Section 2 - Cell Death Flashcards
(131 cards)
1
Q
cell death general method
A
-stress triggers (often oxidative stress)
-stress sensors (proteins)change their function and location upon chemical modification
2
Q
Main players of cell death (6)
A
- stress triggers
- stress sensor proteins
- mitochondria
- Bcl2 family proteins
- Caspases
- multimeric protein complexes
3
Q
mitochondria is _____ but is also the target of ______ (ex. _____)
A
-central sensing station
-many death-controlling mechanisms
-release of cytochrome C
4
Q
Bcl2 family proteins are _____ of cell death. They control mitochondria via ______
A
-executors and regulators
-calcium and formation of pores
5
Q
Caspases are ____ of cell death
A
gateways and executors
6
Q
ROS lead to formation of ______, but mitochondrial permeability transition also ______.
A
-mitochondrial pores unrelated to PTPC
-triggers the same mechanism
7
Q
Proteins that control ROS have a central function in ______
A
maintenance of mitochondrial functions and cell death
8
Q
p66 does what?
A
increases ROS production by interfering with electron flow by holding on cytochrome C and cause “traffic jams”
9
Q
AIF (______) acts as a ______ (like ____) and does what?
A
-Apoptosis Inducing Factor
-chaperone for OXPHOS
-C9Orf72
-induces DNA damage in the nucleus upon apoptosis onset
10
Q
RNA interference vs. CRISPR-cas 9
A
RNAi is temporary, can be used to protein function
CRISPR is permanent, can observe cell adaptation
11
Q
RNA interference summary
A
-short dsRNA cut by dicer to form siRNA
-RISC is assembled with siRNA
-RISC + siRNA binds to mRNA and cause mRNA degradation
12
Q
RNAi knockdown of AIF induces a ____
A
DCF signal (increase ROS production)
13
Q
AIF gene is on what chromosome? effect on males?
A
-X-chromosome
-mutation of AIF allele in males results in complete knockout
14
Q
AIF is released from mitochondria after ___
A
apoptotic signal and cleavage
15
Q
AIF function
A
AIF maintains normal amounts of mitochondrial complex I, by acting as a folding-scaffold during the import of inner mitochondrial membrane proteins
16
Q
in absence of AIF, what happens?
A
lower amounts of complex I lead to electron traffic jam and ROS production
17
Q
Upon mitochondrial dysfunction and prolonged oxidative stress, AIF becomes _____
A
a promoter of DNA damage in the nucleus
18
Q
apoptosis: pro-apoptotic (death) signals do what?
A
-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
19
Q
mitochondria is the central controller of ____ via ____
A
-apoptosis
-AIF, cytochrome C
20
Q
Apoptosis triggers (7)
A
-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)
21
Q
ER stress is ______
A
accumulation of misfolded/unfolded proteins
22
Q
BH3-only proteins exists for ____ and do what?
A
-any stress trigger
-inhibits Bcl2 proteins from blocking mPTP to promote apoptosis
23
Q
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 (_____).
A
-Puma, Noxa
-Bad
-Bim
-Bik, Bim
24
Q
Intrinsic cell death is under the control of Bcl2 family proteins, where ____ is anti-apoptotic, _____ are pro-apoptotic
A
-Bcl2
-Bax/Bak
25
in human tumor cells, anti-apoptotic proteins are _____ while pro-apoptotic proteins are _____
-amplified
-mostly retained though some are deleted
26
erastin is a molecule that ______ which is _____ and kills _____
-targets Ras
-often hyperactive in cancer cells
-cancer cells
27
Annexin V is what?
- a protein associated with PM that indicates apoptosis (apoptosis marker)
28
erastin does not cause ______
apoptotic cell death since it is not associated with caspases or Annexin V activation
29
DFO is what?
deferoxamine = iron chelator
30
DCF is what?
marker of ROS level in cell
31
C11-BODIPY (def.)
fluorescent probe against oxidized lipids
32
erastin kills cells, associated with ______
iron-dependent ROS production and lipid oxidation
33
in presence of erastin and the absence of Fe, what happens?
cells survive
34
erastin follows this death pathway: ____
ferroptosis
35
______, _______, and ______ protect from ROS, hence ferroptosis involves ______
-Glutathione peroxidases
-peroxiredoxins
-peroxisomal catalase
-their inhibition
36
GpX4 is ______
Glutathione peroxidase 4
37
RSL3 is what?
ferroptosis inducer; inhibits GpX4
38
Method for determining ferroptosis inhibitor
1. Create GpX4 KO through tamoxifen treatment
2. Transfection with cDNA library that rescues KO
3. Identify rescuing cDNA (one of them is GpX4)
4. Test for cell survival with ferroptosis inducer RSL3
5. inhibitor blcoks cell death when over expressed in WT and KO
39
FSP-1 is what?
glutathione-independent ferroptosis suppressor that protects from ferroptosis
40
GpX4 KO cells with FSP-1 cDNA do what?
survive
41
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
42
selenium and cystine do what in ferroptosis?
-selenium inhibits ROS production
-both inhibits phospholipid peroxidation (cause membrane rupture), preventing ferroptosis
43
Ferroptosis triggers cell death from _____ that compromises key organellar functions but ______
-lipid and protein oxidation
-does not involve caspases
44
zVAD does what?
inhibit activity of caspases
45
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)
46
TNFa causes ____
cell death (necroptosis)
47
incubation with TNFα and zVAD found a novel inhibitor of necroptosis: ____
- necrostatin-1, an inhibitor of RIPK
(coined term: “necroptosis”)
48
RIPK1 & ALS
-RIPK1 causes axonal degenration by promoting inflammation and necroptosis in ALS
49
____ and ____ slows ALS symptoms in mouse (SOD1 mutation)
-RIP3K KO
-necrostatin (inhibits RIPK1)
50
SOD1 mutation increases amount of ______
necrosis-associated proteins
51
necroptosis is active in _____
human ALS spinal cord samples
52
Necroptosis is a central mechanism that leads to _______ in ALS (upon SOD1 mutation).
axonal degeneration, neuronal death
53
for necroptosis, caspase-8 is associated _____
with the mechanism, but its catalytic activity is not required
54
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
55
DNA damage steps
-sensors: sense DNA
-transducers: tranduce signal of sensors (ex. ATR/ATM)
-effectors: causes DNA repair, arrest, apoptosis
56
ATM (transducer) signals to p53 (effector) causing _____ after DNA damage
apoptosis
57
ATM is activated by ____
phosphorylation on a serine residue (like a lot of kinases)
58
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
59
HMGN1 is what?
a nucleosome binding protein that enhances ATM activation
60
Ways to assess protein phosphorylation
-Reduction of protein mobility
-Incorporation of 32P
-Detection by phospho-specific antibodies
-Spot shift in 2D gel
61
stable transfection (ie. ____) happens when ______
-transformation
-cDNA is integrated into host chromosome
62
after p53 is activated, _______ increases
transcription of down-stream targets
63
How to recognize toxic or pro-apoptotic proteins: _____
-They cannot be stably overexpressed
-Transient overexpression causes cell death
64
PIDD is what?
a death-domain containing protein that is induced by p53 AND PROMOTES APOPTOSIS
65
PIDD interacts with ___ to form ____
-RAIDD (related to FADD) and caspase-2
-PIDDosome
66
PIDDosome was detetcted with ____
gel filtration under native conditions (individual components if complex was found in high MV fractions)
67
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)
68
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)
69
Inflammatory caspases form complexes _______. These complexes are called ______.
-reminiscent of apoptotic caspases (inflammation + apoptosis share similar activation pathway)
-inflammasomes
70
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
71
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
72
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)
73
Fever mediated by ____ that increases _____ and leads to _______
- IL1β
-mitochondrial metabolic reactions (increased heat production)
-vasoconstriction (decreased heat loss)
74
Deregulated inflammasome activity is associated with ______
several inflammatory diseases
75
Inflammasome causes production of ______
IL-1B and IL-18 (Interleukins)
76
Parts of Inflammasome
NLRP3, pro-caspase 1
77
TXNIP (____) links _____
-Thioredoxin-interacting protein
-oxidative stress to inflammasome activation
78
TXNIP is a protein linked to ______
insulin resistance
79
inflammasome activators like ____ induced the _____ from ___ in a ____ manner and allow it to bind ____
-uric acid crystals
-dissociation of TXNIP
-thioredoxin
-ROS-sensitive
-NLRP3
80
TXNIP deficiency impairs ____
activation of NLRP3 inflammasome and the subsequent secretion of interleukin 1B (IL-1B)
81
TXNIP -/- or NLRP3 -/- mice show ____
glucose tolerance and insulin sensitivity
82
ROS + inflammasome
ROS transfers TXNIP from ROS-absorbing thioredoxin (inhibits TXNIP-thioredoxin interaction) to NLRP3 to increase production of IL-1B
83
K+ effect on inflammasome
K+ efflux (movement out of the cell-low [K+] in the cell) can trigger NLRP3 activation without other activators
84
Pyroptosis (def.)
cell death triggered by inflammasome
85
inflammasome activation can lead to pyroptosis causing ______
-release of IL-1B, IL-18, energy
-degradation of DNA
-PM rupture
86
Gout is a disease that cause _____ due to ____
-inflammation in extremities
-high levels of meat we eat
87
Gout caused by phagocytosis of ____, leading to activation of _____ and release of ____, causing ____
-MSU crystals
-inflammasome
-IL-1B
-neutrophil recruitment
88
Drug that inhibits inflammasome + Gout
-in the presence of MSU crystals, no inflammation
89
consequence of drugs that limits inflammasome activity
immunosuppression
90
autophagy (" ____") (def.)
-self-eating
-Ubiquitous process that results in the breakdown of membrane-enclosed cytoplasm within the lysosome in response to stress conditions/starvation
91
autophagy general mechanism
cell consumes its structure by merging autophagosome + lysosome (electron dense)
92
autophagy in the liver
-stimulation of intracellular protein degradation due to starvation,
-results from a decrease in glucogenic amino acids, notably glutamine.
93
different types of autphagy
-chaperone-mediated autophagy
-microautophagy
-macroautophagy (focus)
94
PMSF is what?
lysosomal protease inhibitor
95
vacoule of yeast cells are analogous to _____
lysosome of human cell
96
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)
97
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
98
Inability to trigger autophagy leads to _____ of mutant yeasts in nitrogen starvation medium
cell death
99
Apg1p has a large _____ domain and it is analogous to _______
-protein kinase
-C.elegans unc-51
100
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
101
Apg1 is a what?
kinase, whose autophosphorylation is turned off under conditions of starvation. It then becomes active.
102
peripheral protein is where? when treated with buffer
pellet (associated with membrane)
103
Apg14p is a _____ membrane protein; Apg6p is a _____ membrane protein with a weaker association
-peripheral
-peripheral
104
Apg6p and Apg14p are necessary for______ and form a ______ upon starvation.
-autophagy
-membrane-bound protein complex
105
Human Beclin 1 shares some identity with _____ and is part of a _____
-Atg6/Vps30
-PI3K complex based on Atg14
106
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
107
Beclin has ___ domain
BH3 only (promotes cell death)
108
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
109
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
110
The amount of LC3-II is correlated with _______.
the extent of autophagosome formation
111
autophagy: _____ is part of a complex that phosphorylates largely unknown substrates.
Atg1 (Apg1/ULK1)
112
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
113
autopahgy: ______ promotes isolation membrane elongation and closure with the help of ____
-LC3 (Apg8/Atg8)
-microtubules
114
Autophagosomes are formed shortly after ______ as seen with _____
-birth (after first few hours)
-GFP-LC3 transgenic mice
115
Autophagosome formation is required for survival of _____. It helps them survive ________.
-mouse newborns
-the nutrient starvation encountered just after birth
116
autophagic cell death is a misnomer, rather, autophagy promotes _______
-cellular survival through the elimination of toxins
117
types of organelle-specific autophagy
-Mitophagy
-ER-phagy
118
problems with mitophagy can cause ______
Parkinson disease
119
Parkinson: Damaged mitochondria emits ROS and prevents _______
proteasome/ubiquitin-mediated elimination of PINK1 (PTEN-induced putative kinase 1)
120
Damaged mitochondrion accumulates _____ on the surface
PINK1
121
PINK1 recruits _____ upon autophosphorylation
autophagy receptors
122
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
123
Dual role of ubiquitination in mitophagy
-degradation signal for PINK1
-recognition signal for autophagotic degradation of mitochondria
124
Parkin moves onto mitochondria upon _____, which then leads to the interaction with _____
-opening of PTPC
-PINK1 and mitophagy
125
PINK1 ______ prevents its degradation through ubiquitination
-oxidation
126
PINK1 _____ triggers autophosphorylation, followed by the activation of ______ that disrupts stable association of ubiquitin, preventing degradation
-internal disulfide bond formation
-PINK1 kinase
127
ER-localized autophagy receptors mediate _____
-degradation via the interaction with LC3
128
LIR (_____) target ER for _____
-LC3-interacting region
-degradation through autophagosome
129
RTN3 regulated turnover of ______
endoplasmic reticulum via selective autophagy
130
Starvation in minimal medium leads to remodeling of the ER seen by the ____
-overlap of RTN3L with the lysosomal marker LAMP-1
131
Starvation time-course shows elimination of ER proteins ______ in _____-dependent manner
-Rtn1a, Climp-63
-Rtn3
