Lecture 6 - Apoptosis Flashcards
(34 cards)
Apoptosis: what is it, what is it typically caused by, and how easy is it to stop?
An evolutionarily conserved mechanism to remove unwanted or damaged cells while avoiding collateral damage to the surrounding cells
Caspase-driven cell death occurs by limited proteolysis of specific substrates
Genetic deletion of individual caspase substrates does not prevent cell death - they can prevent some of the specific phenotypes observed, such as DNA fragmentation (CAD) or blebbing (gelsolin), but the cells still die
Caspases: what are they, what do they do, and what does their deletion result in?
Tightly controlled proteases that drive apoptosis and other cellular processes through quick and efficient modification of proteins
Genetic deletion of individual caspase substrates does not prevent cell death - they can prevent some of the specific phenotypes observed, such as DNA fragmentation (CAD) or blebbing (gelsolin), but the cells still die
Apoptosis: what does it aim to do?
- Kill only the right cells (robust regulation)
- Kill cells quickly (rapid activation)
- Dispose of the remains of the cells (generation of signals)
Role of cell death in homeostasis
Millions of cells are made per second (3.8), and so millions of cells must also die per second to maintain homeostasis
Reasons why cells may die:
* Reach the end of their natural life
* Become damaged or acquire mutations (UV irradiation, ROS, etc)
* Become stressed by toxins
* Can be infected and killed by the immune system
Loss of correct regulation of cell death may lead to:
* Excess cell death following ischemia/reperfusion
* Degenerative conditions
* Persistence of mutated cells (cancer)
* Survival of virus-infected cells
Types of cell death
- Necrosis
- Apoptosis
- Autophagy
Role of apoptosis during development
Genetically controlled process that removes redundant cells - we don’t have webbed feet or hands because the cells that would cause webbing are destroyed by apoptosis
Example in other species - C. elegans has 1090 cells in its adult form, and 131 cells die during development (always the same 131 cells)
Membrane blebbing: what is it, what is it driven by, and what is the general mechanism behind it?
Modification of the cytoskeleton to form membrane blebs that can be phagocytosed
Substrates controlling actin dynamics
Cleavage activates PAK
Cleavage of ROCK1 by caspase 3 removes its autoinhibitory domain, resulting in constitutive inhibition of myosin phosphatase, and increased acto-myosin contractility, using up ATP to drive membrane blebbing
Gelsolin: what does it do, and what may be its role in membrane blebbing?
Regulates actin depolymerisation
Cleavage results in a constitutively active N-terminal actin cleaving fragment that drives membrane blebbing
Caspases: what are they, what do they do, how do they do this, and what is the exact mechanism?
Caspase-driven cell death occurs by limited proteolysis of specific substrates
Caspases cleave iCAD to form CAD ,and then CAD cleaves internucleosomal DNA between the histones
Different peptidases and their functions: endopeptidases and cysteine proteases
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- Cleave internal peptide bonds
- Cysteine provides the catalytic nucleophile
Phagocyte recognition: how is it caspase-dependent?
Caspase 3/7 cleave inactive XRP8 to active XRP8 which causes lipid scrambling in the membrane which results in phosphatidylserine (a lipid only present in the inner membrane of a healthy cell) to the outside acting as a signal for phagocytes
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Caspase cascade example
Signal - receptor - initial caspase - executioner caspase - biological substrate
Signal - receptor - caspase 9 - caspase 3 - caspase 7 - biological substrates (CAD)
Executioner caspases: what are they, what are they activated by, and what are the examples?
Dimers that are endopeptidases and cysteine proteases
Activated by proteolytic cleavage by initiator caspases and/or the mitochondria
Caspase 3, 6, and 7
Executioner caspase activation: what is the molecular mechanism behind it?
Dimer before and after cleavage - cleavage results purely in a conformational change to reveal the active site
H237, C285, and R341 (histidine, cysteine, and arginine) are cleaved, and they rearrange to form the active site
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Caspase-substrate binding: what conditions are involved
- The binding of at least 5 amino acids around the scissile bond (R-CO-NH-R trans configuration) in an extended conformation
- An invariant aspartate at P1
- Other specificity-determining residues flanking aspartate
Caspase-mediated cleaving
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IAPs: what are they, what are their domains, and what do they do?
Inhibitor of apoptosis proteins
BIR domain - CARD - RING E3 ligase
Recognise activated caspases that are inappropriately activated and deactivate them
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E3 ligases: what are they, what do they do, and what are some examples, and what signalling pathways are they involved in?
Enzymes that form protein scaffolds to allow for binding of molecules used in signalling pathways
- XIAP (X-linked inhibitor of apoptosis protein) - used in the NLR signalling pathway
- cIAP1 (cellular inhibitor of apoptosis 1) - used in the NLR signalling pathway
- cIAP2 (cellular inhibitor of apoptosis 2) - used in the NLR signalling pathway
- Riplet - used in the TLR signalling pathway
- TRIM25 (Tripartite Motif Containing 25) - used in the TLR signalling pathway
Granzymes
Induce cell death through either CAD or BID pathways
(i)CAD: what is it, what pathway is it involved in, and what does its activation result in?
(Inhibitor of) caspase-activated DNase
Part of the pro-caspase-3 pathway
CAD production, causing cleavage of DNA, damaging DNA and inducing cell death
BID: what is it, what is it produced by, and what is the molecular mechanism behind its activation?
BH3 Interacting Domain Death Agonist
Granzymes induce its production
- BID cleaved by granzymes
- Truncated BID disrupts mitochondrial outer membrane
- Cytochrome C released
- Caspase 9 activated
- DNA cleavage - cell death induced
XIAP: what is its mechanism of action?
Bind to executioner caspases (ie 3) and inhibit them through ubiquitination to deactivate them through proteasome degradation
Caspase activation: how is it watched in real-time?
FRET sensors based on recognition sequences allow real-time activation to be followed
Caspase 1
Inflammatory caspase:
- Cleaves pro-IL-1β into IL-1β
- Cleaves pro-IL-18 into IL-18
- Cleaves Gasdermin D, which then enters the plasma membrane and creates a pore for the exit of IL-1β and IL-18 as well as promoting pyroptosis
- Pyroptosis is a type of programmed cell death that is characterized by inflammation and cell lysis, triggered by inflammasomes(lytic form of cell death)
