CPPS 325 2025 VERGE signalling #19&20&21 UPR to upload (1) Flashcards
What is acidosis in the context of the signaling lecture?
Acidosis refers to slight drops in pH that result in increases in trkA receptor protein localized to membranes of sensory neurons and increased receptor activation in response to NGF challenge.
What effect can be abolished in the presence of proton-sensitive channel blockers?
The effect of acidosis on trkA receptor activation can be abolished.
What does acidosis increase in terms of trkA receptor?
Acidosis increases the level of trkA receptor phosphorylation/activation in the absence of exogenous NGF challenge.
What may the rapid mobilization of trkA receptor to the membrane indicate?
It may be an adaptive response that contributes to the rapid sensitization of sensory neurons under inflammatory conditions.
What does UPR stand for?
Unfolded Protein Response.
What is the primary role of the UPR?
To restore proteostasis by engaging an adaptive stress response to the accumulation of misfolded proteins.
What are the three major transducers/ER stress sensors involved in the UPR?
- PERK
- IRE1α
- ATF6
What happens when a cell undergoes ER stress?
The cell attempts to repair itself by producing new proteins, leading to a competition for BiP/GRP78.
What is the function of BiP/GRP78 in the UPR?
BiP/GRP78 associates with unfolded proteins and transducers of the UPR, maintaining them in an inactive state under unstressed conditions.
What does PERK do upon activation?
Decreases the general protein synthesis rate through phosphorylation of the initiation factor eIF2α.
What mRNA does eIF2α phosphorylation specifically increase the translation of?
ATF4 mRNA.
What does ATF4 control the expression of?
- Genes involved in amino acid metabolism
- Autophagy
- Antioxidant responses
- Apoptosis
What role does IRE1α play in the UPR?
Controls selective mRNA decay and processes the mRNA encoding XBP1.
What does the active form of IRE1α regulate?
Stress responses mediated by JNK, ERK, and NFκB.
What happens to ATF6 upon ER stress induction?
It is processed at the Golgi apparatus, releasing its cytosolic domain that translocates to the nucleus.
What is ERAD?
ER-Associated protein Degradation Pathway.
What is the primary function of proteasomes?
To degrade unneeded or damaged proteins by proteolysis.
What are proteins tagged for degradation with?
Ubiquitin.
What is the role of ubiquitin ligases?
Catalyze the tagging reaction of proteins for degradation.
True or False: The UPR is only activated under extreme stress conditions.
False.
Fill in the blank: The inability of cells to deal with _______ is now recognized as the underpinning for many human diseases.
misfolded proteins
What do chaperone proteins like GRP78 aim to achieve in the ER?
Increase folding capacity and prevent protein aggregation.
What is the consequence of sustained ER stress?
Ultimately leads to the death of the cell.
What does the activation of the UPR aim to restore?
Proteostasis.
What is necessary for the expression of genes involved in protein folding?
Unfolded Protein Response (UPR)
UPR is crucial for various cellular processes including secretion and phospholipid biosynthesis.
What are the three main pathways of the UPR/ER stress response?
- Ire1
- ATF6
- PERK
What type of protein is Ire1?
Type I transmembrane-receptor kinase/endoribonuclease
Ire1 is activated by dimerization and has both kinase and RNase domains.
How does Ire1 maintain an inactive state under unstressed conditions?
By binding to BiP/GRP78
BiP/GRP78 is an ER-localized protein chaperone.
What triggers Ire1’s activation?
ER stress leading to reduced free BiP
The absence of free BiP allows Ire1 to oligomerize and activate its RNase function.
What does activated Ire1 cleave to generate a transcriptional activator?
XBP1 mRNA
This process removes a 26-nucleotide intron to produce XBP1s.
What is ATF6 in unstressed cells?
An ER transmembrane protein
ATF6 is associated with BiP/GRP78 in this state.
What happens to ATF6 upon ER stress?
It translocates to the Golgi for activation
This activation involves cleavage of its cytosolic portion.
What is the role of the cytosolic domain fragment of ATF6?
It acts as a transcription factor (ATF6f)
ATF6f promotes the transcription of UPR-related genes.
What are the two phases of UPR response?
- Phase 1: Adaptive
- Phase 2: Apoptosis
What occurs during Phase 1 of the UPR response?
Cell regains homeostasis through adaptive responses
These include inhibiting translation and promoting mRNA decay.
What triggers apoptosis in Phase 2 of the UPR response?
Prolonged ER stress
This phase eliminates irreversibly damaged cells.
What role does the BCL-2 protein family play in ER stress?
They control ER stress-induced apoptosis
BH3-only proteins regulate activation of pro-apoptotic factors like BAX and BAK.
What does sustained PERK signaling lead to?
Upregulation of CHOP
CHOP is a pro-apoptotic transcription factor.
What are the three ER stress sensors activated during UPR?
- ATF6
- IRE1
- PERK
What is the function of BiP/GRP78 under physiological conditions?
Prevents activation of UPR sensors
This chaperone binds to UPR sensors to maintain them in an inactive state.
What does ATF6 induce upon activation?
Expression of ER chaperones and XBP1
This is crucial for enhancing the cell’s ability to manage unfolded proteins.
What does activated IRE1 do to XBP1 mRNA?
Induces splicing to produce XBP1s
This spliced version regulates transcription of ERAD-related genes.
What is the effect of PERK on protein synthesis?
Hampers general protein synthesis
This is achieved through phosphorylation of eIF2α.
What does K do when activated?
Hampers general protein synthesis via the phosphorylation of eIF2α and enables the translation of ATF4.
What is the role of the endoplasmic reticulum-associated protein degradation (ERAD)?
Sequesters and eliminates damaged or incorrectly folded proteins.
What are the classical implications of the UPR?
Linked to protein-folding stress under both physiological and pathological conditions.
What additional functions does the UPR have?
Regulates various processes including lipid and cholesterol synthesis, metabolism, energy homeostasis, inflammation, and cell differentiation.
How does the UPR relate to peripheral nerve repair?
Short term involves drop in pH, calcium wave, and injury discharge; longer term involves retrograde axonal signals, translational and transcriptional changes.
What is Luman/CREB3?
An important retrograde axonal signal that regulates axon regeneration through regulation of the UPR.
What can axons synthesize or activate after injury?
Proteins that serve as retrograde signals or initiate repair processes at the injury site.
What happens to proteins localized in the axonal ER membrane during injury?
They can be cleaved and become functionally activated in response to nerve injury.
What role does Luman/CREB3 play in sensory neuron regeneration?
Regulates the intrinsic ability of a sensory neuron to regenerate an axon.
What happens when Luman expression is knocked down in injury-conditioned neurons?
They do not regenerate an axon as well.
What is the effect of knocking down Luman in uninjured neurons?
Does not affect their ability to grow axons/neurites.
What is the significance of GRP78/BiP in the context of the UPR?
It plays a vital role in sensing and responding to cellular perturbations.
What does Luman/CREB3 localize to?
The axonal and neuronal ER membrane.
What occurs to Luman/CREB3 following axon injury?
It is cleaved and retrogradely transported to the nucleus to regulate gene expression.
What experimental technique was used to visualize Luman in neurons?
Tagging Luman with RFP and GFP and using dual immunofluorescence.
What does the presence of yellow-orange indicate in the dual immunofluorescence visualization?
Colocalization of Luman and GRP78 in the same axonal structures.
What happens to the GFP signal in the axon after injury?
Decreases, while GFP in the cell body and nucleus increases.
What is the outcome of treating uninjured sensory neurons with Luman siRNA?
Does not affect 24 hr neurite outgrowth.
What does treating injury-conditioned sensory neurons with Luman siRNA do?
Significantly reduces 24 hr axon regeneration/growth.
What is a key factor in the regenerative axon growth process?
Luman/CREB3 acts as a retrograde injury signal.
What is the role of the ER in cellular responses?
Vital for sensing and responding to cellular perturbations by altering protein and cholesterol biosynthesis.
What is the role of Luman in sensory axon regeneration?
Luman regulates the intrinsic ability of a sensory neuron to regenerate an axon by regulating the UPR and cholesterol biosynthesis.
UPR stands for unfolded protein response.
What does UPR stand for?
Unfolded Protein Response.
UPR is a cellular stress response related to the endoplasmic reticulum.
What is the significance of ER stress in axon regeneration?
The exact role of ER stress in assisting or hindering axon regeneration is not fully understood.
ER stands for endoplasmic reticulum.
What triggers the UPR in DRG and axons after sciatic nerve crush injury?
Sciatic nerve crush injury triggers the UPR in DRG and axons.
DRG stands for dorsal root ganglia.
What is BiP in relation to axons?
BiP is also known as GRP78 and is involved in the UPR response in axons.
GRP78 is a molecular chaperone in the endoplasmic reticulum.
What experimental method was used to study the regulation of UPR by Luman?
Microarray with 84 UPR-associated genes was used to assess whether Luman was regulating UPR.
This method helps in examining the expression of multiple genes simultaneously.
What was the effect of Luman knockdown on cholesterol biosynthesis genes?
Knockdown of Luman was associated with reduced expression of genes in the cholesterol biosynthetic pathway.
Regenerating axons require significant amounts of lipids/cholesterol.
What does the treatment with low dose tunicamycin (2 ng/ml) in vitro trigger?
It triggers the UPR at a level akin to injury and rescues impaired axon outgrowth associated with Luman knockdown.
Tunicamycin blocks N-linked glycosylation of proteins.
What is the primary function of GRP78?
GRP78 acts as a molecular chaperone in the endoplasmic reticulum, crucial for the UPR.
It plays a role in protein folding and quality control.
How does GRP78 behave differently in cancer cells compared to normal cells?
In cancer cells, GRP78 can migrate from the ER lumen to the cell surface, influencing tumor growth and progression.
This aberrant localization can promote various cancerous pathways.
What is csGRP78?
csGRP78 is a non-transmembrane peripheral membrane protein found in several cancer cell lines.
It is associated with several membrane proteins including GPI-anchored proteins.
What mediates the cell surface localization of GRP78?
Several proteins facilitate the cell surface localization of GRP78 during ER stress conditions.
This is significant for its role in cancer biology.
What is the consequence of ER stress on GRP78 localization?
Under conditions of ER stress, GRP78 can escape ER retention and pass through the Golgi apparatus.
This allows GRP78 to be expressed on the cell surface.
What is the relationship between Luman and axon outgrowth?
Knockdown of Luman decreased total axon outgrowth, which could be partially rescued by the UPR inducer tunicamycin.
This indicates Luman’s role in supporting axon regeneration.
What effect does tunicamycin have on Luman expression after siRNA treatment?
Tunicamycin does not elevate Luman expression after Luman siRNA treatment.
This is crucial for determining the mechanism of axon outgrowth rescue.
What is the function of the KDEL sequence in GRP78?
The KDEL sequence targets GRP78 to the ER lumen, preventing its release from the ER.
It also reroutes GRP78 back to the ER if it is released.
In which types of cancers has the expression of csGRP78 been reported?
The expression of csGRP78 has been reported in several cancers including:
* leukemia
* pancreatic cancer
* colorectal cancer
* breast cancer
* prostate cancer
csGRP78 is a variant of GRP78 found on the cell surface, associated with various cancer types.
What role does cell surface GRP78 (csGRP78) play in tumor cells?
csGRP78 contributes to:
* tumor cell motility
* cell-matrix adhesion
These functions are critical for cancer progression and metastasis.
Which cell proliferation pathways are stimulated by csGRP78?
csGRP78 stimulates the following cell proliferation pathways:
* PI3K/AKT
* MAPK
Activation of these pathways promotes tumor cell growth.
What effect does blocking csGRP78 with an anti-GRP78 antibody have?
Blocking csGRP78 with an anti-GRP78 antibody protects against cell invasion of a human colon adenocarcinoma cell line.
This suggests that csGRP78 may promote tumor cell migration and invasion.
True or False: Blocking csGRP78 has no off-target effects.
False
Many off-target effects complicate the translation of blocking csGRP78 into therapeutic strategies.
