MT4- COPII etc Flashcards
sequence of events in ER if correct folding?
- glucosylase I and II trim two terminal glucoses.
- ER chaperones recognise eg. Calretriculum and ER57 to aid folding.
- If correct folding, terminal glucose removed from Mannose and packages into a vesicle for export at ER exit sites.
What is the Unfolded protein response?
This is a stress induced pathway which stops protein synthesis but increases synthesis of ER proteins and ER to respond to the overload of unfolded proteins. In response to too much protein for chaperones to deal with.
Yeast UPR?
Yeast upregulates over 300genes in response.
Overview of the UPR?
- After activating UPR, misfolded protein sensors are activated.
- These activate ER stress signals IRE1, PERK and ATF6. IRE1 causes mRNA splicing so initiates translation of genes. PERK reduces other protein translation by P translation initiator factors, but selects TF’s. ATF6 regulates proteolysis releases gene regulaory proteins. All upregulate TF.
- Downstream effect of all to upregulate genes to increase ER folding capacity.
ER stress signal proteins?
ATF6, IRE1, PERK. These are present on the ER membrane and assosicated with ER chaperone called BiP, held an inactive state. But when lots of unfolded protein BiP chaperone has a higher affinity for these so dissociates, and they can then go on to cause their downstream effects.
ER stress/oxygen deprival affect on cell survival?
Certain amount can cause cell survival mechanism by UPR, but prolonged too long or too strong can cause cell death signals. Both are activated but depends on the balance between.
Downside of the UPR?
Cancer cells can use to activate angiogenic factors in tumour to increase O2 and glucose to it.
How does ATF6 upregulate UPR genes?
Bip has a higher affinity for this so dissociates from ATF6. This enables ATF6s translocation to golgi. S1P and S2P proteases cleave AFT6 into cytosolic fragment which can then migrate to the nucleus and upregulate UPR genes.
This is done as a summative effect, so lots of proteins misfolded, lots of BiP dissociates, so lots of ATF8 translocation, lots of UPR genes activated.
Cholesterol homeostasis?
Regulate cell cholesterol synthesis, depending on diet intake. SREBP in SER regulates genes for cholesterol synthesis.
High cholesterol? Low?
High cholesterol: SCAP cholestol sensor (SREBP cleavage activating protein) ensures SREBP TF associates with an Insig ER chaperone and keeps it in the ER.
Low: SCAP allows dissociation from Chaperone, SREBP translocates to golgi, cleaved by proteases (same that cleave ATF6) so form can translocate to nucleus and act upon genes.
WHat is autophagy?
Autophagy is used by cells under stress to engulf their own cytoplasm and organelles. Forms an autophagosome around organelles to fuse with lysosomes to be degraded.
What can activate autophagy?
The ER often activates e,g can engulf extra ER made during UPR.
Cancer cells can use to get nutrients by lysing cells and releasing their nutrients.
Alzheimers cause?
Mutations can cause cleavage of the protein at different points e.g. 42AA instead of 40, and so create folding problems.
CRT? -/- impacts?
Calreticulin in ER binds to misfolded protein and prevents their export. Binds to sequester Ca.
Cardiac problems.
CNX -/- impacts?
Calnexin TM ER chaperone, quality control and assisting folding.
large myelinated fibres- dysmyelination
Glucosidases function?
Glucosidase I and II trims the first two glucose residues before folding, and then II trims the terminal glucose after chaperones ensure correct folding. UGGT adds the terminal glucose back on if incorrect folding so they are recognised by chaperones to give them another chance at folding.
Difference between CNX, CRT K/O and BiP?
All ER chaperones.
BiP fatal, suggests essential for all cultured cells, wheras CNX and CRT more tissue specific.
CNX- Dysmyelination
CRT- cardiac problems
3 examples of ER folding diseases?
CFTR.
Emphysema
Hypothyroidism
If downregulate which chaperone, helps CFTR DF508?
Hsp90 co-chaperone Aha1 downregulation by SiRNA.
This is an ATPase regulator.
Increase in not only band B but also band C (suggesting golgi translocation)
-with a 60% Aha1 K/D, 50-60% reduction in HSP90 bound to CFTR in immunoblots was seen. partial rescue in conductance also.
Emphysema cause?
Deficiency in secretion of alpha1-antitrypsin.
Trypsin is a protease thats secreted into the lungs and creates gaps in the tissue, altering permeability. Anti-trypsin is secreted to stop this here.
genetic predisposition but smoking increases risk.
Emphysema symptoms?
Lung tissue (alveoli) digested and gaps appear. Less surface area for gaseous exchange.
Hypothyroidism cause?
Production of mutant thyroglobulin, which is a precursor to the thyroid hormones. As compensation the Er undergoes massive proliferation, but too high UPR can act as a death signal to cells.
ALS/Motor neuron disease?
(either sporadic or familal) Death of motor neurons resulting in muscle wasting.
ERAD impairement, altered trafficking and protein aggregation which can result in autophagy.
ER stress signals associated with UPR.
ALS/Motor neuron disease and UPR?
PERK activation- gives protection (therapy shift towards?)
IREalpha- degeneration by autophagy or apoptosis.
How was COPII orginally found?
Orginally identified in Sec mutants, because without proteins can’t leave the ER to golgi.
Biochemically v different to ER- lipid and protein content v different so can separate.
Ultrastructurally v different- can see under electron microscope
COPII function?
Coat vesicles for cargo from ER to Golgi.
Function of Sar1?
small GTPase- initiates coat formation and budding following recruitment by sec12
Function of Sec12?
GEF-on ER membrane- recruits Sar1 to initiate coat formation.
What is a GTPase?
Enzyme that has GTP/GDP bound. In active state has GTP bound but then hydrolyses GTP to GDP as energy. as it activates another enzyme e.g. a GAP.
GEF removes the GDP to add GTP again.
WHat is a GEF?
Guanine exchange factor- removes GDP from a GTPase and add GTP to activate.
