Section 2 - Cell Death Flashcards
cell death general method
-stress triggers (often oxidative stress)
-stress sensors (proteins)change their function and location upon chemical modification
Main players of cell death (6)
- stress triggers
- stress sensor proteins
- mitochondria
- Bcl2 family proteins
- Caspases
- multimeric protein complexes
mitochondria is _____ but is also the target of ______ (ex. _____)
-central sensing station
-many death-controlling mechanisms
-release of cytochrome C
Bcl2 family proteins are _____ of cell death. They control mitochondria via ______
-executors and regulators
-calcium and formation of pores
Caspases are ____ of cell death
gateways and executors
ROS lead to formation of ______, but mitochondrial permeability transition also ______.
-mitochondrial pores unrelated to PTPC
-triggers the same mechanism
Proteins that control ROS have a central function in ______
maintenance of mitochondrial functions and cell death
p66 does what?
increases ROS production by interfering with electron flow by holding on cytochrome C and cause “traffic jams”
AIF (______) acts as a ______ (like ____) and does what?
-Apoptosis Inducing Factor
-chaperone for OXPHOS
-C9Orf72
-induces DNA damage in the nucleus upon apoptosis onset
RNA interference vs. CRISPR-cas 9
RNAi is temporary, can be used to protein function
CRISPR is permanent, can observe cell adaptation
RNA interference summary
-short dsRNA cut by dicer to form siRNA
-RISC is assembled with siRNA
-RISC + siRNA binds to mRNA and cause mRNA degradation
RNAi knockdown of AIF induces a ____
DCF signal (increase ROS production)
AIF gene is on what chromosome? effect on males?
-X-chromosome
-mutation of AIF allele in males results in complete knockout
AIF is released from mitochondria after ___
apoptotic signal and cleavage
AIF function
AIF maintains normal amounts of mitochondrial complex I, by acting as a folding-scaffold during the import of inner mitochondrial membrane proteins
in absence of AIF, what happens?
lower amounts of complex I lead to electron traffic jam and ROS production
Upon mitochondrial dysfunction and prolonged oxidative stress, AIF becomes _____
a promoter of DNA damage in the nucleus
apoptosis: pro-apoptotic (death) signals do what?
-open channel (not PTPC) in mitochondria allows for cytochrome C (and Smac/DIABLO) release into cytoplasm
-cytochrome C binds to Apaf-1 then procaspase 9 (initiator
procaspase) which forms caspase 9 binds to from apoptosome
-apoptosome activates executioner procaspases 3,6,7 to form caspases 3,6,7 which cleaves death substrates
mitochondria is the central controller of ____ via ____
-apoptosis
-AIF, cytochrome C
Apoptosis triggers (7)
-DNA damage
-UV light
-Deprivation of growth factors
-Excess cytosolic and mitochondrial calcium
-ER stress
-chemicals that lead to any of the above conditions
-Death receptors (extrinsic apoptosis)
ER stress is ______
accumulation of misfolded/unfolded proteins
BH3-only proteins exists for ____ and do what?
-any stress trigger
-inhibits Bcl2 proteins from blocking mPTP to promote apoptosis
BH3-only proteins control the progression of cell death from specific stimuli such as DNA damage (_____), cytokine deprivation (_____), calcium influx to mitochondria (____) or ER stress from proteasome block (_____).
-Puma, Noxa
-Bad
-Bim
-Bik, Bim
Intrinsic cell death is under the control of Bcl2 family proteins, where ____ is anti-apoptotic, _____ are pro-apoptotic
-Bcl2
-Bax/Bak
in human tumor cells, anti-apoptotic proteins are _____ while pro-apoptotic proteins are _____
-amplified
-mostly retained though some are deleted
erastin is a molecule that ______ which is _____ and kills _____
-targets Ras
-often hyperactive in cancer cells
-cancer cells
Annexin V is what?
- a protein associated with PM that indicates apoptosis (apoptosis marker)
erastin does not cause ______
apoptotic cell death since it is not associated with caspases or Annexin V activation
DFO is what?
deferoxamine = iron chelator
DCF is what?
marker of ROS level in cell
C11-BODIPY (def.)
fluorescent probe against oxidized lipids
erastin kills cells, associated with ______
iron-dependent ROS production and lipid oxidation
in presence of erastin and the absence of Fe, what happens?
cells survive
erastin follows this death pathway: ____
ferroptosis
______, _______, and ______ protect from ROS, hence ferroptosis involves ______
-Glutathione peroxidases
-peroxiredoxins
-peroxisomal catalase
-their inhibition
GpX4 is ______
Glutathione peroxidase 4
RSL3 is what?
ferroptosis inducer; inhibits GpX4
Method for determining ferroptosis inhibitor
- Create GpX4 KO through tamoxifen treatment
- Transfection with cDNA library that rescues KO
- Identify rescuing cDNA (one of them is GpX4)
- Test for cell survival with ferroptosis inducer RSL3
- inhibitor blcoks cell death when over expressed in WT and KO
FSP-1 is what?
glutathione-independent ferroptosis suppressor that protects from ferroptosis
GpX4 KO cells with FSP-1 cDNA do what?
survive
FSP-1 does this to protect from ferroptosis
turns ubiquinone/a-tocopheryl (vitamin E) radical to ubiquinol/a-tocopherol which functions as a lipid antioxidant, preventing lipid peroxidation
selenium and cystine do what in ferroptosis?
-selenium inhibits ROS production
-both inhibits phospholipid peroxidation (cause membrane rupture), preventing ferroptosis
Ferroptosis triggers cell death from _____ that compromises key organellar functions but ______
-lipid and protein oxidation
-does not involve caspases
zVAD does what?
inhibit activity of caspases
In necroptosis, ____ forms a complex with ____ that is not dependent on ______
-FAS receptor
-RIP kinase
-fomation of the DISC complex (of which FADD (adaptor protein) is an essential component); ip with FasL (ligand)
TNFa causes ____
cell death (necroptosis)
incubation with TNFα and zVAD found a novel inhibitor of necroptosis: ____
- necrostatin-1, an inhibitor of RIPK
(coined term: “necroptosis”)
RIPK1 & ALS
-RIPK1 causes axonal degenration by promoting inflammation and necroptosis in ALS
____ and ____ slows ALS symptoms in mouse (SOD1 mutation)
-RIP3K KO
-necrostatin (inhibits RIPK1)
SOD1 mutation increases amount of ______
necrosis-associated proteins
necroptosis is active in _____
human ALS spinal cord samples
Necroptosis is a central mechanism that leads to _______ in ALS (upon SOD1 mutation).
axonal degeneration, neuronal death
for necroptosis, caspase-8 is associated _____
with the mechanism, but its catalytic activity is not required
necroptosis steps
-FADD + TNFa + RIPK1 + caspase 8 form complex
-capsase 8 is inhibited
-RIPK1/2 & MLKL form necrosome that migrate to PM and allows Ca++ to enter cell = cell death
DNA damage steps
-sensors: sense DNA
-transducers: tranduce signal of sensors (ex. ATR/ATM)
-effectors: causes DNA repair, arrest, apoptosis
ATM (transducer) signals to p53 (effector) causing _____ after DNA damage
apoptosis
ATM is activated by ____
phosphorylation on a serine residue (like a lot of kinases)
DNA damage is detected by sensor and repair proteins such as_____ that trigger the phosphorylation of _____. These then phosphorylate downstream targets that can be either protective (e.g.,_____) or destructive (e.g., ____). P53 activates transcription of_____, which then forms the ______
-HMGN1
-ATM and ATR
-BRCA1
-p53
-PIDD
-PIDDosome, an apoptotic complex based on the adaptor RAIDD and caspase-2
HMGN1 is what?
a nucleosome binding protein that enhances ATM activation
Ways to assess protein phosphorylation
-Reduction of protein mobility
-Incorporation of 32P
-Detection by phospho-specific antibodies
-Spot shift in 2D gel
stable transfection (ie. ____) happens when ______
-transformation
-cDNA is integrated into host chromosome
after p53 is activated, _______ increases
transcription of down-stream targets
How to recognize toxic or pro-apoptotic proteins: _____
-They cannot be stably overexpressed
-Transient overexpression causes cell death
PIDD is what?
a death-domain containing protein that is induced by p53 AND PROMOTES APOPTOSIS
PIDD interacts with ___ to form ____
-RAIDD (related to FADD) and caspase-2
-PIDDosome
PIDDosome was detetcted with ____
gel filtration under native conditions (individual components if complex was found in high MV fractions)
Methods to detect protein complexes: ______
-Chemical crosslinking and detection of high molecular weight protein complex
-Co-immunoprecipitation
-Protein-protein interaction assays based on FRET
-BioID (id with biotin and idenfy with mass spec.)
-Gel filtration chromatography
-Two hybrid system (fused proteins should cause activity if they normally interact- ex. transcription)
Caspases specifically associate with distinct_____, similar to BH3 only proteins: caspase-8 is part of the _____, caspase-2 is part of the ______ and caspase-9 is part of the______, caspase-1 is part of _____
-stress triggers
-DISC complex (necroptosis)
-PIDDosome (DNA damage)
-apoptosome (apoptosis)
-inflammasome (pyroptosis)
Inflammatory caspases form complexes _______. These complexes are called ______.
-reminiscent of apoptotic caspases (inflammation + apoptosis share similar activation pathway)
-inflammasomes
Inflammasomes are activated by the arrival of _______; ______ are integral signaling components for their activation.
- pathogen-associated molecular patterns and danger-associated molecular patterns (DAMPs/PAMPs)
-ROS
Inflammation is triggered by _________ resulting in ______ often resulting in ______
-infection or tissue stress and injury
-the coordinated delivery of neutrophils to the site of infection and injury
-collateral damage
inflammation involves vasoactive compounds that first trigger ________ , ______ and ______ that trigger swelling, pain and fever (via pyrogenics), among others
-vasoconstriction and later vasodilation (e.g., histamine)
-chemokines (e.g., IL8)
-cytokines (e.g., IL1b)
Fever mediated by ____ that increases _____ and leads to _______
- IL1β
-mitochondrial metabolic reactions (increased heat production)
-vasoconstriction (decreased heat loss)
Deregulated inflammasome activity is associated with ______
several inflammatory diseases
Inflammasome causes production of ______
IL-1B and IL-18 (Interleukins)
Parts of Inflammasome
NLRP3, pro-caspase 1
TXNIP (____) links _____
-Thioredoxin-interacting protein
-oxidative stress to inflammasome activation
TXNIP is a protein linked to ______
insulin resistance
inflammasome activators like ____ induced the _____ from ___ in a ____ manner and allow it to bind ____
-uric acid crystals
-dissociation of TXNIP
-thioredoxin
-ROS-sensitive
-NLRP3
TXNIP deficiency impairs ____
activation of NLRP3 inflammasome and the subsequent secretion of interleukin 1B (IL-1B)
TXNIP -/- or NLRP3 -/- mice show ____
glucose tolerance and insulin sensitivity
ROS + inflammasome
ROS transfers TXNIP from ROS-absorbing thioredoxin (inhibits TXNIP-thioredoxin interaction) to NLRP3 to increase production of IL-1B
K+ effect on inflammasome
K+ efflux (movement out of the cell-low [K+] in the cell) can trigger NLRP3 activation without other activators
Pyroptosis (def.)
cell death triggered by inflammasome
inflammasome activation can lead to pyroptosis causing ______
-release of IL-1B, IL-18, energy
-degradation of DNA
-PM rupture
Gout is a disease that cause _____ due to ____
-inflammation in extremities
-high levels of meat we eat
Gout caused by phagocytosis of ____, leading to activation of _____ and release of ____, causing ____
-MSU crystals
-inflammasome
-IL-1B
-neutrophil recruitment
Drug that inhibits inflammasome + Gout
-in the presence of MSU crystals, no inflammation
consequence of drugs that limits inflammasome activity
immunosuppression
autophagy (“ ____”) (def.)
-self-eating
-Ubiquitous process that results in the breakdown of membrane-enclosed cytoplasm within the lysosome in response to stress conditions/starvation
autophagy general mechanism
cell consumes its structure by merging autophagosome + lysosome (electron dense)
autophagy in the liver
-stimulation of intracellular protein degradation due to starvation,
-results from a decrease in glucogenic amino acids, notably glutamine.
different types of autphagy
-chaperone-mediated autophagy
-microautophagy
-macroautophagy (focus)
PMSF is what?
lysosomal protease inhibitor
vacoule of yeast cells are analogous to _____
lysosome of human cell
blocking protein degradation with protease inhibitor PMSF, what happens in mutant yeast?
apg1-1 mutant accumulates no autophagic bodies in the vacuole (seen as speckled pattern= degradtion)
apg1-1 mutant accumulates ______; therefore degradation of proteins is _____, detected via absence of _____
-no trichloroacetic acid (TCA)-soluble protein fragments in PMSF
-inhibited
-small peptides that cannot be precipitated with TCA
Inability to trigger autophagy leads to _____ of mutant yeasts in nitrogen starvation medium
cell death
Apg1p has a large _____ domain and it is analogous to _______
-protein kinase
-C.elegans unc-51
Apg1 presence is required for the formation of _____ and the ability to
autophagic bodies (indicated by darker accumulations under microscope)
-keep growing following exposure to starvation
Apg1 is a what?
kinase, whose autophosphorylation is turned off under conditions of starvation. It then becomes active.
peripheral protein is where? when treated with buffer
pellet (associated with membrane)
Apg14p is a _____ membrane protein; Apg6p is a _____ membrane protein with a weaker association
-peripheral
-peripheral
Apg6p and Apg14p are necessary for______ and form a ______ upon starvation.
-autophagy
-membrane-bound protein complex
Human Beclin 1 shares some identity with _____ and is part of a _____
-Atg6/Vps30
-PI3K complex based on Atg14
Apg1 (human form called_____) phosphorylates _____, that allows for the generation of ___ by ____ when in complex on the ER. This alters membrane properties and triggers the formation of ______
-ULK1
-Beclin-1 on two N-terminal serines (Atg6)
-PI3P
-Atg14
-an isolation membrane/autophagosome
Beclin has ___ domain
BH3 only (promotes cell death)
Microtubule-associated protein 1 light chain 3 (LC3), a homologue of____ is essential for_____ in yeast, is associated to the _____ after processing.
-Apg8p
-autophagy
-autophagosome membranes
Two forms of LC3, called LC3-I and -II, are produced post-translationally in various cells. LC3-I is ____ , whereas LC3-II is _______ .
-cytosolic
-membrane bound on autophagosomes due to lipidation of LC3-I
The amount of LC3-II is correlated with _______.
the extent of autophagosome formation
autophagy: _____ is part of a complex that phosphorylates largely unknown substrates.
Atg1 (Apg1/ULK1)
autophagy: ____ is one of the substrates of Atg1 and is part of a _____
-Beclin1 (Apg6/Atg6)
-PI3K complex localized to the ER that triggers formation of the isolation membrane
autopahgy: ______ promotes isolation membrane elongation and closure with the help of ____
-LC3 (Apg8/Atg8)
-microtubules
Autophagosomes are formed shortly after ______ as seen with _____
-birth (after first few hours)
-GFP-LC3 transgenic mice
Autophagosome formation is required for survival of _____. It helps them survive ________.
-mouse newborns
-the nutrient starvation encountered just after birth
autophagic cell death is a misnomer, rather, autophagy promotes _______
-cellular survival through the elimination of toxins
types of organelle-specific autophagy
-Mitophagy
-ER-phagy
problems with mitophagy can cause ______
Parkinson disease
Parkinson: Damaged mitochondria emits ROS and prevents _______
proteasome/ubiquitin-mediated elimination of PINK1 (PTEN-induced putative kinase 1)
Damaged mitochondrion accumulates _____ on the surface
PINK1
PINK1 recruits _____ upon autophosphorylation
autophagy receptors
PINK1 on mitochondrial surface recruits ______, which triggers ______ that result in the recognition of ______
-Parkin (ubiquitin ligase)
-mono-ubiquitination of mitochondrial surface proteins (PINK1)
-ROS-emitting mitochondria by autophagosomes
Dual role of ubiquitination in mitophagy
-degradation signal for PINK1
-recognition signal for autophagotic degradation of mitochondria
Parkin moves onto mitochondria upon _____, which then leads to the interaction with _____
-opening of PTPC
-PINK1 and mitophagy
PINK1 ______ prevents its degradation through ubiquitination
-oxidation
PINK1 _____ triggers autophosphorylation, followed by the activation of ______ that disrupts stable association of ubiquitin, preventing degradation
-internal disulfide bond formation
-PINK1 kinase
ER-localized autophagy receptors mediate _____
-degradation via the interaction with LC3
LIR (_____) target ER for _____
-LC3-interacting region
-degradation through autophagosome
RTN3 regulated turnover of ______
endoplasmic reticulum via selective autophagy
Starvation in minimal medium leads to remodeling of the ER seen by the ____
-overlap of RTN3L with the lysosomal marker LAMP-1
Starvation time-course shows elimination of ER proteins ______ in _____-dependent manner
-Rtn1a, Climp-63
-Rtn3
