Protein Misfolding

Question 1

Describe random coil model

Answer 1

Has properties of a chain with random flight
Steric limitations
Doesn’t favour a particular conformation, meaning all the conformations have the sane probability
Has spectroscopic properties of unfolded proteins. Higher 15N, lower 1H

Question 2

Describe molten globule states

Answer 2

Hydrophobic collapse, resulting in liquid-like sidechains

Question 3

Describe hydrogen exchange labelling

Answer 3

Solvent is switched to D2O, so H is more likely to be exchanged at the NH sidechains on the outside in contact to water.
NH groups in H-bonds or a tightly packed structure are protected
NMR or mass spec can map positions of these NHs

Question 4

What levels of UPR take place in cells?

Answer 4

Short term- reduced protein load
Medium term- Increased machinery processing unfolded proteins, e.g ER size increases and secretory pathway
Long term- cell death

Question 5

What is the direct binding model of UPR?

Answer 5

IRE-1 and PERK bind unfolded proteins in the ER and dimerise/oligomerise to form a binding site

Question 6

What is the BiP binding model of UPR?

Answer 6

Hsp70 (BiP) binds IRE-1, PERK and ATF6
With more unfolded protein, free BiP decreases, so BiP dissociates, inducing IRE-1 and PERK to dimerise.
ATF6 is released from the ER and moves to golgi where it is proteolysed
IRE-1 can dimerise without unfolded protein, but BiP prevents this unwanted action

Question 6

How can IRE-2 action lead to cell death?

Answer 6

Phosphorylated IRE-1 attracts TRAF2 which allows JAK signalling and possibly cell death an insulin resistance

Question 6

Describe IRE-1 action

Answer 6

1. Unfolded protein is sensed, causing dimerisation in the ER
2. Unfolded protein autophosphorylates in the cytosolic domain, increasing affinity for nucleases and kinase and RNase activity.
3. IRE-1 cleaves XBP1 mRNA and the ends religate, causing a frameshift
   Spliced XBP1 mRNA is a transcripitional activator of ERAD
   Unspliced XBP1 mRNA inhibits UPR

Question 7

Describe PERK signalling

Answer 7

1. In response to unfolded protein, PERK dimerises in the plane of the ER and autophosphorylates
2. eIF2a is phosphorylated, inhibiting eIF2B so eIF2-GTP cannot form
3. Global translation drops
4. ATF4, XBP1 and UPR Genes activated, e.g amino acid transporter that protects against oxidative stress.

Question 8

Describe ATF6 response

Answer 8

1. Upon stress, BiP dissociates from ATF6. No dimerisation
2. ATF6 moves to golgi where S1P and S2P cleave it
3. Cytosolic segment ATF6f moves to nucleus and activates UPR genes
4. e.g XBP1. Also CREBH activates acute-phase response genes encoding inflammatory proteins

Question 9

How is the UPR involved in immune response?

Answer 9

UPR and ERAD cause foreign proteins to degrade and cells present MHC1 as the proteasome produces antigens against it.
Viruses exploit secretory system and UPR blocks transcription and translation, killing cells

Question 10

How is the UPR involved in cell differentiation?

Answer 10

UPR may respond to low phospholipid and high cholesterol for membrane synthesis.
May aid differentiation into cells with secretory functions as it enlarges ER
UPR targeted for cancer therapies, neurodegenerative diseases and viral infections

Question 11

How is the UPR involved in autophagy?

Answer 11

UPR triggers it to clear protein debris

Question 12

Describe autophagy

Answer 12

1. Ubiquitin tags cargo proteins to be degraded
2. Atg8 adaptor protein links this to autophagosome membrane
3. Membrane closes and lysosome fuses
4. Autolysosome matures and material is degraded by low pH