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BIOC 212 > ER protein quality control > Flashcards

ER protein quality control Flashcards

1
Q

What are 2 possible degradation outcomes for proteins fo the secretory pathway?

A
  1. Misfolded secretory proteins → Ub-proteasome system in cytosol
  2. Digestion by proteases inside lysosomes at the end of the pathway
    *No degradation occurs in the ER
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2
Q

Where is the protein quality control checkpoint for all organelles in the secreotry pathway?
Why?

A

ER
Because folding is required to exit the ER to the secretory pathway

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3
Q

What are the different ER chaperones?

A
  1. Bip (HSP70) →ERdj proteins (~DNAJ co-chaperones) and NEF co-chaperones
  2. GRP94 (HSP90) → no co-chaperones (not like its cytosolic equivalent)
  3. Thioredoxin family - PDI and ERp57
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4
Q

What are the proteins of the ER responsible for ER N-linked-Glycosylation

A
  • Calnexin and calerticulin
  • UGGT → UDP-glucose:glycoprotein glycotransferase
  • Glucosidases, mannosidases
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5
Q

How is the ER stress response called?

A

Unfolded Protein Response (UPR)

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6
Q

What is Sec63?

A

Specialized transmembrane DNAJ that recruits BiP to translocating polypeptides → folding and modifications take place during translocation

*Sec63 does not bind the substrate directly

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7
Q

Which proteins does the translocon (Sec61 translocon) interact with in the ER side?

A
  • Signal peptidase
  • OST (oligosaccharyl transferase)
  • Sec63 (DNAJ that recruits BiP to translocon)
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8
Q

What is the ERdj3?

A

ER equivalent of DNAJ → co-chaperone to BiP to assist folding of proteins

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9
Q

What is responsible for catalysation of disulfide bonds in ER lumen?

A

Thioredoxins → PDI and ERp57

They had reactive Cys residues close together → can oxidize substrate to break wrong disulfide bond

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10
Q

Why is it important that disulfide bonds are catalysed and controlled as proteins get into the ER?

A

As proteins get into the ER, they want to make disulfide bonds, but if it happens before the protein is properly folded, stabilizes a misfolded state. Disulfide bonding should stabilize the native state so PDI and ERp57 interact with Cys to prevent them from making prematured or wrong disulfide bonds

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11
Q

What is the process of PDI regeneration?

A

*To direct disulfide bonding, oxidized PDI becomes reduced → has to reoxidize

  1. PDI becomes reduced after oxidizing the substrate
  2. PDI is oxidized by Ero1 protein with cofactor FAD → Ero1 is reduced
  3. Ero1 is regenerated by FAD+ → FAD+ is reduced to FADH2 +
  4. FAD is regenerated by O2 –> FAD+ and H2O2
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12
Q

What are the roles fo calnexin (CNX) and calreticulin (CRT) ?

A

CNX has ~50 kDa lumenal domain + a TM helix anchor
CRT has a lumenal domain, no TM helix, but has a signal for retention in the ER

Both (CRT and CNX lumenal domain):
- recognize glycan pattern on polypeptides (N-linked glycosylations)
- bind to thioredoxin (ERp57) → helps catalyze disulfide bonds as calnexin gives it time

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13
Q

What are 2 important parts of the the Calnexin lumenal domain?

A
  • Glycan binding domain → lectin
  • ERp57 binding site
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14
Q

What does CNX bind to specifically on ER proteins?
What is its role?

A

Binds to glycan with 1 glucose on glycan (2 have been trimmed off)
*No interaction with the protein, only with the glycan
- Keeps the polypeptide in the ER
- Single glucose is a signal for incomplete folding so CNX (chaperone) gives it time to fold?

CNX → chaperone → assists folding + quality control (ensuring only properly folded proteins go furthur in the secretory pathway)

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15
Q

What are the meanings of 1, 3 glucose and trimmed mannose on the N-linked glycan?

A
  • 3 glucose = immature protein → just got into the ER
  • 1 glucose = recognized by CNX and CRT
  • Mannose start to be trimmed off → signal for degredation
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16
Q

What is the role of UGGT?

A
  1. Binds non-native polypeptides → reattaches a glucose to the glycan → CNX can bind again

Native folded polypeptides are not recognized by UGGT

UGGT = UDP-glucose:glycoprotein glyucosyltransferase

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17
Q

What is the role of glucosidase?

A

Glucosidase removes Glucose from native and non-native polypeptides

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18
Q

What is the role of Mannosidase?

A

Mannosidase trims sugars further → UGGT does not recognize shortened glycosylation

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19
Q

What are the steps of the calnexin and calreticulin cycle?

A
  1. CNX keeps polypeptide in ER
  2. Glucosidase removes the last glucose
  3. UGGT restores glucose on misfolded polypeptides → CNX binding

Exit 1 - Folded polypeptides do not have glucose restored and exit to Golgi

Exit 2.1 - Mannosidase trims glycans without glucose → slow, irreversible, proteins retained in ER by chaperones likely to get trimmed
Exit 2.2 - Mannose-binding lectins (EDEM) select short glycans for degradation

If not exited, UGGT restores glucose on it for further folding

20
Q

What is ERAD?
What proteins does it affect?

A

ER associated degradation

ERAD degrades both lumenal (soluble) and TM polypeptides

2 possible substrates:
- Misfolded proteins → quality control before proteins are sent to the rest of the secretory pathway
- Regulated degradation in response to signals → regulates metabolism

21
Q

What are the steps for ERAD?

A
  1. Substrates are recognized and brought to E3 Ub ligase complexes (by EDEM, chaperones)
  2. E3 complex poly-Ub substrate, and retro-translocates (dislocates) substrate into cytosol
  3. Substrate is deglycosylated, and degraded by proteasomes
22
Q

What is the first step of ERAD?

A

Recognition: BiP to ERAD
BiP prevents aggregations of misfolded substrates (binds to exposed hydrophobic patches)
Bip (binds substrate) + specialized DNAJ (ERdj5) + lectin (EDEM) = complex:
- ERdj5 → J domain (interacts with BiP) + thioredoxin domain (catalyzes breakage of disulfide in substrate (reverse of PDI))
- EDEM → recognizes short glycans for degradation (trimmed mannose, no glucose)

Complex targets the substrate to E3/protein translocator complex

23
Q

What is ERdj5?

A

The ER equivalent of DNAJ, co-chaperone to BiP (ER version of HSP70)

J domain → interacts with BiP
Thioredoxin domain → catalyzes breakage of disulfide bonds on substrate (has to HS side chains to interact with S-S on substrate)

24
Q

How is the target protein transported/interacts with E3 ligase in ERAD?

A

Transmembrane E3 ligases (HRD1 and gp78) → form complexes with substrate recognition adaptors:
- mifolded lumenal proteins (SEL1)
- mannose-binding lectins (EDEM)
- chaperones (BiP)
- misfolded TM proteins (erlin1/2, derlins)

*Substrate recognition adaptors bring misfolded proteins to the TM and interact with E3 ligases

25
Q

What are the characteristics of p97 and its mechanism in ERAD?

A

homo-hexamer or 97 kDa subunits
- Cytosolic protein
- Uses ATPase energy to extract proteins from membrane
- Substrate also threaded through the central pore (hole)

Role in ERAD → pulling or unfolding activity

p97 forms complexes with other proteins:
- Ub-binding adaptors
- peptide:N-glycanase (PNGase) removes glycans
- DUBs remove poly-Ub (will be re-Ub after extraction to complete targeting to proteasome)

*Important because N-linked glycans and poly-Ub are too large to fit through pores (for unfolding)

26
Q

What are HRD1 and gp78?

A

They are E3 ligases → homologous, multiple TM helices
- Poly-Ub as soon as the protein gets in the cytosol

27
Q

Why is disulfide breakage by Thioredoxin domain of ERdj5 important for degradation of ER proteins?

A

Proteins can be translocated if have disulfide bonds → too bulky

28
Q

What are the characteristics and roles of the AAA protein family?

A

Large family of ATPase, usually hexameric rings

Unfoldase subunit of the proteasome 19S regulator

29
Q

What is the ERAD process in 7 steps?

A
  1. Misfolded polypeptides are recongized by lectins and chaperones (BiP, ERdj5, EDEM)
  2. Disulfide bonds broken by thioredoxin DNAJ
  3. Adaptators bring misfolded polypeptides to E3 ligase (at membrane) → lectins, chaperones, lumenal and TM adaptors
  4. TM E3 ligase poly-Ub substrates and start retro-translocation (HRD1 and gp78)
  5. de-Ubiquitination
  6. Substrates are de-glycosylated (PNGase)
  7. p97 ATPase helps extract substrates from membrane (AAA-family)
  8. Re-Ubiquitinated by Cytosolic E3 mechanisms (CHIP)
  9. Substrates are recognized by proteasome or shuttling receptor
30
Q

How is the stress response specific to ER proteins called?

A

Unfolded protein response (UPR)

31
Q

What factors activate the ER unfolded protein response?

A

Activate by accumulation of unfolded proteins in ER

These factors can cause accumulation of misfolded proteins in the ER:
- Reductive stress → breaks disulfide bonds
- Glycosylation inhibitors
- Loss of calcium

32
Q

What is upregulated in case of UPR?

A

Transcription of ER chaperones
Transcription of ERAD components
Lipid synthesis → allows the ER to expand in size

*Cell death is induced if response is insufficient
*Signaling has to cross the ER membrane for transcriptional upregulation

33
Q

What are the 3 UPR signaling pathways?
What are they important for?

A

Signaling is important to upregulate transcrition of ER chaperones, ERAD components in UPR conditions

3 parallel pathways:
- IRE1 → regulated mRNA splicing initiates translation of proteins
- PERK → reduction of proteins entering the ER
- ATF6 → regulated proteolysis in golgi apparatus releases

34
Q

How does IRE1 work? What is its importance?

A

Important sensor for misfolded proteins in the UPR signaling pathway
- Has a lumenal domain (sense misfolded proteins), kinase domain + RNase domain (in cytosol)
- Dimerizes in response to UPR

Autophosphorylates → activate RNase activity → splices out 26 base intron → frameshift allows XBP1s (spliced) to be translated efficiently
*XBP1u (unspliced) translated at very low levels

XBP1s = transcription factor that upregulates UPR genes → ERAD, chaperones, lipids

35
Q

What is the process of IRE1 activation/prevention from activation?

A

2 IRE1 proteins bind a misfolded protein together → dimerization

BiP (HSP70) binds inactive IRE1 and prevents dimerization
BiP binding to unfolded proteins release IRE1 → can form dimer

36
Q

How does PERK work? What is its importance?

A
  • Has lumenal domain + kinase domain (acts on the cytosol, from sensor in the lumen)
  • dimerizes upon stress and autophosphorylated → same activation mechanism as IRE1

Phosphorylated dimer → phosphorylate eIF2alpha → inhibit translation → reduce ER protein folding load, except for ATF4

37
Q

What is ATF4?

A

Not the same as ATF6
It is a special mRNA that is not inhibited by PERK, can be expressed in eIF2alpha conditions
ATF4 = transcription factor:
- expression of more XBP1
- CHOP → activates apoptosis genes
- decision → recover from stress or commit cell death
- usually promote cell death

38
Q

What is the integrated stress response? (ISR)

A

Affects cytosol AND ER
*Many protein can phosphorylate eIF2alpha
Phosphorylated eIF2alpha is inactive (can’t bind to GTP) → inhibit translation:
- Decrease amounts of unfolded new proteins
- special mRNAs including ATF4 are still translated to promote cell death

39
Q

How does ATF6 work? what is its importance?

A

Signaling pathway in response to presence of misfolded proteins in the ER → Activating Transcription Factor 6

  1. Under normal conditions, BiP is bound to it at the ER membrane
  2. When unfolded protein accumulate → BiP dissociates from it
  3. Dissociation of BiP allows ATF6 to be transported to the Golgi (vesicular transport/part of the membrane it bound to detaches and forms a vesicle)
  4. Golgi proteases cleave off the cytoplasmic domain - ATF6(N)
    ATF6(N) = soluble TF that regulates UPR genes, binds to ER stress response elements (ESRE)
40
Q

Why is AFT6 transported to the Golgi when BiP dissociates from it?

A

Because BiP was masking its Golgi export signal, which is now exposed

41
Q

What proteins are transcribed in the presence of ATF4? (still transcribed after PERK is activated)

A
  • XBP1
  • CHOP
  • cell death
42
Q

What is the most common disease mutation is CFTR that causes Cystic Fibrosis? By what process does it affect the final protein?

A

Deletion of Phe in NBD1 (∆F508) → disrupts hydrophobic core of NBD1 → disrupts folding → mutant NBD1 can’t interact correclty with TM helices or NBD2 → mutant CFTR is retained in the ER and degraded instead of trafficking to the plasma membrane

43
Q

What is the structure of CFTR and its characteristics?
Which characteristics are important for translocation of CFTR?

A

Chloride channel that maintains hydration in lung airways
- 12 TM helices
- 2 cytosolic nucleotide-binding domains (NBD1/2)
- Cytosolic N-terminus and loops

Translocation:
- TM1 and TM7 → Signal Anchors (Charges around TM1 and TM7 determine orientation in the membrane)
- N-linked glycan attached during translocation

Most common disease mutation = deletion of Phe in NBD1 → disrupts hydrophobic core of NBD1

44
Q

How does the CFTR ER associated degradation occur?

A

*CTFR is a TM protein

  1. Misfolded mutant CFTR is selected for ERAD by E3 ligases → TM E3 ligase complexes in the ER (gp-78-RMA1)
  2. HSP70-CHIP complexes in the cytosol
  3. Specialized co-chaperones also promoted ERAD
  4. p97 helps to extract mutant CFTR from membrane
45
Q

What is special about the C-term of Calreticulin?

A

Has KDEL-COO(-) → sequence for retention of proteins within the ER (ER retrieval signal)

BIOC 212 (15 decks)

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