Lecture 11

Question 1

what is UPR

Answer 1

unfolded protein response
ER proteins
can promote cell death if stress too severe
repress making proteins to reduce load = repress gene expression pathway
but increases activation of expression of chaperones

Question 2

how is upr activated

Answer 2

accumulation of unfolded proteins in er
reductive stress - breaks disulfide bonds
glycosylation inhibitors
loss of calcium

Question 3

describe what happens during upr

Answer 3

transcription of er chaperones, erad components, lipid synthesis = upregulated, allows er to expand in size = make er bigger so misfolded proteins will not aggregate
induces cell death - apoptosis if response insufficient
limited to er, no over lap with hsr
Signalling has to cross er membrane - tm communication system - be encoded by nucleus of cell

Question 4

how many upr signalling pathways

Answer 4

3

Question 5

describe upr signalling pathways

Answer 5

IRE1 = rely on dimerization of protein and their phosphorylation, both activate transcription of proteins (transcription factors), activate transcription of ER components
PERK = same as IRE1
ATF6 = more direct pathway = stays as monomers but ptm = clevage of protein - must be first transported to golgi, cleavage product = transcription factor

Question 6

describe IRE1 and XBP1 - gen

Answer 6

IRE1 = monomer, stays bc bip
binds unfolded proteins then dimerizes
phoshorylates itself = ptm
cleavage domain
XPB1 recognizes intron and excises then can code functional protein
splicing event in cytosol
then goes to nucleus

Question 7

describe IRE1 and XBP1 - 6 steps

Answer 7

1. IRE1 has a lumenal domain, kinase and RNase domains
2. IRE1 dimerizes in response to unfolded proteins
3. Autophosphorylation activates RNase activity
4. XBP1u (unspliced) is translated at very low levels
5. IRE1 splices out 26 base intron, frameshift allows XBP1s (spliced) to be translated efficiently
6. XBP1s is a transcription factor that upregulates UPR genes

Question 8

describe IRE1 activation

Answer 8

direct binding of unfolded protein by 2 IRE1 causes dimerization
BiP(HSP70) bind inactive IRE1 and prevents dimerization
BiP binding to unfolded protein releases IRE1 to form dimers
both mechanisms activate IRE1

Question 9

describe PERK - gen

Answer 9

tm monomer then dimer
self phosphorylates
eIF2a = also phosphorylated, usually recognizes met = if phosphorylated then no transcription initiation
so mrna escapes
can be translated fast, mrna already in cytosol, effort of cell to repress expression of proteins
if stress response continues = activates cell death

Question 10

describe PERK - 5 steps

Answer 10

1. PERK has a lumenal and a kinase domain
2. PERK dimerizes upon stress and autophosphorylates = same activation mechanism as IRE1
3. Phosphorylates translation factor to inhibit translation
4. Certain mRNAs are not inhibited = ATF4 transcription factor
5. ATF4 = expression of more XBP1, CHOP – activates apoptosis genes, decision: recover from stress or commit to cell death

Question 11

what is eIF2alpha

Answer 11

translation initiation factor

Question 12

what is ISR

Answer 12

integrated stress response

Question 13

when is ISR used

Answer 13

er stress
viral infection
amino acid starvation
heme deprivation
proteasome inhibiton
UV radiation

Question 14

describe ISR

Answer 14

Translation initiation factor eIF2α turns on general translation
Phosphorylated eIF2α is inactive
eIF2α Kinases respond to different stresses to
inhibit translation= decrease amounts of unfolded new
proteins, special mRNAs including ATF4 are still translated to promote cell death
PERK – ER stress
Other kinases – proteasome inhibition (HSR) starvation, viral infection, etc

Question 15

describe AFT6 gen

Answer 15

only visible in stress
has motif –> transport from ER –> golgi
but usually hidden by chaperones
part in cytosol becomes transcription factor
then cleavage and activates expression

Question 16

describe AFT6 - 6 steps

Answer 16

1. ATF6 is normally transmembrane at the ER - BiP binding site masks ER exit signal
2. Upon stress, BiP is competed away by unfolded proteins
3. ATF6 is transported to Golgi
4. Golgi proteases cleave off cytosolic domain ATF6(N)
5. ATF6(N) is a soluble transcription that upregulates UPR genes

Question 17

describe CFTR

Answer 17

cystic fibrosis tm regulator
mutation in proteins of secretory pathway
lungs not well hydrated = dry much mucous, accumulates bacteria
chloride channel that maintains hydration in lung airways
mutation causes loss of CFTR function in cystic fibrosis
first hereditary disease to have gene identified and sequenced
er translocation, chaperone assisted folding, erad

Question 18

describe CFTR sequence

Answer 18

1480 aas
12 tm helices
2 cytosolic nt binding domains (NDB)
cytosolic N terminus and loops (in cytosol so type 2)

Question 19

describe CFTR sequence translocation

Answer 19

TM1 and TM7 act as signal anchors
charges around TM1 and TM7 determine orientation in membrane
other tm helices are threaded in and out
N linked glycan attached during translocation
calnexin

Question 20

describe CFTR structure

Answer 20

Most common disease mutation is deletion of Phe in NBD1 (ΔF508)
Mutation disrupts hydrophobic core of NBD1 and its folding
Mutant NBD1 cannot interact correctly with TM helices or NBD2
Mutant CFTR is retained in the ER and degraded, instead of trafficking to the plasma membrane
hydrophobic collapse wont happen = protein wont fold in cytosol = affects structure so ER degrades, no chloride transport or hydration of lungs

Question 21

describe CFTR folding

Answer 21

Normal CFTR requires cytosolic chaperones to complete its folding
DNAJ and HSP70
Transfer from HSP70 to HSP90 via complex by TPR co-chaperone HOP
Other HSP90 co-chaperones help complete folding
CFTR goes through calnexin cycle because of N-linked glycan (adds polysaccharides, mannose trimmed = erad, tm protein = pulled out to er to be degraded)

Question 22

describe CFTR ERAD

Answer 22

Misfolded mutant CFTR is selected for ERAD by E3 ligases
HSP70-CHIP complexes in the cytosol
TM E3 ligase complexes in the ER (gp78-RMA1)
Specialized co-chaperones also promoted ERAD
p97 helps to extract mutant CFTR from membrane