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ANAT 212 > Lecture 10 > Flashcards

Lecture 10 Flashcards

1
Q

how does the ER sustain protein quality control

A

N linked glycosylation - only asparagine
asp-aa-ser-threo polysaccharide = always the same
PTM

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2
Q

where are all secretory pathways synthesized and folded

A

at the ER

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3
Q

what are degraded in ER

A

misfolded secretory proteins

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4
Q

how are misfolded secretory proteins degraded at er

A

ub proteasome system
only pathway for transport of secretory proteins back to cytosol

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5
Q

where else can proteins be degraded - secretory pathway

A

lysosomes at end of pathway
digestion by proteases inside lysosome

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6
Q

what is the ER

A

protein quality control checkpoint for all organelles in the secretory pathway

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7
Q

do proteins of secretory pathway ever touch the cytosol

A

NEVER
but
Exception = not folded proteins properly = will seep out to cytosol to be degraded

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8
Q

name ER chaperones

A

BiP (HSP70) = ERdj proteins (DNAJ cochaperones), NEF cochaperones
GRP94 (HSP90) - no co-chaperones
thioredoxin family - PDI and ERp57

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9
Q

name parts of ER N linked glycosylation

A

calnexin and calreticulin
UGGT (UDP-glucose:glycoprotein glycotransferase)
glucosidases, mannosidases, lectins (glycans binding)

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10
Q

name parts of ER misfolded protein degradation

A

degradation takes place on cytosolic proteasomes
folding is necessary to exit er to secretory pathway

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11
Q

what is the ER stress response

A

unfolded protein response = UPR

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12
Q

describe BiP cochaperones

A

Substrate-binding DNAJ (ERdj3) assists folding
Translocon interacts with proteins that act on nascent polypeptides
folding and modification takes place during translocation

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13
Q

describe how translocon interacts - BiP and cochaperones

A

signal peptidase
OST = oligosaccharyl transferase
sec63= specialized TM DNAJ that recruits BiP to translocating polypeptides - does not bing substrate directly

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14
Q

describe protein disulfide isomerase

A

ER lumen is an oxidizing environment, but spontaneous disulfide formation is inefficient and/or incorrect for folding

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15
Q

describe how disulfide formation happens - gen

A

catalyzed by thioredoxins = helps disulfide bonding, chaperones, 2 cysteines
PDI and ERp57 - have 2 reactive Cys residues close together and can oxidize substrate
if proteins not folded properly = still wants to make v strong disulfide bonds but its bad so regulated by PDI and ERp57 - want cysteines stabilizing native state to be formed

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16
Q

describe disulfide isomerization - gen

A

Oxidized PDI catalyzes formation of disulfide bonds in substrate= PDI becomes reduced (A)
Reduced PDI aids rearrangement of disulfide bonds during folding (B) = first disulfides may not be correct for native state, native disulfides are most stable
in both reactions = PDI forms mixed disulfide intermediates with substrate

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17
Q

describe disulfide isomerization - A

A

PDI = engaged in disulfide bonds =one of PDI and one of protein
direct disulfide bonding of 2nd cyst then makes specific disulfide bond
multistep
reducing pdi = must reoxidize PDI so can direct disulfide bonding of proteins - wants to always be available and active

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18
Q

describe disulfide isomerization - B

A

specific order must be followed so protein folds properly
PDI = helps correct wrong disulfide bonding
helps break disulfide bonds by making disulfide bonds to own cysteines = stepwise

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19
Q

describe PDI regeneration - gen

A

a chemical cascade regenerates oxidized PDI
oxidation potential in ER is maintained by controlled by enzymes = PDI, Ero1

20
Q

describe PDI regeneration - 4 steps

A

1- PDI becomes reduced after oxidizing the substrate
2- PDI is oxidized by Ero1 protein with cofactor FAD; Ero1 is reduced
3- Ero1 = regnerated by FAD
4-FAD = regenerated by O2 (only needs oxygen)

21
Q

what is CNX and CRT

A

calnexin = cnx
calreticulin = crt
highly homologous

22
Q

describe CNX

A

~50kda lumenal domain and a TM helix anchor - always attached to er membrane facing lumen

23
Q

describe CRX lumenal domain and CRT

A

Recognizes glycan pattern on polypeptides (N linked glycosylation) - glycan binding domain = lectin
binds to thioredoxin (ERp57)

24
Q

describe CRT

A

lumenal domain but with no TM helix
has signal for retention in ER

25
Q

describe calnexin binding

A

N linked glycan = branched mannose polymer with 3 glucose residues - always the same polysaccharide
glucose and mannose are trimmed off step by step
CNX specifically binds the glycan with 1 glucose - keeps polypeptide in ER, single glucose is a signal for incomplete folding but is eventually removed

26
Q

describe calnexin binding - informally

A

3 glucose = immature protein that just entered er
IN er = ENZYMES trim glucose - 1st and 2nd off
then when one glucose left = recognized by cnx and crt
going to hold protein by binding polysaccharide with only one glucose and gives time to fold
cnx and crt do not interact with protein they interact with this ptm but only when has one glucose
give time for protein to fold
once released = glucose trimmed then only mannoses left = then if mannoses trimmed = protein seeped out into cytosol and degraded by proteasome = bc if less mannoses = indicates it didnt fold properly

27
Q

describe ER N LINKED GLYCOSYLATION ROLE IN PROTEOSTASIS - uggt

A

UDP-glucose:glycoprotein
glucosyltransferase
Binds non-native polypeptides and reattaches a glucose to the glycan – CNX can bind again
Glucosidase removes Glc from native and non-native polypeptides Mannosidase trims sugars further- does not recognize shortened glycosylation

28
Q

describe ER N LINKED GLYCOSYLATION ROLE IN PROTEOSTASIS - uggt - iNFORMALLY

A

glucose trimmed, one left + mannose –> calnexin binds ptm and thioredoxin ERp57 helps with disulfide bonding –> glucosidase = trimmed, unfolded = hydrophobic aaas on surface = recognized by UGGT –> if successfully folded = exit from er –> if not = adds another glucose and goes through another cycle
given many cycles since v taxing to make protein = given many chances

29
Q

describe calnexin and calreticulin cycle

A
  1. CNX keeps polypeptide in ER
  2. Glucosidase removes the last Glc
  3. UGGT restores Glc on misfolded polypeptides – CNX binding
  4. Folded polypeptides do not have Glc restored and exit to Golgi
  5. Mannosidase trims glycans without Glc = slow, irreversible and proteins retained in ER by chaperones likely to get trimmed
  6. Mannose-binding lectins (EDEM) select short glycans for degradation
30
Q

what is not recognize by uggt

A

Native folded polypeptides are not recognized by UGGT

31
Q

what is ERAD

A

ER associated degradation

32
Q

what does ERAD degrade

A

both lumenal (soluble and transmemebrane polypeptides)
many substrates are misfolded proteins = quality control, before proteins are sent to rest of secretory pathway
also regulated degradation in response to signals - regulates metabolism

33
Q

name 3 steps of ERAD

A

1- substrates are recognized and brought to E3 ub ligase complexes (of misfolded)
2- E3 complex polyubs substrate and retrotranslocates (dislocates) substrate into cytosol (kinda like pore = brings er lumen/membrane proteins from er to cytosol)
3- substrate is deglycosylated and degraded by proteasomes *ubiquitination degradation

34
Q

what is step 1 of erad

A

recognition - BiP to ERAD

35
Q

describe recognition - BiP to ERAD

A

Substrates which cannot fold are prevented from aggregating by BiP
BiP binds substrate in complex with specialized DNAJ (ERdj5) and
lectin (EDEM)= ERdj5: J domain and thioredoxin domain, catalyzes breakage of disulfide in substrate, reverse of PDI
Complex targets substrate to E3
want to make not bulky = fold as much as possible

36
Q

describe erad steps - generally

A

lectin= recognizes polysaccharides when no 3 glucose and trimming mannoses
disulfide isomerases = breaks there
E3 ub ligase = polyubs protein = want to be degraded - targeted
N glycanase cuts polysaccharides
AAA atpase helps pull polypeptide out - through retrotransposon
like in between 1st and 2nd polyubs

37
Q

describe targeting and export - substrate adaptors and E3 ligases

A

TM E3 LIGASES (HRD1 and gp78) form complexes with substrate recognition adaptors - many adaptors allow misfolded proteins to be targeted = misfolded lumenal proteins (SEL1L), mannose binding lectins (EDEM), misfolded TM proteins (erlin 1/2, derlins), chaperones (BiP)
other interactions = derlins, p97 receptors

38
Q

describe retrotranslocation - pore

A

still in question if retrotranslocon requires a true pore
regulated opening and closing is pore like
but large enough to allow n linked glycans
bigger so can fit protein - get through retrotranslocon to cytosol

39
Q

describe retrotranslocation - polyubs

A

polyubiquitination in cytosol needed

40
Q

describe retrotranslocation - what performs functions

A

large tm E3 ligase complexes are thought to perform all of these functions
HRD1 and gp78 E3 ligases = homologous, multiple TM helices

41
Q

what is retrotranslocon assisted by

A

cytosolic protein p97/VCP - pulling or unfolding activity

42
Q

what is only in the cytosol

A

n end rule
chip
SCF E3 ligases

43
Q

describe p97 mechanism

A

n linked glycans and polyub too large to fit through pore
helps pull protein out of er
p97 forms complexes with other proteins = ub binding adaptors, peptide: N glycanase (PNGase) removes glycans (before goes into aaa atpase), DUBs remove poly ub - but E3s re ub after extraction to complete targeting to proteasome
hexamer = limited diameter so if have poly ub wont fit into shape

44
Q

describe AAA proteins

A

large diverse superfam of atpases with many diff functions
usually hexameric rings

45
Q

describe p97 and AAA proteins

A

unfoldase subunits of proteaseome 19s regulatory
p97: homo hexamer of 97kda subunits = uses atpase energy to extract proteins from membrane, substrate is also threaded through the central pore (hole)
ub then de ub –> aaa atpases (unfolded here) –> polyubs –> goes to proteasome

46
Q

describe erad summary - 7 steps

A
  1. Misfolded polypeptides are recognized by lectins and chaperones
  2. Disulfide bonds are broken by a thioredoxin DNAJ
  3. Adaptors bring misfolded polypeptides to E3 ligases – lectins, chaperones, lumenal and TM adaptors
  4. Transmembrane E3 ligases polyubiquitinate substrates and start retro-translocation
  5. p97 ATPase helps extract substrates from membrane
  6. Substrates are de-glycosylated (PNGase)
  7. Substrates are recognized by proteasome or shuttling receptors

ANAT 212 (34 decks)

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