Lecture 3: Necrosis and Autophagy Flashcards
What is necrosis?
Necrosis is the unregulated cell death.
• It is caused by factors external to the cell or tissue such as toxins or trauma.
• Necrotic death is prolonged. It doesn’t depend on apoptotic CED-3 caspase.
• Necrosis is assisted by the same engulfment machinery in C. elegans but different ones in mammals.
• Necrosis can cause a chain reaction. Release of lysozymes can damage its neighbours, which in turn undergo necrotic cell death.
How does apoptosis compare to necrosis?
Apoptosis and necrosis are more of a spectrum. Cells can undergo apoptosis, apoptosis like PCD, necrosis like PCD or necrosis. Necrosis like PCD is necroptosis or inflammation.
• Active vs passive
• Fast vs slow
• Tidy vs messy
• Irreversible vs reversible
• Cell and shrink vs expand
• Membrane blebbing vs membrane leaking
• Nuclear condensation vs organelles expand
• Mitochondrial MOMP vs PMT
• Caspase driven vs caspase independent
• Needs ATP vs ATP independent
• PS externalised vs lysosomal release
• Phagocytic engulfment vs delayed or no phagocytosis
• Non-inflammatory vs inflammatory (danger).
What is the calpain-cathepsin hypothesis for necrosis?
- Ion channel malfunction leads to excess calcium.
- Further release of calcium from stores like the ER leads to activation of calcium activated proteases called calpains.
- Calpain activity leads to lysozyme rupture.
- The rupture releases cathepsins (potent proteases active at low pH) and other lytic enzymes resulting in widespread damage.
What is necroptosis?
Necroptosis is programmed necrosis.
• It is caspase independent.
• It depends on RIP3 and RIP1 kinases.
• Inhibited by necrostatin which is an RIP3 inhibitor.
• Cells collapse rather than swell but membrane blebbing doesn’t occur.
• We don’t know the mechanisms.
• Caspase 8 inhibit necroptosis. It may sequester RIP1 and/or degrade RIP3.
What is autophagy?
Autophagy means self-eating. It allows the cell to recycle components.
• Triggered by nutrient depletion e.g. ATP or glucose or growth factor depletion.
• Can also be cause by imbalances such as excess vacuoles or membrane systems; or by some types of cellular damage like hypoxia or pathogen damage.
• Autophagy is characterised by the formation of double membrane vesicles (autophagosomes). They are the recycling bins of the cell.
• A phagophore engulfs the material for degradation. The autophagosome is completed.
• The lysozyme then fuses with the outer membrane of the autophagosome.
• The lysosomal enzymes degrade inner vesicle and its contents. The nutrients return to the cytoplasm.
• Autophagy can be used to remove developmental determinants. P granules are concentrated into the primordial germ cells and removed from somatic cells by autophagy. Persistence of P-granules in somatic cells allows efficient mutant screening for new autophagy factors.
• 32 Atg genes were found in S. cerevisiae. Most of them have homologues in vertebrate cells. They function in the same way. It’s highly conserved and it’s more ancient than apoptosis or necrosis.
• There are at least 3 more core autophagy proteins conserved in all animals, but absent from yeast. There are also differences reflecting different stress and nutritional responses.
What are some special types of autophagy?
There are specialised types of autophagy.
• Mitophagy is the removal of old or damaged mitochondria. It is activated by reactive oxygen species or hypoxia. The half life of mitochondria is about 10 days. Healthy mitochondria constantly import PINK1 which is then degraded at the inner membrane. The import depends on mitochondrial membrane potential. Depolarised mitochondria can’t import PINK1 which builds up on the outer membrane and recruits a ubiquitin ligase called parkin. Many membrane proteins are ubiquitinated which acts as a mitophagy signal. Mitophagy an also remove mt from RBCs and to take out paternal mt from a fertilised egg.
• Xenophagy is the removal of intracellular bacteria. P62 binds polyubiquinylated bacteria and the autophagy protein (LC3/Atg8). Captured bacteria are delivered to lysosomes and they’re degraded. Some bacteria can survive and exploit this niche.
• Reticulophagy is the removal of excess ER. After drug overdose, the liver cell smooth ER (site of detoxification) becomes hypertrophied. It occupies most of the cytoplasm. The massive increase in SER is reversible.
• Pexophagy is the removal of excess peroxisomes by specialised autophagy.
How does autophagy work on a molecular level?
There are multiple proteins involved in autophagy.
• ATG1 kinase is a key control point. It is normally inhibited.
• When inhibition stops ATG13 and ATG`7 are phosphorylated. An active ATG1/ATG13/ATG17 complex forms. This complex translocated to the ER.
• ATG6 (beclin) normally binds to Bcl-2. However, ATG6 binds to ATG14, Vps15 and PtdIns3K to form a complex. This is vesicle sorting related.
• These two complexes target other ATG proteins to the preautophagosome structure (PAS).
• Atg12 is changed by Atf7 (an E1 ubiquitin activating enzyme).
• Atg12-Atg10 become E2 which is a ubiquitin conjugating enzyme.
• Atg12 then covalently forms a bond with Atg5.
• Atg12-Atg5-Atg16 forms a complex (E3 ubiquitin ligase).
• These form oligomers which are the recruited to phagophore outer surfaces.
• This complex promotes Atg8 being conjugated to phosphatidylethanolamine to form Atg8-PE.
• Atg8 may determine phagophore size. Atg8 is removed from the outer surface of the mature autophagosome by protease Atg4.
• Fusion with the lysozyme then becomes possible. It’s mediated by Atg14 interaction with t-SNARE complex.
• Autophagy requires substantial membrane recruitment. Atg9 integral membrane protein may shuttle membrane from other vesicles like ER and mt during phagophore expansion.
• Atg18/WIPI recruits and assists Atg9 membrane shuttling.
How is autophagy induced?
Autophagy must integrate multiple signals.
• TORC1 (target of rapamycin complex I) inhibits autophagy. Rapamycin induces autophagy.
• Rheb-GTP activates mTROC1. Rheb G-protein means Ras homologue enriched in brain.
• mTORC1 inhibits ATG1.
• Insulin and amino acid pools activate Rheb-GTP.
• AMPK inhibits Rheb-GTP and activates ATG1. AMPK is activated by low ATP/AMP.
• PKA inhibits ATG1 and ATG8. PKA is paradoxically activated by low glucose.
• Hypoxia activates BNIP3, which displaces Bcl-2.
• ER stress is caused by the unfolded protein response. It leads to JNK and other kinases activated beclin.
