signals
2. AKT/PKB released and phosphorylate BAD
3. BAD usually displaces BLC-2 to allow in cytochrome C and lead to cell death
4. Therefore phosphorylated BAD can’t displace BCL-2 so there is no cell death

https://canvas.sgul.ac.uk/courses/2414/pages/mechanisms-of-disease-ii-cell-damage-and-cell-death-sdl?module_item_id=90324

Question 26

What is TP53?

Answer 26

TP53 is a transcriptional factor activated by DNA damage