Section 3 - Interorganeller Stress Flashcards
(125 cards)
1
Q
Heat-shock proteins (HSPs) (def.)
A
constitutively expressed molecular chaperones that guide the normal folding, intracellular disposition and proteolytic turnover of many of the key regulators of cell growth and survival
2
Q
HSP expression increase in response to ?
A
-heat/temperature change
-inflammation
-other protein-denaturing stressors
3
Q
HSP expression increases in response to protein denaturing stressors as an _____
A
evolutionary conserved response to restore normal protein-folding environment and enhance cell survival
4
Q
in tissues exposed to heat, proteins are ____
A
unfolded and aggregrate
5
Q
source of heat in the cell
A
mitochondria, peroxisomes
6
Q
HSP are mostly ____ and they help _____
A
-chaperones
-newly synthesized proteins fold
7
Q
HSP protect proteins from ______
A
cellular stresses that can cause unfolding and aggregation
8
Q
HSP target proteins to ______, prevent and clean up ______
A
-degradation
-protein aggregates
9
Q
______ are a hallmark of neurodegenerative diseases
A
protein aggregates
10
Q
proteins are folded to reach ____
A
energy minimum
11
Q
large proteins require ___ to reach energy minimum
A
energy inputs
12
Q
as protein chain forms more intrachain contacts, it lowers its _____ (2)
A
-internal free energy & reduces conformational freedom
13
Q
heat shock is lethal to cells injected with ____
A
antibodies against Hsp70
14
Q
generation of antiserum/antibody
A
1) transformation of bacteria with cDNA
2) produce proteins
3) inject proteins into mouse to make antibodies
15
Q
Hsp70 requires binding and hydrolysis of ____ to ____
A
-ATP
-achieve folding
16
Q
protein binding to Hsp70 occurs when _____, a state promoted by _____. release requires _____
A
-ADP-bound
-J-proteins (they remove P from ATP)
-ATP hydrolysis
17
Q
ER equivalent of HSP70
A
GRP78/BiP
18
Q
Ulk1 (part of _____) interacts with ____ but not its _____
A
-autophagy
- Hsp90 (and Cdc37)
- kinase-dead mutant form
19
Q
inhibitor of Hsp90
A
geldanamycin + variant: 17AAG
20
Q
The inhibition of Hsp90 with the geldanamycin variant 17AAG does what?
A
degrades Ulk1
21
Q
The effect of Hsp90 inhibition on autophagy
A
-can’t trigger autophagy
22
Q
effect of Hsp90 degradation on mitography
A
Hsp90 degradation blocks CCCP-mediated mitophagy
23
Q
Hsp90/Cdc37 and Ulk1 kinase activity are needed to _____
A
undergo mitophagy
24
Q
Heat shock proteins in cell
A
Hsp70
Hsp90
25
Hsp70 job in cell
-anti-aggregation
-membrane translocation
-intracellular disposition
26
Hsp90 job in cell
ligand binding, phosphorylation, dimerization
27
Overview of protein folding in the ER
-protein translocated into ER through Sec61 complex
-BiP/GRP78 (ER-localized Hsp70) binds to translocated protein in ATP-dependent manner
-co-translational folding follows glycosylation
-calnexin binds to glycosylated proteins to promote proper disulfide bond
-disulfide bond formation is mediated by PDI,ERp57
28
oxidation of proteins in ER forms _____ using ____
-disulfide bonds
-PDI in oxidized form
29
Ero1 does what?
oxidizes PDI (reduced to oxidized form) in ER, uses O2
-Ero1 goes from oxidized to reduced
30
ROS production in ER occurs when _____
substrates or enzymes involved in protein folding (ex. PDI, Ero1) are not present in correct amounts
31
Em405/Em440 ratio in ER indicates _____. The ___, the better
-oxidizing environment of ER
-higher
32
SERCA is what? function
-sarco/endoplasmic reticulum Ca2+-ATPase
-transport calcium from the cytosol into the sarcoplasmic reticulum
33
Calcium depletion via SERCA inhibition with this drug (______) leads to the _____. Hence this inhibits ______.
-TG=thapsigargin
-reduction of the ER lumen, but not of the cytosol
-pro-oxidants within the ER
34
ATP depletion by ____ inhibits ___ activity
-hypoxia, cut off glucose supply
-BIP
35
Block of glycosylation by _____ inhibits ___ activity
-tunicamycin
-Calnexin
36
Reduced calcium content by _____ inhibits ___ activity
-thapsigargin
-Calnexin, PDI
37
Reduction of the ER environment by ____ inhibits ___ activity
-DTT
-PDI
38
sequence conserved in BiP/GRP78/94 of mamalian and yeast promoters that is import for the induction by unfolded proteins in ER
unfolded protein-response element
39
when ER is stressed, BiP does what? then what happens?
-dissociates from ER folding sensors
-unfolded protein response occurs
40
UPR (_____) uses three ER sensor proteins to do what?
-unfolded protein response
-signal to nucleus
41
Three ER sensor proteins involved in URE + function
-IRE1 (induction of chaperones)
-PERK (translation inhibition)
-ATF6 (induction of chaperones, CHOP)
42
ER sensor proteins are regulated by ______. Loss of BiP binding causes _____
-BiP binding
-dimerization (IRE1, PERK) or ER-Golgi migration (ATF6)
43
Upon activation, IRE1 becomes _____, PERK exposes a _____ and ATF6 migrates to the _____
-an endonuclease (for XBP1)
-cytosolic kinase domain (for eIF2a)
-Golgi, where a cytosolic transcription factor domain is released
44
XBP1 transcription is induced by _____ and spliced by ____ in response to ____ to produce _____
-ATF6
-IRE1
-ER stress
-highly active transcription factor
45
The XBP-1 protein is only expressed at its full-length size (bigger) under ______.
-ER stress conditions
46
The XBP-1 splicing mechanism
Excision of 26bp allows translation of the 50 kDa XBP-1 protein that is (among other things) required for B cell development; yeast Hac1 splicing highly similar
47
Ire1p makes_____ during ER stress, to which the _____
-foci
-mRNA of XBP1 attaches
48
PERK can do what?
phosphorylate eIF2α, the eukaryotic translation initiation factor 2 subunit 1 (eIF2α), but also autophosphorylate
49
in vitro assay, PERK activity leads to _____
protein synthesis shutdown
50
Kinase dead PERK can't do what?
shutdown protein synthesis
51
PERK autophosphorylation is tied to _____
-ER stress
52
translocon component ___ acts in ER turnover during ____
-Sec62
-stress recovery
53
Depletion of Ca2+ from the ER through treatment with _____ induces _____. Following removal of the stress, _____
-cyclopiazonic acid (inhibits ER filling)
-UPR-associated ER chaperones
-ER homeostasis is re-established
54
BafA1: bafilomycin A1 blocks _____
lysosome/autophagosome acidification and hence cathepsins (proteases)
55
Herp is what?
an ER membrane protein upregulated by ER stress, is implicated in regulation of ERAD
56
Calnexin action during ER stress recovery
forms clusters within puncta (clumps)
57
Calnexin clustering requires ____
Sec62 portion of translocon
58
Sec62 interacts with ____ during ER stress recovery
LC3 (autophagy)
59
Condition of cell that activates PERK
hypoxia
60
the mitochondria and the ER make _____ that mediates _____
-extensive contacts
-calcium signalling
61
lesser known partner of GFP
aequorin
62
Histamine-triggered opening of IP3Rs demonstrates that ______
mitochondria must lie close to the mouth of ER Ca2+ release
63
IP3R is what?
calcium channel; allows for release of Ca++ from ER into mitochondria
64
calcium shuttling between ER and mitochondria
-3uM ATP or IP3 (release through IP3R) causes release of Ca++ from ER
-calcium uptake by mitochondria
-calcium uptake by serca in ER
(cycle repeats)
65
Mitochondria-associated membranes (MAM) represent a ________
region of the endoplasmic reticulum (ER) which is reversibly tethered to mitochondria
66
SERCA calcium pump and IP3R form a ______
calcium shuttle platform between the ER and mitochondria at the MAM
67
ER-mitochondria calcium cycle is required for ____ in mitochondria
-the Krebs cycle
68
conc of calcium in mitochondria compared to ER
ER: higher
mitochondria: lower
69
Mitochondria calcium uptake increases during _____
exercise
70
MCU (_______) -mediated calcium flux is necessary for _____
-mitochondrial calcium uniporter
-peak muscle performance during exercise
71
Calcium release from the ER is essential for normal mitochondrial respiration, as seen by ______
reduction of respiration in full IP3R KO
72
Energy crisis from mitochondrial Ca2+ deficiency activates ____
autophagy
73
inhibitor of IP3Rs
XeB (xestospongin B)
74
XeB effect on cancer cells
-causes energy crisis which activates AMPK but only kills malignant cancer cells
75
The connection between the ER and mitochondria becomes tighter after ______, showing role for ____
-24h of ER stress
-protein folding and apoptosis
76
evidence of lipid shuttling between organelles: PS synthase (makes phospatidylserine) is found in _____. PS decarboxylase (makes ______) localizes to ____
-MAM
-PE
-mitochondria
77
Multiple contacts exist between _____. Many of their functions include _____, similar to what happens between the ER and mitochondria.
- virtually all organelles
- lipid and calcium homeostasis
78
interactome (def.)
-is the whole set of molecular interactions in a particular cell, almost like one big organelle
79
Lipid synthesis takes place mostly at which organelle?; the bulk of their synthesis has been localized to the _____
-the ER
-mitochondria-associated membrane (MAM)
80
Lipid bodies are considered _____ that have a distinct ______. They can be generated in _____ with high concentrations of ______
-organelles
-set of proteins involved in their biogenesis and regulation
-all cell types so far
- fatty acids
81
lipid bodies usually store ____ but in some cell types they have ____
-fatty acids
-specialized functions
82
lipid bodies specialized function in liver cells
storage of vitamin A precursor
83
lipid bodies specialized function in eyes
-storage of precursor for rhodopsin (needed for retinal pigment)
84
lipid bodies specialized function in mammary epithelial cells
production and secretion of milk fat globules
85
lipid bodies specialized function in eosinophils
storage of arachidonic acid
86
adipocytes lipid droplets are rich in _____ while other cells usually contain _____
-cholesterol
-next to none
87
lipid droplet creation
1) neutral lipids are synthesized between the leaflets of ER membrane
2) Mature lipid droplet then bud from ER membrane to form an independent organelle coated by monolayer of phospholipids and members of PAT-domain family of proteins
88
PAT-domain family of proteins
perilipin, ADRP and TIP47-related protein-domain family of proteins
89
lipid bodies originate from ____
ER
90
The association of PAT proteins (______) with lipid bodies is _______ coordinated
-perilipin, adipophilin, TIP47
-spatially and temporally
91
Lipid bodies remain associated with the_____ to facilitate _____. They can also be found associated with other organelles such as ______.
-ER membranes and mitochondria
-lipid exchange
-endosomes
92
proteins involved in lipid trafficking
ORP5 and ORP8
93
ER transmembrane proteins ________ both contain ____ domains that specifically interact with _____ and ORD domain which is the ______
-ORP5 and ORP8
-PH
- PI4P/phosphatidylserine
- lipid binding domain
94
TIRF (_____) allows to observe _____
-total internal reflection fluorescence
-less than 200nm thick slices on the surface of cells
95
co-expression of PI4K and ORP5 leads to ____
higher amounts of PI4P on PM and more ORP5 near the PM
96
the double knockdown of ORP5/6 results in PS _____
no longer at PM
97
PI4P used as signal at ER-PM contacts to establish ______. Machinery depends on the lipid transfer proteins _____
-PS enrichment on the inner leaflet
-ORP5 and ORP6
98
PI-3P (_____) functions as signal for ______
-Phosphatidylinositol 3-phosphate
-membrane bending (e.g. autophagosome)
99
PIP and PIP2 can be further phosphorylated to give _____
-3-phosphorylated phosphatidylinositol by the PI-3 kinase enzymes.
100
The 3-phosphorylated phosphatidylinositol serves as a _____
-scaffold for the activity of the kinase Akt/PKB
101
PI-3P acts a signal to block _____
-apoptosis via Akt and Bcl2 family protein Bad
102
Trophic factor binding leads to activation of____ through _____. This then causes sequestration of _____. This prevents _____
-Akt
-PI-3 kinase
-Bad with 14-3-3 protein after Bad phosphorylation
-cell death
103
PTEN (______)is found frequently ____ in tumours
-tumor suppressor gene
-mutated
104
PTEN function
phosphatase (dephosphorylate PIP3 to make PIP2 which results in inhibition of the Akt signaling pathway, which plays an important role in regulating cellular behaviors such as cell growth, survival, and migration)
105
Plentiful energy stops apoptosis, autophagy and promotes protein synthesis with ____
two mTOR complexes sitting at the central control point.
106
mTOR stands for _____
mammalian target of rapamycin
107
rapamycin does what?
inhibits mTOR; immunosuppressant(used for people who have organ transplants); anti-proliferative drug
108
mTOR undergoes activation upon ____ and inactivation upon _____. Inhibition of mTORC1 stops _____. mTORC2 is most important for ______.
-hyperactive PI3K (cancer)
-an energy crisis (blocked respiration or glycolysis that makes AMP/ATP unfavorable and activates AMPK)
-cancer growth
-cell structure
109
mTOR activates signal transduction pathways leading to the activation of _____
T helper cells
110
_____ is one protein required for rapamycin toxicity in yeast. Without it, cell will do what in the presence of rapamycin?
-FKBP
-survive
111
TOR2 (mTOR in ___) is what?
-yeast
-essential phosphatidyinositol linase homolog needed for G1 progression
112
TOR controls ____
translation initiation and early G1 progression
113
TOR2 is required for organization of _____
actin cytoskeleton, preventing growth
114
_____ is a prerequisite for growth
-Normal cytoskeleton
115
TCP20 is what?
an actin chaperonin
116
cell with TOR2 deleted has _____; phenotype is rescued by _____
-aberrant actin cytoskeleton,; random actin dots
-TCP20
117
T17N mutants = ______
Q61L mutants = ______
-permanently bound to GDP, also called kinase-dead
-permanently bound to GTP, also called dominant active
118
TOR2 activates _____ via GEF _____ which makes them ____
-RHO1, RHO2
-ROM2
-GTP-bound/active
119
active RHO1/RHO2 do what?
remodel actin cytoskeleton
120
active RHO1/2 is inactivated by _____
Sac7p (Rho GAP)
121
Tor2p absence can be rescued by _____
Rho1p or Rho2p
122
Rapamycin acts by forming a _____
toxic complex with its intracellular receptor FKBP12, and this complex then binds and inhibits TOR directly
123
in cells, there are two TOR complexes (______) and ____
-TORC1 (has TOR1/2) and TORC2 (has only TOR2)
-only one is rapamycin sensitive
124
TORC2 controls ____ and is rapamycin ______
-actin cytoskeleton
-insensitive
125
In S. cerevisiae, FKBP12 is encoded by the ____, and FPR1-rapamycin specifically binds and thereby inhibits ____
-FPR1 gene
-TOR1 and TOR2, but not the TOR2-based TORC2 complex
