Topic 8 - Apoptosis Flashcards
What is Apoptosis?
- programmed cell death (but not the only kind - necrosis: premature death of cells in living tissue by autolysis)
- greek word for “fallin off”
On what organism was apoptosis reintroduced as a legitimate pathway
- idea proposed in the 70s, only 20 years later was it agreed upon based on C. elegans study
What is the difference between Apoptosis and Necrosis
Necrosis: swelling, bursting, mess from inflammatory response
Apoptosis: shrink and condense - cytoskeleton collapses - nuclear envelope disassembles - chromatin condenses - Blebs (apoptotic bodies) - Macrophages engulf these Blebs
When is programmed cell death critical?
- during early development; when unwanted structures are eliminated
- further regulates cell numbers and does a quality control
- ex. frogs lose their tails, their fingers reshape
The purpose of Apoptosis is to…
- eliminate abnormal cells; assisted by the immune system by checking for cells with self antigens
What does rapid turn over refer to?
- the rapid response to an infection leads to more cells produced that can be supported by survival factors – a challenge in cancer and infections
What is the TUNEL Technique?
- apoptosis can be biochemically recognizable via observing fragmented DNA through gel electrophoresis
- Tdt-mediated dUTP nick end labelling
- this refers to knicking DNA at linker regions between nucleosomes
- in this technique the Terminal Deoxynucleotidyl Transferase (Tdt adds labelled dUTP to 3’ -OH of DNA
How does phosphatidyl serine function with apoptosis?
- this PS is typically on the cytoplasmic side of the membrane
- during apoptosis though, it will flip to the extracellular side to function as a signalling molecule to macrophages
What is unique about the PS apoptotic method?
- PS-dependent phagocytosis inhibits the production of inflammation inducing signals (cytokines) by the phagocytic cell
- it provides a signal for macrophages who do not respond to self antigens
What role does mitochondria play in apoptosis?
- during apoptosis the mitochondria will loose the electron potential across the inner membrane (the site for cytochrome C)
- this cause the relocation of cytochrome C enzyme from the intermembrane space to the CYTOSOL
How can you identify the location of cytochrome C to determine the status of the cell?
- a cytochrome-C-GFP and a mitochondrial Stain will co-localize the position of the cytochromce
Is cytochrome c released slowly over time or rapidly?
- Cytochrome C is released rapidly within 5 minutes
- it precedes flipping by the phosphatidyl serine
What are the Caspases?
- family of proteases with a cysteine in their active sites which cleave specific aspartic acid AA***
- they are synthesized as Pro-caspases which require proteolytic cleavage performed by other caspases to activate themselves
What intracellular machinery is responsible for apoptosis?
- apoptosis machinery is always present in the cell but in the INactive form
- a family of proteases called Caspases with cysteine in their active sites but cleaving the target at the aspartic acids
What components form the caspase?
- adapter binding domain
- protease domain with cleavage sites
- require the activation from an adapter protein
Who are the initiator Caspases? how are they activated?
- Caspase 2,8,9,10
- an external signal will lead to the formation of the procaspases into activation complexes by cleaving themselves
Who do Caspase 2, 8, 9, 10 activate via cleavage?
- Executioner Caspases are: Caspase 3,6,7
- these will perform as Caspase Cascade = Death Signal
What are the targets of the Executioner Caspases?
- nuclear laminas
- cytoskeletal components
- cell-cell adhesion proteins
- DNA endonuclease (cleaves the regions between nucleosomes)
What sort of cascade is the Caspase Cascade?
- Caspase cascade is irreversible
- it amplifies the proteolytic cascade
What is the Extrinsic apoptotic pathway?
- The apoptotic pathway induced by the immune system
- a killer lymphocyte displaying Fas Ligand will bind the Fas Death Receptor displayed on its membrane
Explain the Fas Ligand/Receptor, the DISC and how the immune system regulated this process?
- Fas Receptors on the infected cell forms homotrimers
- while FasL is part of the TNFalpha signalling protein family (Tumour Necrosis factor)
- in the target cell the DISC complex will form (Death-Inducing-Signalling-Complex) consisting of the: 1. Death domain FADD (Fas-Assocciated-Death-Domain) 2. Adapter domain 3. Death effector domain (Caspase 8)
What is an interesting point about the Death receptors?
- they can also trigger NON-apoptotic pathways which would lead to a survival and inflammation response
Who are the decoy receptors or FLIPs?
- the decoy receptors have ligand binding domain but no death domain
- the FLIP: is the intracellular blocking protein which resembles the initiator procaspase 8 but lack any proteolytic activity - so it functions as a competitior
When would an intrinsic apoptotic pathway be initiated?
- in response to stress, lack of oxygen, or injury to the cell
- for mammals response is dependent on the release of mitochrondrial intermembrane proteins
- Cytochrome C binds and activates the apoptotic protease activatin Factor 1
Who is ApaF?
- Apoptotic Protease Activating Factor 1
- when cytochrome C is released into the cytosol it will bind to Apaf-1
- the active Apaf-1 contains CARD (Caspase-Activating-Recruitment-Domain)
- this will initiate the formation of the apoptosome and recruitment of Caspase 9
- the Apoptosome will activate the Caspase 9 which cleaves the nearby executioner caspases
Apoptosome?
- activated by CARD (Caspase activation recruitment domain) on Apaf-1 (apoptotic protease activating factor-1)
Which protein family regulates intrinsic apoptosis? who form what?
Bcl2 proteins - heterodimers
- B cell Lymphoma
Who are the anti-apoptotic Bcl2 family proteins?
Bcl2, BclXL
Who are the pro-apoptotic Bcl2 family proteins?
- Bax, Bak
Who are the pro-apoptotic BH3-only proteins?
- Bim, Bad, Bid, Puma, Noxa
The balance between what determines if a cell lives or dies?
- pro-apoptotic and anti-apoptotic activity
The two pro-apoptotic Bcl2 protein regulators?
- apart of the BH123 family
Bax: cytosolic and translocates into the mitochondrial membrane with an apoptotic signal
Bak: tightly bound to the outer mitochondrial membrane ALWAYS
How are Bak and Bax activated?
- they are dependent on the being activated by the pro-apoptotic BH3 only (Bad, Bim, Bid)
- these 3 link apoptotic activity to stimulate intrinsic apoptosis
Who is apart of the anti-apoptotic Bcl2 family? and what do they do?
Bcl2 and BclXL who both site on the mitochondrial membrane
- these two will inhibit the BH123 family (Bax, Bak)
- they preserve membrane integrity, prevent release of mitochondrial proteins & release Ca2+ from the ER
Who does the intrisic pathway proceed?
- requires the anti-apoptotic Bcl2 proteins to be inactivated by the inhibitors from the BH3 only Bcl2family
The BH3-only proteins function in what way?
- Bim, Bid, Bad directly interact with Bax and Bak to assist in their activation
- Bim: JNK (MAP kinase) activation leads to intrinsic pathway
- Puma and Noxa: after DNA damage, after increased p53, puma and Noxa are transcribed and intrinsic pathway is activated
- Bid: Caspase 8 will cleave Bid, tBid will inhibit the Bcl2 and triggers apoptosis via BH123
- Bad and Bim: form heterodimers with anti-apoptotic Bcl2 and Bcl-XL for pro-apoptotic activity
BH3 only proteins regulate the intrinsic pathway
- If no survival signal received, a MAP kinase cascade will turn on BH3-Bim for apoptosis
- the transcription of genes encoding BH3 only proteins Puma and Noxa will trigger the intrinsic pathway
- The extrinsic pathway can activate the intrinsic pathway - active Caspase8 cleaves Bid, where Bid translocates to the outer mitochondrial membrane where it inhibits anti-apoptotic Bcl2
A triple knockout of Puma, Puma Bim, and Puma Bim Bid compared to a Wildtype examined what?
- the Wild Type had normal Caspase 3 activity while reduced
What are the IAPs?
- the Inhibitors of Apoptosis
- who bind and inhibit activated caspases by setting a threshold (some may poly-ubiquitylate caspases)
- these IAPs set an inhibitory threshold that must be overcome before apoptosis can be active again
Who are the Anti-IAPs?
- these IAPs will translocate the IAP’s to block the anti-apoptotic threshold
What are the Three Survival Factors?
A) Increased production of the Anti-Apoptotic Bcl2 Proteins - will block oligomerization thus blocking apoptosis
B) Inactivation of pro-apoptotic BH3-oly Bcl2 proteins - this will inhibit Bad: which will release active Bcl2 which prevents oligmerization blocking apoptosis
C) the inactivation of the anti-IAP: the last kinase will phosphorylate HID via an anti-IAP which inhibits IAP
