Cell Death (Unit 7) Flashcards
(41 cards)
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
