Cell Death Flashcards
what are the four cell death pathways?
- apoptosis
- necroptosis
- pyroptosis
- ferroptosis
cell death in disease
-too little: abnormal development and cancer where the division > death
-too much: neurodegeneration, HIV/AIDS, and normal aging
discovering cell death
-growth factors required for neurons to live
-lysosomes- LMP- organelle in cell that kills proteins
-BCL-2- B cell lymphoma- first oncogene discovered and keeps cells alive
-caspase- protease responsible for death –> cells that die require caspase
programming/regulated cell death (PCD/RCD)
-developing, normal physiological processes and type of regulated cell death (any kind of gene-dependent death)
-if a cell contributes to its own death –> programmed
what happens generally when a cell decides to die?
cell has to decide to die –> amplify death signal –> death effector
does the dying cell actively contribute to its own death?
-no- accidental and could be instant death or murder
-yes- programmed or regulated cell death that is cell autonomous and cell suicide
“apoptosis” based on cell morphology
-healthy cell then ligation of portal vein –> we see necrotic death then cells destroyed themselves by blebbing together and being engulfed
-apoptosis is non-inflammatory compared to necrosis
classic apoptotic morphology
cell on the right has been blebbed together and nuclei have been broken up
how did we map the apoptosis pathway?
-WT c. elegans
-131 cells die during development and ran screen for worms that didn’t have any death –> turned out to be those that had mutant in caspase
caspase nomenclature
-“c”- cysteine amino acid at the active site
-“asp”- cleaves its substrates after aspartate/asp/D
-“ase”- standard for enzymes
-14 mammalian caspases –> only 3, 8, and 9 are involved in apoptosis
apoptosis pathway
BH3 –| BCL-2 –| BAX –> MOMP produces cytochrome C –> Aparf1 apoptosome –> caspase 3 –> execution (no lysis)
discovery of the first human apoptosis regulator- BCL-2
-go to patients whose tumors had classic chromosomes translocations and clone out genes next to the breakpoint –> you’ll find new oncogenes
-BCL-2- doesn’t behave like a regular oncogene since it’s an inhibitor of cell death
BCL-2 homology motifs (BH)
-BH3- facilitates interaction between pro- and anti-death in BCL-2 family membranes
-“BH3-only” proteins- mostly unrelated to the BCL-2 family
-BH3 is required for cell death activity of BCL-2 homolog BAX and BH3-only proteins
BCL-2 is intact and likely overexpressed by strong enhancer- BCL-2 proteins have deep groove b/c that’s where the BH3 interacts
BH3 memetics occupy the same groove –> led to generation of venetoclax that sits in BCL-2
BCL-2 proteins regulate mammalian apoptosis (intrinsic pathway)
-BCL-2, BCLX, and BCL1- anti-death proteins have homologs that are pro-apoptotic
–> BCL-2 and BAX are opponents
-BH3-only proteins have several stress signals- can activate death proteins and inhibit anti-death proteins –> lynch pins of pathway
-if BAX wins, it goes to the mitochondria and oligomerizes on outer mitochondrial membrane and pokes a hole and releases cytochrome C from IM space
-cytochrome C serves as co-factor to drive assembly of apoptosome (Aparf1 apoptosome) –> recruits caspase 9 then cleaves and activates caspase 3 –> makes key cuts in cells that makes them look morphologically apoptotic
what happens in a mouse without apoptosis?
- mouse took out all three of the BH3-only proteins and those animals have webs between their toes
- KO all three of the pro-death proteins (BAX/bak/bok) and mouse has too many neurons
- make a version of cytochrome C- only works in the ETC
- KO components of the apoptosis path like Apaf-1, caspase 9, and caspase 3 –> brain has a lot of neurons
extrinsic and intrinsic apoptosis pathways converge on caspase 3
-TNFRI binds to its ligand RIPK 1/3 and activates caspase 8, which activates caspase 3
-extrinsic- caspase 8 and intrinsic (mitochondrial)- caspase 9 –> both amplify the death signal, leading to downstram caspase 3
-caspase 3 chops up several substrates in the cell
caspase-3 causes apoptotic morphology and death
-activates other caspases
-inactivates Rb
-cleaves things that make cells recognized by their neighboring cells
how do you detect apoptosis?
-cytochrome C tagged with GFP and phosphatidylserine (eat me) signal flips from inner to outer leaf of PM
-DNA turns blue when the cell membrane becomes permeable (apoptosis –> necrosis)
TUNEL assay labels the ends of DNA tha are cleaved up when they cut by an enzyme that gets activated through caspase pathway into these nucleosomes
-specific for apoptosis? yes if you’re watching cells, they have apoptopic morphology and they expose phosphotidylserine but for necrotic cell since you have fragmented DNA –> yes or no depends on how you read the gel
we don’t have that many proteins that inhibit caspases
- mimic caspase 8
- caspase inhibitor XIAP with loop that sits in the active site of caspase
what would be a possible treatment strategy for leukemia patient with a tumor overexpressing BCL-2?
XIAP inhibitor- inhibit anti-death protein
necroptosis
-you don’t see apoptotic cell death bodies and they eventually rupture
-begins with death receptor from extrinsic apoptosis that leads to caspase 8 then 3 and apoptosis
-TNF-alpha has a pro-survival fnction- activates NFKB and other signaling pathways –> also plays a role in necroptosis
-crosstalk downstream of receptor from apoptotic pathway via caspase 8, which can inhibit necroptosis
-RIPK1 activates RIPK3- when kinase amplifies and makes huge complex, it activates pore-forming protein (MLKL) and goes up to the plasma membrane from inside the cell and pokes a hole
what treatment would cause patients with IBD to get worse?
caspase-8 inhibitor –> now you can’t use caspase 8 to cut up RIPK1
pyroptosis
-inflammatory pathway
-we get bombarded by all kinds of microbes and pathogens –> need DAMPs, PAMPs, and commensals and we have to distinguish these from our own microbiome
-proteins activated by signals and oligomerize into machine (inflamosome) that activates caspase 1 (non-apoptotic)
-caspase 1 cleaves gasdermins that are inside cells and have to be activated by cleavage from caspase 1 to form channel that inserts in PM
-channel is how we get pro-inflammatory cytokines out of the cell like IOIBeta
-all three pathways so far have effector at the end
ferroptosis
-does not have a pore former
-pro-survival mechanisms
-cells need glutamine that they pump out to bring Cysteine, which they turn into Cystine –> need glutathione for reducing selenocysteine, not put into proteins in usual manner
-handful of proteins that have selenocysteine and one of these is caspase 12 and gluthathione peroxidase four (GPX4)
-selenocysteine is encoded by a stop codon
-need glutathione peroxidase b/c it’s an enzyme that fixes our lipids
-regulated death b/c it occurs in a specific way
which gene makes the blue cells divide/grow faster?
-cMYC- oncogene that primarily acts as TF driving cell proliferation (classic oncogene) vs BCL-2 is anti-death oncogene
–> cMYC
which gene leads to more blue cells although both cells grow at the same rate?
BCL-2- anti-death gene
which gene(s) could be elevated in blue Myc-expresssing cells, which divide faster, but have equal cell numbers?
RIPK and MLKL
where did cell growth originally come from?
-PCD/RCD in unicellular species –> do all cells undergo regulated cell death?
-took 100 flasks and inoculated them with yeast and neglected them
-at first you get a lot of cells then they run out of food then some of the flasks recovered and the rest died
-how did some survive? those that were willing to die before they ran out of food might be the ones that ultimately survive
-repeated with death resistant strain and all the flasks used up all the media and everyone died
-what’s the death pathway? not known
-experiment with yeast suggested that PCD/RCD may benefit the population
bacterial and fungal gasdermins reveal ancient mechanism of cell death and innate immunity
-gasdermins is protein for pyropoptosis pathway
-bacteria have a gasdermin that you can’t find by usual sequence gazing
-do they have anything to do with cell death? when bacteria get infected by bacteriophage that is ultimately going to kill them, those first bacteria activate suicidal death using gasdermins and prevents phage from finishing its replication cycle and spreading
-viruses make counter attacks
