Lecture 1 - cell death: apoptosis Flashcards
Ion concentrations: internal and external concentrations of sodium (Na⁺) in a eukaryotic cell
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
Ion concentrations: internal and external concentrations of potassium (K⁺) in a eukaryotic cell
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
Ion concentrations: internal and external concentrations of calcium (Ca²⁺) in a eukaryotic cell
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
Ion concentrations: internal and external concentrations of chloride (Cl⁻) in a eukaryotic cell
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
Ion concentrations: internal and external concentrations of hydrogen (H⁺) in a eukaryotic cell
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
Ion concentrations: internal and external concentrations of magnesium (Mg²⁺) in a eukaryotic cell
Extremely low (1mM) internal concentrations for use in enzymes, DNA, and other cellular processes; however, concentration is even lower (0.5mM)
Death receptor (DR4): what is it, what can it do, and what possible treatments can it be used for?
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
Three types of cell death
Necrosis, apoptosis, and autophagy
Necrosis
- Swelling of cell
- Loss of plasma membrane integrity
- Release of contents into surrounding tissue
Apoptosis: what is its process and why does it happen?
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
Autophagy
- Maintenance of plasma membrane integrity
- Organelles are broken down and reused as nutrients
What is a part just as important as cell death within cell death?
Releasing signals that affect how the body responds
How are biological effects stimulated?
Signal -> receptor -> enzyme -> enzyme -> biological effect
How are transcriptional effects stimulated?
Signal -> receptor -> kinase -> kinase -> biological effect
Can be reversed - phosphatases remove phosphates added by kinases
How are biological substrates cleaved?
Signal -> receptor -> proteinases -> cleaved proteinases -> cleaved biological substrate
Can be reversed - phosphatases remove phosphates added by kinases
What proteins control apoptosis?
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
CAD: what is it, what is it bound to, and what does it do?
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 …
How does the apoptotic cell tell macrophages it needs to be destroyed?
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
iPLA2: what is it, what does it cleave, and what does it do?
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
Xkr8: what is it, what does it do, and what does it do?
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
Capsases: what are the two types and how are they naturally expressed?
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
How are caspases activated?
Signal -> receptor -> initiator caspase -> cleaved executioner caspase -> cleaved biological substrate
Main initiator/executioner caspases
Caspase-9
Caspase-3 and caspase-7
Proenzyme executioner caspase structure
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
Proenzyme initiator caspase: structure, caspase-9 concentration and dimerisation kd, and its two large pro-domains.
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
APAF-1: what is it, what does it bind to to activate it, and what does it do?
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
What does the apoptosome do?
Binds Caspase-9 by the CARD, causing high local concentration of Caspase-9, resulting in dimerisation and activation
Cytochrome c: where is it obtained from, what does it usually do
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
Bcl-2: what is it, what does it do and
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)
Bax what is it, what does it do and
Bcl-2 associated X proteins
Antagonises Bcl-2 and promotes cell death
Bcl-2 family: the types, what they do, what the examples are, and their similarities?
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)
What factors affect BH3 activation and what types of oncogenic mutations interact with BH3?
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
P53: what is it, how is it activated, what is its role in cancer, and what difficulties does its mutation have in cancer treatments?
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.
How was a drug devised to kill cancer cells?
- 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 (??)
Overall apoptosis process
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
