lecture 6 Flashcards
apoptosis
what does apoptosis do
physiological method to eliminate unwanted/unnecessary cells
example of apoptosis
during development, paws/hands have extra tissue which is webbing between digits; to get rid of it is apoptosis is triggered
define apoptosis
controlled, polite cell death
define necrosis
bad; uncontrolled cell death
what happens in necrosis
cells burst, release intracellular contents into extracellular space (which is bad because harmful enzymes spill out into space which is dangerous and can trigger more necrotic death)
what else can necrosis trigger
can trigger immune response by promoting inflammation; white blood cells infiltrate which is damaging to normal tissues
what happens in apoptosis
cells don’t burst and release; they are broken into small components so they dissasemble
describe apoptosis (how regulated it is)
highly regulated; everything in cell remains contained in barrier; b/c job of machinery is to make sure stuff remains where its supposed to be
how does stuff get taken away in apoptosis
when broken into chunks, things like macrophages come in and phagocytose, clean up dead cell, make sure tissue operates as healthily as possible
irreversible injury
cell can no longer function, structural integrity is disrupted
what happens in irreversible injruy
cell needs to die; prefers to do so through apoptosis
what happens in normal cell injury
cells usually bounce back; thru homeostasis they upregulate or downregulate certain genes
when does necrosis happen
if you have a massive injury, happens very quickly, not enough time for cells to undergo apoptosis
what are signs of reversible cell injury
reduced oxphos (E production) and depletion of ATP –> sign that mitochondria is injured
physical injury –> cell swelling
morphological changes to cell as injuries accumulate –> ER, mitochondria, cytoskeleton change function and appearance
what’s the end result if these things are allowed to progress
cell death
how can u tell necrosis happens
cell burst
how can u tell apoptosis happened
cellular fragmentation, not bursting
what does apoptosis depend on / what molecular mechanisms allow it to occur
depends on intracellular proteolytic cascade mediated by caspases
important mediator of apoptotic process
caspase
what happens when apoptosis is triggered
apoptotic signal –> caspase becomes active, initiate apoptotic signaling cascade resulting in morphological changes like cell fragmentation & packing into smaller pieces for engulfment by macrophages
what are caspases
proteases
what happens when caspases are activated
bind and cleave other proteins within cytoplasm
what is caspase / how does it transmit signal
caspase is a signaling protein, transmits signals by cleaving downstream proteins
how are caspases activated
cleave themselvse
describe inactivated caspases
monomers floating around in cytoplasm; intact, full length
what happens when cell decides to undergo apoptosis
apoptotic signal arrives, signal is gonna assemble an adaptor complex –> a dimer
what is the adaptor complex
2 identical dimers that can each bind and recruit a caspase to the complex
what is the job of adaptor
to bring caspases close enough together so they can cleave each otehr
what is this cleavage analagous to
trans autophosphorylation
what happens after the dimerization event hapens
proteins enzymatic activity gets close enough to act on their neighbors (in RTKs thats a phosphate group added) (in caspases, one cleaves the other)
what does cleavage do
initiates activation
what is this cleavage process called, and where is it in
cross cleavage; common to all mechanisms of caspase activation
what happens once cleaved
active caspase dimers are released, labeled as mature active caspases
what do mature active caspases do
cleave other proteins downstream in apoptotic pathway
initiator caspases
caspases that are initially activated; these are caspases that get things going
what do initiator caspases do
start process in response to upstream signal; go and activate executioner caspases next
executioner caspases
go and cleave all downstream targets needed for apoptosis to occur
what’s on top of next step of pathway
active initiator caspase that’s been cleaveed
what do active initiator caspases do next
goes and cleaves executioner caspases to activate them
how are executioners different from initiator caspases
executioners are already dimerized, but inactive
why don’t executioners do cross cleavage
if they did, they wouldn’t be able to exist as inactive dimers b/c they would just cleave each other
who cleaves executioner caspases
initiator caspases
what happens when initiator caspases cleave executioner caspases
similar rearrangement; executioners go and cleave multiple types of cell proteins
what does this process of cleavage by executioners result in
process of apoptosis
how many pathways needed for apoptosis to occur
MANY; we are just looking at one specific oen
how many targets do executioners have
multiple
how much does one set of initiator caspase activate
multiple dimers of executioner caspase; not a one to one
what does each downstream step represent
amplification where one upstream protein can activate multiple copies of downstream proteins amplifying signal
describe example pathway of what an executioner caspase is gonna do
dna cleaved into regular sized fragments
what is one hallmark of apoptosis
dna structure of cells undergoing apoptosis is cleaved in regular sized fragments
what does this dna cleaving allow you to determine
help determine when cell undergoes apoptosis vs necrossi
what is the end result of this specific pathway
it cuts DNA b/w histones [histones are regularly spaced in genomic DNA, cuts regularly spaced, so you get spaced out DNA ladders]
what happens if this dna cleavage pathway happens in healthy cells
NEVER want this to happen; cells will die if you cleave DNA ; hella rightly regulated
what are key players in executioner caspases controlling cleavage of DNA
CAD proteins, caspase activated DNase
what is activated by executioner caspase
CAD
what is CAD activity
DNase activity; cuts DNA
what happens when there’s no apoptosis
CAD is inhibited by the inhibitor iCAD –> held in place
what does active executioner caspase do to CAD
it cleaves iCAD or inhibitor of CAD; CAD is ready
what happens when iCAD / CAD inhibitor is degraded
CAD is released and becomes active
what can CAD do when its active
cleaves DNA between histones, resulting in regular DNA pattern
what is net result of inhibiting the inhibitor
activation of protein
what is the normal resting state
it’s held in inactive inhibited state; to activate you inhibit the inhibitor
what does CAD degrade
degrades DNA between histones to begin orderly destruction of nuclear contents
what does CAD convert
converts genomic DNA into ladder of regularly sized pieces (experimental hallmark of cells undergoing apoptosis)
why is immune system good at triggering apoptosis
immune cells kill cancer cells; T cells kill tumor cells; tumor cells have ways to evade apoptotic signals, so immune system needs to get good at triggering apoptosis
two pathways of apoptosis triggered by immune cells
extrinsic and intrinsic pathway
extrinsic pathway
cell receives apoptosis signal from outside / external environment
example scenario of extrinsic pathway
imagine a virus infected cell that’s gonna be taken out by T cell
what are key players in extrinsic pathway
caspase 8, adaptor protein: FADD, Fas death receptors
what is caspase 8
initiator caspase of extrinsic apoptotic pathway
what are adaptor proteins
bring inactive initiator caspases together so they can close cleave each other to become activated
what is specific adaptor protein in extrinsic apoptosis
FADD protein
what is the other key thing in extrinsic apoptosis
needs to be a receptor involved since signal is coming from outside; receptor need to be able to recognize signal to transmit info across hydrophobic barrier of plasma membrane
specific receptor in extrinsic pathway
Fas death receptor
describe receptors in cell when it’s normal and not undergoing extrinsic apoptosis
death receptors at surface; inactive, not bound
describe adaptor proteins in cell when it’s normal and not undergoing extrinsic apoptosis
adaptor proteins in cytoplasm; folded up & inactive, not bringing anything together
describe caspases in cell when it’s normal and not undergoing extrinsic apoptosis
caspase 8 is monomer, floating around cytoplasm, inactive
what comes from top
signal; killer lymphocyte or killer T cell
job of killer T cell
go in and trigger death of cells (cancer cells, infected or damaged cells, etc.)
how does killer T cell trigger death
expresses fast ligand on its surface –> contact dependent signaling
describe fast ligand
ligand is a trimer; binds to 3 Fas death receptors
what does fast ligand bind to
Fas death receptors
what happens once fast ligand activates fas death receptors
can now bind and unfold FADD adaptor proteins
what happens when FADD adaptor proteins are unfolded
they are active can bind to initiator caspases
what do adaptor proteins do
bring caspases close enough together so they can cleave each other to activate
what happens when caspase 8s are cleaved
release as dimers (caspase-8 dimer), go and activate executioner caspases
what are executioner caspases in extrinsic pathways
caspases 3 and 7
what happens after to killer T cell
job is done; gonna go and find other affected cells
general features that show up in apoptosis pathways
adaptor proteins, cross-cleavage, initiator caspases activate downstream executioners
what do adaptor proteins do
bring initiator caspases close enough together for cross cleavage
what is specific to extrinsic pathway
caspase 8, trimers of receptors, signal comes from T cell, specific FADD adaptor proteins
where does extrinsic pathway occur
contained within cell
what is intrinsic pathway
even more polite way to die; cell decides for itself to die for betterment of group
what is central regulatory node of intrinsic apoptotic pathway
mitochondria
what happens to mitochondria in intrinsic pathway
mitochondria can’t function as efficiently, ATP production drops, cell homeostasis goes out the window
what happens if ATP production drops
cell is metabolically not active enough and dies
what is key factor cell is gonna listen to in inactive pathway
when ATP can’t be produced in enough abundance
what does intrinsic pathway of apoptosis depend on
mitochondria
how do cells know mitochondria isn’t working properly
if mitochondria is leaky
what happens if mitochondria membrane is leaky
proton gradient of oxphos is gonna be destroyed –> ATP production is gonna crater; apoptosis
what should mitochondrial membrane be for regular cell
intact
how does cell know when mitochondria membrane is leaky
cytochrome c
what’s up w/ cytochrome c in healthy mitochondria
cytochrome C always held within mitochondria; never exposed to cytoplasm [good thing; membrane is intact and fully producing ATP]
how is mitochondria damaged
UV radiation, triggers oxygen radicals that damage membranes and make them leaky
where is cytochromes not supposed to be
in cytoplasm (green)
what is trigger for intrinsic apoptotic pathway
cytochromes in cytoplasm (supposed to be in membrane)
describe damaged mitochondria in signaling pathway
cytochrome c released into cytoplasm
what detects cytochrome c in cytoplasm
adaptor protein Apaf1
job of adaptor protein
assemble initiator caspases so they are close enough to cleave each other
what does Apaf1 do
binds cytochrome c
what does Apaf1-cytochrome c binding do
triggers conformational change in adaptor that allows it to assemble into oligomerized form
what does cytochrome c do (important)
cytochrome c binds one copy of adaptor, triggers conformational change that allows it to form higher order oligomer
what happens once oligomer is formed
recruits initiator caspases
what is caspase for intrinsic pathway
caspase 9
what happens after cross-cleaving (intrinsic)
activated caspase 9 dimers are released, cleave & activate executioner caspases & trigger apoptosis
what happens in some situations w/ intrinsic pathway (in diagram apoptotic stimulus pointing at mitochondria)
some signaling pathways purposefully cause release of cytochrome c (even tho mitochondria is healthy)
what pathway can trigger intrinsic pathway
PI 3-kinase pathway AKA survival signaling pathway
what regulatory proteins have the job of controlling if cytochrome c is released or not from mitochondria
Bcl2 family (Bcl2, Bak, Bad)
how do Bcl2 family of proteins regulate intrinsic apoptosis
by controlling mitochondrial permeability
3 varieties of Bcl2 fam
anti-apoptotic fam proteins, 2 pro-apoptotic fam proteins
name the anti-apoptotic fam protein
Bcl2
name the pro-apoptotic proteins
Bak and Bad
what do these apoptotic proteins do
control permeability of mitochondria in response to upstream signals
what happens when Bak is activated
cell is gonna wanna undergo apoptosis (b/c it’s pro apoptotic)
where is Bak found
outermembrane of mitochondria
describe inactive Bak
exists as monomers in outer membrane of mitochondria
describe inactive Bak again
membrane is intact, no cytochrome c is leaking out, bak is inactive
what does bak directly control
permeability of outermembrane
what happens when apoptotic stimulus acts on Bak
activation of Bak, it clusters into an oligomer that forms a channel thru which cytochrome c floods cytoplasm
what does Bak channel cytochrome cytoplasm trigger
Apaf1 adaptor protein complex, caspase 9 activation, all steps necessary for intrinsic apoptosis
what about Bcl2
anti-apoptitic; when active it sits in outer membrane of mitochondria
what does Bcl2 do
Bcl2 binds Bak and blocks cytochrome channels from forming
what is Bcl2 similar to
safety mechanism; when safety is engaged, Bak can never fire
what happens when you take out Bcl2
remove safety, when trigger is pulled Bak makes channels –> apoptosis
basically what does Bak do
pro-apoptotic; forms channels and increases permeability of membrane
basically what does Bcl2 do
anti-apoptotic; blocks Bak and formation of channel
what is 3rd apoptotic regulator protein
Bad; pro-apoptotic
what does active Bad do
inhibits Bcl2
what does Bad inhibiting Bcl2
inhibits inhibitor of Bak; causes activation of Bak and apoptosis
what does PI 3-kinase signaling pathway
prevents apoptosis
what does trans autophosphorylation of RTK provide
docking site for PI 3-kinase
what does PI 3 kinase do
phosphorylates PI (4,5)P2 into PI(3,4,5)P3; which provides docking site in plasma membrane for downstream kinases PDK1 and AKt
what does PDK1 do
helps activate Akt
what happens when AKt is activated
released from plasma membrane, goes into cytoplasm
what does Akt phosphorylate
Bad
what does Bad phosphorylation do
releases active apoptosis inhibitory protein
what happens when Bad is phosphorylated
can’t inhibit the inhibitor Bcl2 (meaning Bcl2 is free to inhibit apoptosis)
what happens to Bcl2 when PI 3 Kinase pathway is active
Bcl2 can bind to Bak and prevent cytochrome channel from forming (cytochrome can’t go into cytoplasm)
describe PI 3 kinase pathway
survival pathway; if in right env. cells don’t need to go in apoptosis
what happens if survival signal for PI-3-kinase pathway is withdrawn or cell is in wrong place and needs to self-destruct
Bad is gonna be de-phosphorylated, can inhibit the inhibitor Bcl2 and allow Bak to trigger apoptosis (thru cytochrome channel formation)
what does this survival signaling pathway regulate
intrinsic mechanism of apoptosis
when do cells stay alive
when pathway is active
why do we need survival signaling
we have more neurons early in dev. than as adult; more cells than the survival factor can keep alive
describe abundance of survival signal
kinda limited; some cells are gonna die so it leaves cells that remain to enjoy benefits of limiting survival signal
what causes apoptosis via PI3-kinase mechanism acting on regulation of intrinsic apoptotic pathway
apoptosis adjusts number of nerve cells to size of target; more nerve cells than survival factors released by target cells
