Lecture 1 - cell death: apoptosis

Question 1

Ion concentrations: internal and external concentrations of sodium (Na⁺) in a eukaryotic cell

Answer 1

Relatively low (1-2mM) internal concentrations due to the Na⁺/K⁺ pump, sodium is used to maintain resting membrane potential and nerve impulse transmission; much higher (145mM) external concentrations which are used in depolarisation

Question 2

Ion concentrations: internal and external concentrations of potassium (K⁺) in a eukaryotic cell

Answer 2

Extremely high (150mM) internal concentrations for use in cell excitability, resting membrane potential, and muscle contraction; external concentrations are much lower (5mM) for use in repolarisation in muscle and nerve cells

Question 3

Ion concentrations: internal and external concentrations of calcium (Ca²⁺) in a eukaryotic cell

Answer 3

Extremely low (0.1µM) internal concentrations for use in muscle contraction, enzyme activation, and signal transduction pathways, etc; external concentration is extremely larger (1-2mM) and is brought into the cell when needed

Question 4

Ion concentrations: internal and external concentrations of chloride (Cl⁻) in a eukaryotic cell

Answer 4

Relatively low (5-10mM) internal concentrations to maintain cell volume and pH; external concentration is much higher (125mM) and is used to maintain a gradient for chloride channels and maintain osmotic balance

Question 5

Ion concentrations: internal and external concentrations of hydrogen (H⁺) in a eukaryotic cell

Answer 5

Extremely low (0.01x10⁻⁷M) internal concentrations to keep a pH of 7 for cellular and enzyme activities; concentration can vary outside of the cell for altering pH

Question 6

Ion concentrations: internal and external concentrations of magnesium (Mg²⁺) in a eukaryotic cell

Answer 6

Extremely low (1mM) internal concentrations for use in enzymes, DNA, and other cellular processes; however, concentration is even lower (0.5mM)

Question 7

Death receptor (DR4): what is it, what can it do, and what possible treatments can it be used for?

Answer 7

Potential targets for anti-cancer drugs as they are involved in apoptosis pathways and can be targeted to induce apoptosis in cancer cells

Potential therapeutic approach in cancer treatment

Question 8

Three types of cell death

Answer 8

Necrosis, apoptosis, and autophagy

Question 9

Necrosis

Answer 9

• Swelling of cell
• Loss of plasma membrane integrity
• Release of contents into surrounding tissue

Question 10

Apoptosis: what is its process and why does it happen?

Answer 10

ATP-dependent death:
* Cell shrinkage
* Cytoskeleton collapses
* Loss of nuclear membrane
* Chromatin condenses and DNA is cleaved into fragments
* Membrane blebs which break off into apoptotic bodies
* Cell surface alters to attract phagocytes
* These changes require energy – apoptosis uses ATP

It protects from infected cells, damaged cells, or unwanted cells
Apoptosis will minimise collateral damage to the tissue

Question 11

Autophagy

Answer 11

• Maintenance of plasma membrane integrity
• Organelles are broken down and reused as nutrients

Question 12

What is a part just as important as cell death within cell death?

Answer 12

Releasing signals that affect how the body responds

Question 13

How are biological effects stimulated?

Answer 13

Signal -> receptor -> enzyme -> enzyme -> biological effect

Question 14

How are transcriptional effects stimulated?

Answer 14

Signal -> receptor -> kinase -> kinase -> biological effect

Can be reversed - phosphatases remove phosphates added by kinases

Question 15

How are biological substrates cleaved?

Answer 15

Signal -> receptor -> proteinases -> cleaved proteinases -> cleaved biological substrate

Can be reversed - phosphatases remove phosphates added by kinases

Question 16

What proteins control apoptosis?

Answer 16

The key signalling enzymes controlling apoptosis are caspases (endopeptidases)

Cysteinyl ASPartate proteinASES cleave substrates at the aspartate - cleaving after an aspartate is unusual - often resulting in function activation

Question 17

CAD: what is it, what is it bound to, and what does it do?

Answer 17

Caspase-activated DNAase

Often bound with ICAD (Inhibitory caspase-activated DNAase)

When ICAD is cleaved, CAD is released and dimerises with another CAD and cleaves DNA at the histones, resulting in a …

Question 18

How does the apoptotic cell tell macrophages it needs to be destroyed?

Answer 18

Caspase-3 cleaves both iPLA2 and Xkr8, causing them to activate and destroy the cell

iPLA2 is cleaved (turned off?? on??) and cleaves phosphatidylcholine and sends lysophosphatidylcholine from the membrane to recruit macrophages

Xkr8 gets cleaved (turned on) and then causes phosphatidylserine to be flipped in the membrane, becoming a macrophage receptor

Question 19

iPLA2: what is it, what does it cleave, and what does it do?

Answer 19

Phospholipase A2

When iPLA2 is cleaved (turned off?? on??), it cleaves phosphatidylcholine and sends lysophosphatidylcholine from the membrane to recruit macrophages

This causes macrophages to move towards the cell to destroy it

Question 20

Xkr8: what is it, what does it do, and what does it do?

Answer 20

A membrane-bound enzyme

Xkr8 gets cleaved (turned on) and then causes phosphatidylserine, a phospholipid only found in the inner membrane normally, to be flipped in the membrane, becoming a macrophage receptor

This allows macrophages to detect the cell and destroy it

Question 21

Capsases: what are the two types and how are they naturally expressed?

Answer 21

Executioner caspases: small subunit, large subunit, small pro-domain

Initiator caspases: small subunit, large subunit, large pro-domain

Enough in the body to kill every cell, usually expressed as inactive proenzymes - activated during apoptosis

Question 22

How are caspases activated?

Answer 22

Signal -> receptor -> initiator caspase -> cleaved executioner caspase -> cleaved biological substrate

Question 23

Main initiator/executioner caspases

Answer 23

Caspase-9

Caspase-3 and caspase-7

Question 24

Proenzyme executioner caspase structure

Answer 24

Proenzyme executioner caspases are dimers that
contain a loop which means it is inactive but cleavage by initiator caspases causes rearrangement of the active site - activation

Question 25

Proenzyme initiator caspase: structure, caspase-9 concentration and dimerisation kd, and its two large pro-domains.

Answer 25

Monomers activated by induced dimerisation * Caspase-9 concentration of caspase 9 ~ 20 nM * Kd for dimerisation ~ 50 µM - cytosolic caspase 9 is an inactive monomer The large pro-domain is used in the activation mechanism DED - Death Effector Domain CARD - CAspase Recruitment Domain

Question 26

APAF-1: what is it, what does it bind to to activate it, and what does it do?

Answer 26

Apoptotic Proteinase Activating Factor is usually folded, preventing its CARD from binding to and activating Caspase-9's CARD, but when it's bound by cytochrome c, it unfolds and forms a heptamer (7 subunit structure) - an apoptosome

Question 27

What does the apoptosome do?

Answer 27

Binds Caspase-9 by the CARD, causing high local concentration of Caspase-9, resulting in dimerisation and activation

Question 28

Cytochrome c: where is it obtained from, what does it usually do

Answer 28

The mitochondria The electron transport chain - transporting electrons between complex III and IV Its effect in ETC and apoptosome formation are distinct: APAF-1 gene mutation does not affect respiration

Question 29

Bcl-2: what is it, what does it do and

Answer 29

B cell lymphoma gene 2 Reciprocal translocation between chromosomes (14;18) The Bcl-2 family of proteins controls apoptosis by mediated mitochondrial outer membrane permeabilization (MOMP) Keeps cells alive - problem in cancer (cells not killed)

Question 30

Bax what is it, what does it do and

Answer 30

Bcl-2 associated X proteins Antagonises Bcl-2 and promotes cell death

Question 31

Bcl-2 family: the types, what they do, what the examples are, and their similarities?

Answer 31

Is Bcl-2 the family name?? Is there another name? Apoptotic - make holes in the outer mitochondrial membrane: Bax, Bak, and Bok Anti-apoptotic - block OMM permeabilisation: Bcl-2, Bcl-XL, Bcl-W, and Mcl-1 BH3-only proteins - either inhibit the anti-apoptotic proteins or activate the pro-apoptotic: PUMA, Bik, Bim, Bad, and Bid The A and AA types contain the same structure except the anti-apoptotic type contains a part of the domain called BH4 while BH3-only... (Contains only BH3... duh)

Question 32

What factors affect BH3 activation and what types of oncogenic mutations interact with BH3?

Answer 32

Growth regulators, cell damage, DNA damage, In cancers, if there is an excess stimulant, BH3 proteins won't be activated enough to promote cell death If lymphoma cells have expressed excess A and AA proteins, then the BH3 effect is diminished as there are too many A and AA proteins to regulate

Question 33

P53: what is it, how is it activated, what is its role in cancer, and what difficulties does its mutation have in cancer treatments?

Answer 33

A tumour suppressor that is a transcription factor, upregulating BH3 production Activated as a response to cell damage Mutated in 50% of cancers - Chemotherapy drugs given to cancer patients try to activate BH3-only proteins in order to kill cancer cells but if p53 is mutated, the tumour cells don’t respond.

Question 34

How was a drug devised to kill cancer cells?

Answer 34

* NMR spectroscopy identified small molecules that bound in the same region as the BH3-domain * The low-affinity molecules were combined in a larger drug to form a high-affinity binding drug * Drug was tested and improved lots and finally administered - killing cancer cells * Venetolax (??)

Question 35

Overall apoptosis process

Answer 35

Signal -> Bcl-2 sensor -> Bcl-2 protein -> Bcl-2 effector -> APAF-1 activation -> Caspase-9 activation -> Caspase-3/6/7 activation -> iPLA2 and Xkr8 activation -> macrophage recruitment -> cell death