Week 3 Flashcards
What is ER stress
Occurs whenever the protein-folding capacity of the ER is overwhelmed
What is the rough ER associaated with
Biosynthesis of secretory and membrane proteins protien glycosylation and maturation and folding
What does the smooth ER associated with
lipid biosynthesis
What are the two organelles calcium is stored
ER and mitochondria
What is cehmical stimuli of ER stress
Biochemical inhibitors which can influence N-linked protien glycosylation, calcium homeostasis, vesicular transport
What are pathological conditions
Viral infenctions
Human disease
What does the drug Tunicamycin do?
Inhibitor of N-linked protien glycosylation in the ER
What is the dolichol phosphate
A long-chain polyisoprenoid lipid that is embedded into the ER membrane
What happens when the oligosaccharide precursor is synthesized
Flips to the ER lumen and becomes available for N-glycosylation fo relevant proteins
What is N-glycosylation
Covalent beta linkage of the precursor to the amide nitroge of the aspargine residue
What enzyme catalyzes core oligosaccharide
N-acetyleglucosaime
What does Tunicamycin act as
Tight binding competitive inhibitor close in comparison to UDP prevent the transfer of N-acetylglucosamine 1 phosphate to dolichoil monophosphate
What else can Tunicamycin do
Induce apoptosis
What type of inhibitor is Thapsigargin
Non-competitive of sacro-endoplasmic rectiulum Ca Atpase.
What are SERCAs
ATP driven ionic pumps which move Ca ions agianst gradient from the cytosol to the endoplasmic rectium remain high CA in the ER lumen
Where can Thapsigargin bind
Enzyme and enzyme substrate complex reduicng velocity of Ca
What is the Ca ions in the cytoplasm
100 nM
What is the cell concentration in ER
1 micro meters
What does calcium ions act in
Signal transduction and the increase in the cytoplasmic calcium activates a variety of signaling systems in cells
What is the homeostatic gradient of Ca is respobsible for
Essential for proper functioning fo the ER which important for proper folding of proeteins occurs only at a high local concentration of a Ca
What happens when cells are treated with Thapsigargin
Inhibit SERCA as a result of Ca ions will diffuse from the ER lumen into the cytoplasm
What is Brefeldin A
A macrocyclic lactone that inhibits vesicular transport between ER and Golgi
What is BFA
Targets and blocks the recruitment of a protein factor ARF1
What is ARF1
The small GTPase ADP-ribosylation factor 1which you need for COP1-coated vesicles
What is COP1 vesicles
They mediate retograde transport of KDEL with soluble proteins which are needed for ER to carrry out it’s function
What is KDEL
It is a sorting signal sequence at the C-terminal end of proteins which have to be located in the ER
What happens if COP1 transport is inhibited
The ER luminal proteins will not be returned to the ER and their recycling will be inhibited which increases the amount of unfolded proteins inducing the ER stress
What are some diseases releated to ER stress
neurodegeneration, stroke , bipolar disorder, cardiac, cancer, diabetes, muscle degeneration
What is the unfolded protein response
When an accumulation of unfolded proteins in the ER activates a series of homeostatic responses
What do cells do under mild stress conditions
Cell survival: refolding or elimination of misfolded proteins activate UPR mechanisms
What do cells do under severe stress
They trigger apoptosis
What is BiP
and Hsp70 molecular chaperone also known as GRP78 a sensor for the ER stress pathway
What does BiP do in unstressed cells
Mediates proper folding of proteins and keeps signalling molecules inactive
What happens in with BiP in stressed cells
BiP preferentially binds to the unfolded proteins over the luminal domain of effectors the signalling molecules can signal to start the UPR cascade
What are the three major effector branchers of the UPR
Insoitol- requring enzyme 1 (IRE1)
Protein kinase RNA-like ER kinase (PERK)
Activating transcrption factor 6 (ATF6)
What do the three major effector branches of the UPR
Block general protein translation
Increase chaperone expression
Enhance ER-associated protein degradation
What are the three domains of IRE1
Transmembrane domain
ER luminal domain
Cytosolic domain
What is located in the ER luminal domain of IRE1
Binding BiP and an oligomerization site
What is apart of the cytosolic domain
A protein serine/threonine kinase domain (phosphorylated different types of protiens)
endoribonuclease (RNAse) domain (cuts RNA)
What is IRE1 considered
A bifunctional enzyme
What are IRE1 substrates
IRE1 itself (trans-autophosphorylation)
mRNA molecule which encodes a transcription factor called XBP1
What is the homolog of XBP1 in yeast
HAC1
What is the process of singlaling of IRE1 in mild ER stress
BiP doesn’t bind too IRE1 which allows it to trans-autophosphorylation on the cytsoloic tail once is phosphorylates it lead to allosteric activation of the adjacent RNAase which then goes to splice a 26 intron from the unspliced XBP1. XBP1 now moves the nucleus and activates the transcprtion genes in UPR
What is the process of signalling by IRE1 in severe ER stress
IRE1 oligomerizes and activates each other. The pathways it then goes to activate are 1. signalling pathways leading to apoptosis 2. promote degradation of miRNA targeting pro-apoptotic genes 3. promote the cleavage of various ER-localized mRNA
What is the difference between oligomerized IRE1 and IRE1 dimers
Oligomerized IRE1 induced cell apoptosis while dimers induce cell adaptation
How much longer is unspliced XBP1
26 nucleotides longer
What test is used to detect XBP1
Cells are treated with thapsigargin and XBP1s is expressed more in treated cells in PCR
What atare the three domains of PERK
- Transemembrane domain
- ER luminal domain (BiP and oligomerization)
- Cytosolic domian, Kinase and RNase
What are PERKS substrates
PERK itself and elF2 alpha
What is the signaling by PERK in association with Bip in unstressed cells
PERK is in a repressed state through an association with BiP
What is the signaling by PERK in stressed cells
BiP release and dimerization of PERK which leads to trans-autophosphorylation of the cytoplasmic domain PERK than goes to phosphorylate elF2 which inihbits the elF2 activity and hence slows down global protein translation and ATF4 is upregulated can either be pro surival or prod death pathways
What are pro survival genes
XBP1 chaperones and antioxidant enzymes
What are pro death gene
which encodes a transcrption factor that inihibit the expression of antiapoptic BCL-2 and upregulate BIM mitohcondridal dependent apoptotic pathway
What makes the structure of ATF6 different thatn IRE1 and PERK
The c terminal is in the ER luminal
What are the three domains of ATF6
- Transmembrane domain
- ER luminal domain Bip binding domain and golgi localization sequences an golgi localization sequences
- Cytosolic domain which contains domains characterstic for transcrption factors bZIP and transcrption activation domain
What is apart of the Golgi Localization Sequences
GLS1 and GLS2
What can BiP bind too in ATF6
Only GLS1 and GLS2
What is the signaling by ATF6 in unstressed cells
ATF6 is maintained in the ER through its association with BiP that binds to GLS1
What is the signalling pathway of cells under ER stress
BiP is no longer bound too ATF6 so and GLS1 and GLS2 help move it to the Golgi in the Golgi ATF6 is the processed by Site-1 and Site-2 proteases releasing the cytosolic domain fragment ATF6f moves to the nucleus which can now upregulate UPR genes
How can ATF6 translocation to the ER to the nucleus be detected
Fluroescence microscopy that have the GFP-ATF6 plasmid and you treat it with tunicamycin for different periods of time
What does DAPI bind too
AT clusters
What was observed in the fluroence microscopy
That untreated the ATF6 is located mostly in the cytoplasma and when treated is then found in the nucleus
What is the steps to perform qPCR
Isolate total mRNA from control and treated cells, make cDNA and run qPCR using oligonucleotide primers specific to your gene of interest then you analyzed the expression of the genes
What is BiP
GRP78
What do the UPR response elements
ERSE
ERSE II
UPRE
What do these response elements do
They bind the transcription factors of the ER stress but differes in specificity
Does gene expression alway correlate with the expression of relevant proteins
NO
What is western blotting
You move the electrophoresis transfer it to membrane apply a primary memebrany then the secondary antibody and then it can usally by chemiluminescence
What was upregulated in the western blotting
CHOP sXBP1 phospho-elf2a and BiP
What is imumunodetection
You are able to see where the actual protein is
What is the process of direct antibody imumunodetection
Cells or tissues are fixed on glass slides and treated with specific antibodies the primary antibody is attached to an enzyme this is then detected by adding substrate. The antibody primary antibody is conjugated by fluorescence microscopy
What is the down side of direct detection
Signal amplification is not possible the signal may be difficult to detect if the protein is in small quantities. There is also interference make it harder for the antibody to bind to it’s protien
What is a benefit of the direct imumunodetection
You can use a variety of colors for furoscenets the only limit is how many colors the microscope can detect
What is the indirect method of imumunodetection
It employs a secondary antibody with specificity against the unlabeled primary antibody to amplify the primary signal amplification is possible because of multiple secondary antibodies
What is an advantage of the indirect immunostaining
As a higher specificity of immunodetection because the primary antibody is not chemically modified and can bind more strongley to the protien
