Apoptosis, Autophagy, Necrosis Flashcards
two major pathways of apoptosis (programmed cell death)?
intrinsic: triggered by intracelular stress (oxidative damage, ischemia)
extrinsic: triggered by death ligand (tumor necrosis factor, TNF)
what is the basic progression of intrinsic pathway of apoptosis (naming molecules involved)?
- intracelular stress
- mitochondria cytochrome c
- APAF1
- caspase 9 (this step inhibited by Bcl-2)
- caspase 3
- apoptosis execution by DNA fragmentation
what is the basic progression of extrinsic apoptosis pathway? (naming molecules involved)
- extrinsic signal (death ligand such as TNF)
- FADD
- DISC formation
- caspase 8
- caspase 3
- apoptosis execution by DNA fragmentation
how do pro-apoptotic signals affect mitochondria in intrinsic apoptosis pathway?
pro-apoptotic stimuli increase permeability of outer membrane and promote mobilization of Cytochrome C (usually bound to cardiolipin, CL)
in cytosol, cytochrome C activates APAF1 and proteolytic maturation of caspase-9 and caspase-3
[reminder - cyt c functions as electron shuttle in ETC, but also functions as apoptosis signal]
how do Bcl-2 family proteins influence apoptosis in intrinsic pathway
B-cell lymphoma-2 (Bcl-2) superfamily:
pro-apototic: Bax, Bak - promote permeability of mitochondria and Cyt C release
anti-apoptotic: Bcl-2, Bcl-XL - bind Bax/Bak to inhibit
which Bcl-2 proteins are pro-apoptosis, which are anti-apoptosis?
Bax and Bak are PRO-apoptosis
Bel-2 and Bcl-XL are ANTI-apoptosis and bind/inhibit Bax/Bak
how is Bcl-2 implicated in cancer?
in follicular lymphoma, a chromosomal translation commonly occurs which leads to high levels of Bcl-2 expression
this causes decreased propensity to undergo apoptosis —> increased cancer growth
Bcl-2 overexpression linked to poor disease outcomes in several cancers
how does p53 function to prevent cancer formation? give 3 ways
initiates apoptosis (blocks anti-apoptosis Bcl-2 and Bcl-XL while promoting pro-apoteosis Bak/Bax)
activates DNA repair proteins
arrests growth by holding cell cycle at G1/S checkpoint
how does APAF1 function in intrinsic pathway of apoptosis
APAF1 = apoptotic protease activation factor 1
cytochrome C (released from mito) activates APAF1 by displacing it from CARD (CAspase Recruitment Domain)
CARD domains form cluster of 7 —> apoptosome
*apoptosome cleaves procaspase 9 to form caspase 9 (initiator caspase), which activates cas3/6/7 (executioner caspases)
the molecular “hitmen” are
caspases - enzymes that give rise to morphological and biochemical changes arising form apoptosis
must be CLEAVED to be active
regulated at post-translational level so they can be rapidly activated
group these into apoptosis initiators and effectors:
caspase
2, 3, 6, 7, 8, 9, 10
caspase initiators: 2, 8, 9, 10
caspase effectors: 3, 6, 7
what are the pro-apoptotic receptors in the extrinsic pathway? what do they bind?
DR4 and DR5
bind endogenous Apo2L (apoptosis-inducing ligand 2) and TRAIL (TNF-related apoptosis-inducing ligand)
after activation, what do DR4 and DR5 recruit? (extrinsic apoptosis) What steps occur next?
recruit FADD: Fas-associated death domain
FADD recruits Caspase 8 and/or 10 (initiator caspases) to the DISC (death-inducing signaling complex)
cas-8 and 10 are released into cytoplasm, activate effectors (cas3/6/7)
what are the ligands of each of these TNF-family death receptors of extrinsic apoptotic pathway?
a. FAS
b. DR5
c. TNFR1
d. DR3
FAS binds FasL (Fas ligand)
DR5 binds TRAIL/Apo2L
TNFR1 binds TNFalpha
DR3 binds Apo3L
T/F: extrinsic pathway stimulates apoptosis independently of p53 and mitochondria
TRUE
fill in the blanks of extrinsic apoptotic pathway:
1. ligands bind _____
2. ______
3. caspases _____
4. caspases _____
- ligands bind DEATH RECEPTORS
- FADD/DISC
- caspases 8, 10 (initiators)
- caspases 3, 6, 7 (effectors)
where do extrinsic and intrinsic apoptotic pathways converge?
activation of effector caspases 3, 6, 7
what is a distinctive feature of DNA degradation by caspase-activated DNase(CAD) when detecting apoptosis?
DNA laddering - biochemical marker of apoptosis
what are 2 biochemical markers of apoptosis?
- DNA laddering
- caspase activation (can be measured via expression levels) - must be cleaved to become active
how can TUNEL assay be used to detect apoptosis
TUNEL = Terminal Transferase dUTP Nick-End Labeling)
measurement of DNA fragmentation
TdT (terminal deoxynucleotidyl transferase) adds dNTPs to 3’ end of DNA molecules in absence of a template
in imaging, apoptotic cells appear as blebs
macroautophagy vs. microautophagy vs. chaperone-mediated autophagy
macroautophagy: cytosolic components are sequester within double membrane organelle or autophagosome, which fuses with lysosome (forms autolysosome) for degradation
microautophagy: lysosomes DIRECTLY engulf cytosolic components via invagination
chaperone-mediated autophagy: chaperone protein and cytosolic protein form complex that is recognized by LAMP-2A which translocates it into lysosome
what recognition protein is required for chaperone-mediated autophagy
LAMP-2A: translocates chaperone protein/ cytosolic protein complex to lysosome
4 stages of autophagy
- induction
- autophagosome formation
- autophagosome-lysosome fusion
- autophagosome breakdown
autophagy is inhibited by ___ conditions and stimulated by ____ conditions
starvation STIMULATES autophagy
nutrient-rich conditions INHIBIT autophagy
a key regulator of autophagy induction (step 1) is ____
mTORC1 (mammalian target of rapamycin complex 1)
*pharmacological inhibition of mTOR leads to downstream phosphorylation events which ACTIVATES autophagy-related genes
a new drug is developed that inhibits mTOR. What is the effect of this drug?
mTORC1 is a key regulator of autophagy induction (inhibits)
inhibiting mTOR leads to downstream dephosphorylation and ACTIVATION of autophagy-related genes
inhibit mTOR —> activate autophagy
starvation also inhibits mTORC1 (starvation stimulates autophagy
what occurs during autophagosome formation (step 2 of autophagy) - be specific
formation of a membrane around the targeted portion of the cell
LC3 maturation and lipidation are required
what proteins mediate autophagosome-lysosome fusion?
SNAREs: syntaxin17, SNAP-29, VAMP8
the ____ is the garbage truck of autophagy, the ____ is the dump site
autophagosome = garbage truck, delivers garbage to
lysosome = dump site (incinerator)
what are biological markers of autophagy events and clearance, respectively?
LC3 lipidation - marker of autophagy events
LOW levels of p62 - marker of autophagy clearance
when is autophagy in neuro-degeneration protective?
neuro-degenerative disease cause impaired clearance of protein aggregates
autophagy tries to compensate
5 types of necrosis
- coagulation necrosis - ischemia
- liquefactive necrosis - cell destruction leading to escape of hydrolases
- enzymatic fat necrosis - escape of lipases
- caseous necrosis - bacterial liquefaction
- gangrenous necrosis - ischemic + bacterial liquefaction
what are the morphological features of necrosis? (describe early and late)
early necrosis: cell swelling, chromatin digestion, disruption of membranes
late necrosis: extensive DNA hydrolysis, vacuolation of endoplasmic reticular, organelle breakdown, cell lysis
release of intracelular content after rupture causes inflammation
Which of these is correct? Fix the false statement or make it correct.
a. Proteins which regulate release of pro-apoptotic factors ALL contain BH3 domain
b. Cytochrome C interacts with APAF-1 to form the apoptotic body
TRUE: proteins that regulate pro-apoptotic factors all contain BH3 domain
statement b fixed:
Cytochrome C interacts with APAF-1 to form the APOPTOSOME
Are any of these statements false? If so, which ones?
a. The bcl-2 gene translocation decreases apoptosis
b. Proteins which regulate release of pro-apoptotic factors all contain BH3 domain
c. p53-mediated activation of Bax and Bak leads to release of cytochrome c from the mitochondria
they’re all true yay!
which is true of the TUNEL assay?
a. used for detecting apoptotic bodies
b. common method for detecting autophagosome formation
c. measures DNA fragmentation
d. a biochemical marker for apoptosis
e. used to demonstrate chromatin condensation
TUNEL Assay MEASURES DNA FRAGMENTATION
Which of these is TRUE?
a. mTORC1 can induce autophagy
b. autophagosome breakdown requires lysosomal enzymes such as cathepsin A
c. autophagy impairment is associated with reduced apoptosis
TRUE: b. autophagosome breakdown requires lysosomal enzymes such as cathepsin A
