Cell Death (Unit 7) Flashcards
Why are caspases zymogens
Zymogens are proteins with inactive enzymes
Capases need to be activated to work
What’s one way that caspases activate apoptosis
Inactivation of flippase and activation of scramblase
(both via cleavage)
How are initiator caspases activated?
Procaspases Brought into close proximity and then they permanently cleave eachother
What is meant by the fact that caspases cleave themselves?
They do autocatalysis and cleave other capases
What are the targets of executioner caspases?
Lamins, proteins that keep endonucleases inactive, cytolskletal compoents, cell-cell adhesion components
What binds to caspases to keep them inactive?
An adaptor protein (the prodomain)
Are caspases dimers or monomers?
Starts as one protein
Cleaved into large and small subunits to form heterodimer.
Active form consists of tetramer made from two heterodimers
What is the main way that all apop programs converge?
Caspase activation
What are caspases (based on name)
Proteases with a cysteine
Do caspases only do PCD?
Most do but some also do inflammation or immune response
How do caspases know where to cleave?
Look for signature tetrapeptide motif and cleave after the D (Asp)
What is caspase amplification?
One intiator caspase can activate multiple executioners
When can you no longer turn back apop?
Once executioners are activated
What is the apoptosome?
A wheel-like heptamer that activates initiator caspases
How is the apoptosome formed?
Released CytC (and dATP) binds Apaf1 to induce a shape change which frees its oligomerization domain and its CARD
7 active Apaf1s oligomerize into a wheel with CARD at the middle
It then recruits initiator caspases and activates them
Then, the initatior caspases(now on the wheel) activate executioners
What starts the instrinsic pathway?
Release of proteins meant to be in IMS
Release of CytC
What can the intrinsic activation pathway do to help apop?
It can help to amplify the extrinsic pathway
What 3 things can result in triggering the instrinsic pathway?
Hypoxia
DNA damage
Starvation
How can we do a DNA clevage visualization via ladder on gel electrophoresis (?)
The ladder will have characteristic cuts bc CAD (caspase-activated endonuclease) cleaves DNA in linker regions between nucleosomes
1)Incubate cells with apop agent
2) at diff time points, take out an aliquot and extract DNA
3)Run on agarose gel
4) stain with ethidium bromide to see the ladder
How can we visualize apop by looking at the # of DNA ends?
DNA ends are 3’ OH and 5’ phosphate
Use TUNEL+ to add labelled dUTP to the 3’OH
More fluorescene = more ends
(is this using FRAC???)
How can Annexin 5 be used to visualize apop?
Conjugate it with fluorescence and watch it bind PS after it moves to the outer leaflet
What are some ways to see apop?
-characteristic ladder
-Label DNA ends
-Annexin 5 labelling exposed PS
-loss of mito membrane potential
-release of cytC
Necrotic Cell Death vs Apop
Apop: Programmed, built-in and tightly regulated
Necrosis: Uncontrolled, parts spill everywhere, results in inflammation which then causes damage [happens bc of damage or toxins]
Why should cell death and division be balanced
Too much death : Autoimmunity, neurodegeneration
Too little death: Linked with Cancer
Too much Division: Cancer
How does the extrinsic pathway work?
Must form DISC: Death-Inducing Signalling complex
Death receptors (TNF) on cell surface activated which exposes their death domain
FADD adaptor binds death domain which exposes its DED
Caspase binds with its adaptor proteins -> DISC now fully formed and initatior caspases are activated then released
Give an example of how to activate a cell’s death receptor
Fas ligand on Tc cell
How do cells avoid getting extracellularly apop activated
Fake death receptors with no death domain
how can death receptors activate non-apop intracellular signalling pathways
TNF activates NFkB which works on inflammatory response
What is BCL2 fam?
Share BCL2 domains
-control release of IMS proteins like CytC by binding and inhibiting pro-apop proteins
Which BCL2 fams are pro apop and which are counter?
Pro:
-Bcl2 fam effectors (BH1-3): Bax + Bak
-BH3 only: Bad
Counter:
-Bcl2 fam (BH1-4):
Bcl2 + BclxL
What is MOMP
Mito outer membrane permeabilization
Describe Bcl2 Family effectors (BH1-3)
-what activate them?
-what do they do?
Activated by apop stimuli but usually depends on BH3-only proteins
When active, they form dimers to make pores in OMM thus at least one is required for Apop
where do Bak and Bax come from?
Bak:tightly bound to OMM
Bax: translocates from cytosol
What do BcL2 Family proteins do?
Where do they tend to live and why?
Bind BH3 domain of Bax or Bak in cytsol or on OMM to stop them from forming a pore
Live on OMM (to stop improper IMS protein release) or on nucl or on ER (to stop improper Ca+ relase)
What does Bad do?
Binds BclxL to stop it from binding BcL2 family effector proteins
What are IAPs?
APop inhibiting factors
How are IAPs regulated?
Smac and Omi neutralize them
Apop is an ordered and predictable process that utilizes DNases and protease what are the steps?
Cytoskletal collaspe causes cell to shrink and condense
Nucl envelop dissembled
Chromatin condenses and fragments
Cell surface blebs
Cell breaks up into membrane-enclosed pieces (apoptotic bodies “corpses”)
Phagocytes recognize PS on cell surface and eat
Why do we have PCD?
-Development
-Quality control (kill non-functional or weird cells ex: lymphcoytes after infection, maturing B and T lymphocytes that fail their tests)
-Homeostasis (ex: need continous supply of fresh neutrophils)
What does p53 do?
promotes apop and stops cell cycle
What do survival factors
bind surival factor receptors to either INC bcl2 production (via activating transcription factor)
OR
Inactive BH3 (by activated a kinase that phophorylates it)
