8: Necrosis and Apoptosis 3 Flashcards
extrinsic pathway over view: what happens
death ligand binds to death receptors - adaptors, disc formation, caspase 8 activation, and then caspase 3 execution pathway.
intrinsic pathway overview: what happens
radiation, toxins, hypoxia, etc. cause mitochondrial changes. formation of apoptosome. caspase 9 activation, then caspase 3 activation.
caspase independent pathway: through what? normally located?
AIF: apoptosis inducing factor. AIF normally located in intermembrane space of mitochondria
caspase independent pathway: overview of events
cell receives signal to die, for ex. ROS. AIF release from mit. migrates to cell nucleus, binds to and triggers destruction of DNA and cell death.
misfolded proteins produced when? they trigger?
constantly. trigger a protective stress response aka UPR: unfolded protein response.
prolonged ER stress and UPR activation result in? misfolded proteins can also?
result in activation of cell death pathways. misfolded proteins also aggregate and interact with chaperones and transcription factors.
what proteins play a key role in communication between ER and mitochondria? which specific ones?
BCl2 family: Bcl2, Bcl XL, BAX, BAK, B1-1 and BIK
other ER stress proteins
BAP31, p53 dependent products NOXA and PUMA
ER stress is coupled to?
specific independent pathways, and intrinsic and extrinsic apoptotic pathways
caspases stand for? how many types?
cysteine dependent aspartate specific proteases. 14 different caspases in mammals.
caspases: synthesized as? activated by?
inactiv procaspases. all (except the initial caspase in a cascade) are activated by proteolytic cleavage by another caspase
sequence of events in caspase activation
prodomain cleaved off, capsase divided into small and large subunits. cleaved subunits associated to form active capsase
which caspases asoociated with inflamation? apoptosis?
inflam: 1, 4, 5, 11 - 14. apoptosis: 3, 6, 7 are executioners. 2, 8, 9, 10 are initiators
6 caspase substrates
other capsases. DNase. regulators of survival pathways. regulators of apoptosis like Bcl2, bclXL, XIAP, Bid. kinases like AKT, Raf1. structural proteins like laminin and actin.
apoptosis vs. necrosis: dislocated how? represent?
two processea re temporally dislocated, likely represent two extremes of a continuum.
necrosis process can start only and exclusively when?
the cell dies, and is an irreversible process = no return
A vs. N: cell size and membrane?
A: shrinkage and fragmentation, membrane is blebbed by integrity maintained. N: cell swells, smoothing and lysis of membrane.
A vs. N: mitochondria?
A: increased membrane permeability but structurally ok. N: swells and disordered structure
A vs. N: nuclei and DNA degradation?
A: chromatin clumped and fragmented, DNA degraded regularly every 180 bp, DNA appears in cytosol. N: nuclei swells, membrane disrupted, diffuse and random degradation of DNA
A vs. N: process?
A: DNA-programmed cascade that requires protein synthesis, RNA transcription and ATP. N: no protein synthesis, RNA transcription, is energy independent and see ATP depletion
A vs. N: inducing stimuli?
A: developmental programs and disease processes. N: disease processes
5 assays for apoptosis
cytomorphological changes. DNA fragmentation. detection of caspases, cleaved substrates, regulators and inhibitors. membrane alterations. mitochondrial assays.
tunnel staining: stands for? does what? problem?
terminal deoxynucleotidyl transferase mediated dUTP nick end labeling. labels fragmented ends of DNA. detects apoptosis, but DNA will break in necrosis too.
how to measure DNA fragmentation
gel electophoresis: compare with DNA ladder to see it if breaking every 180 bp
gel electrophoresis: what do you see in A vs. N
A: “laddering”: while N you just see a diffuse band near the top (larger size) - no trace of laddering and overall smaller DNA signal
diseases associated with inhibition of apoptosis? increase apoptosis?
inhibition: cancer, autoimmune disorders, viral infections. increased: neurodegenerative disorders, ischemic injuries, AIDS, toxin induced liver disease (alcohol)
