Lecture 2: Mitochondria, Granzymes and Engulfment Flashcards
How do C. elegans and mammals compare?
- Triggers are mainly intrinsic for C. elegans, but both are used in mammals.
- EGL-1 is the activator in C. elegans while mammals use many.
- CED-9 is the Bcl-2 family protein in C. elegans while there are many different signals in mammals.
- Mitochondria play a minor role in C. elegans but a major role in mammals.
- CED-4 is used in C. elegans but Apaf-1 is used in mammals.
- CED-3 is used in C. elegans but many are used in mammals.
How does the Fas ligand pathway work?
FasL is a ligand which can induce apoptosis.
• FasL binds to the FasR death receptor.
• FasR trimerizes upon Fas binding.
• This recruits adaptors like the Fas-associated death domains (FADDs).
• FADD is used to recruit procaspase 8 as part of the death inducing signalling complex (DISC). FADD and procaspase 8 both have the death effector domain (DED) which can bind them together.
• Procaspase becomes activated into caspase 8 and it then activates procaspase 3.
• Caspase 8 also turns bid into t-Bid. t-Bid causes Bak/Bax channels to form and release cytochrome C.
• Cytochrome C binds to Apaf-1 to form the apoptosome.
• The apoptosome activates capase 9 which cleaves caspase 7 and caspase 3.
• There are multiple ways to activate caspase 3. If it goes wrong then cancer can occur.
What is the BCL-2 family?
The BCL-2 family has more then 25 members. There are pro-apoptotic and anti-apoptotic proteins.
• They share BCL-2 homology domains.
• The Bax family is pro-apoptotic. Bax and Bak lead to MOMP.
• The BH3 only family includes EGL-1. They are anti-anti-apoptotic.
• Bcl-2 by itself is anti-apoptotic. CED-9 is a homologue.
How does mitochondrial outer membrane permeabilization (MOMP) work? Which proteins are released?
The mitochondrial intermembrane space provides a safe storage space where apoptotic proteins can be sequestered and then rapidly released.
• Pro-apoptotic BCL-2 (bax and bak) proteins are used to permeabilize the membrane.
• Cytochrome C. Cytochrome C and Apaf-1 form the apoptosome. Apaf-1 has CARD (caspase recruitment domain) which resembles DED. A heptamer forms which can recruit procaspase9 and activate it.
• Smac (second mitochondria-derived activator of caspase)/DIABLO (direct IAP-Binding protein with low PI). They are both pro-apoptotic and targeted to the mitochondria. They inhibit IAPs.
• AIF (apoptosis inducing factor). It is a flavoprotein which binds FAD. It increases MOM permeability and enhances caspase action.
• Endonuclease G. Mitochondrion specific endonuclease. It assists in caspase-independent nuclear DNA fragmentation.
How do knockouts of mitochondrial proteins affect mice?
Cell deaths aren’t identical. It shows that there are redundant mechanisms so cells will be affected to different extents.
• Apaf-1: mice can sometimes survive to adulthood. They have a great excess of neurones.
• Cytochrome c: embryos survive to midgestation.
• Smac/DIABLO: mice are viable and normal. Cells have normal apoptosis.
• Endonuclease G: mice are viable and normal.
What is the role of p53 in MOMP?
p53 up-regulates transcription of pro-apoptotic Bax, Bid, Puma and Noxa.
• After DNA damage, 25% of p53 translocates from the nucleus to the mitochondrial membrane.
• We don’t know exactly how it works. It could bind and sequester Bcl-2 or bind and activate Bax and Bak.
• PUMA (p53 up-regulated modifier of Apoptosis) is upregulated by p53. It then dissociates p53-Bcl-xL complexes and liberates p53, so it can bind to Bax.
How does granzyme-triggered apoptosis work?
Killer cells release granzymes which can activate apoptosis.
• Perforins allow the movement of granzymes from a killer cell to the target cell. Perforin KO mice are severely immunodeficient and tumour-prone. The mechanism is still very controversial though.
• Serpins protect the killer cell by irreversible binding.
• Granzyme A activates caspase independent pathways such as histones.
• Granzyme B activates caspase 3 and 7.
How can ATP trigger apoptosis?
ATP is released excessively when spinal nerves are traumatically injured.
• This can trigger excess caspase-dependent apoptosis of adjacent neurones by activating the purinergic P2X7 receptor.
• Brilliant Blue G (food dye) is an antagonist of the receptor.
• Spinally injured rats can have reduced signs of damage if they are given high doses of BBG.
How does phagocytic engulfment work in C. elegans and mammals?
After apoptosis, the corpse must be disposed of by phagocytes.
• Corpses give the “eat me” signal. Cells lose membrane lipid asymmetry. Phosphatidylserine is exposed on the outside surface. Lipid flippase is inactivated and lipid scramblase is activated.
• Circulating proteins recognise PS. Annexin V binds to PS. Labelled Annexin V is used as a label for apoptotic cells both in vivo and in vitro.
• Apoptotic cells also emit signals like ATP and UTP which attract phagocytes.
• CED-7 is a membrane transporter protein that is needed in both the dying cell and the engulfing cell. Other proteins act only in the engulfing cell.
• Two engulfment pathways synergise. Class A uses proteins like ced-1 Class B uses ced-2.
• Single or double mutants within either class A or class B induce similar delays in engulfment. Double mutants of both classes give a much longer delay.
• CED-8 and its homologue Xkr8 are transmembrane proteins. They promote PS exposure after activation by caspase 3 or 7.
• AIF (vertebrate) is released from mt during apoptosis. They promote scramblase activity.
• Recombinant diannexin (annexin V dimer) prevents blood-cell induced apoptosis for transplants by masking PS.
How does membrane permeability transition occur?
Bax and Bak form channels which release cytochrome C. There is a limited initial release. Then mitochondrial permeability transition occurs (mPT). There is a subsequent massive re-structuring and release.
• The inner membrane becomes more permeable.
• Mitochondrial membrane potential is lost. This step isn’t essential. A major target for caspase 3 is complex I.
• mPT depends on adenine nuclear transporter (ANT 1/2), anion channel VDAC1 and cyclophilin D. Cyp D knockout mice have no mPT and exhibit normal apoptosis but abnormal necrosis.
