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BIOC 212 > ER protein quality control > Flashcards

ER protein quality control Flashcards

(45 cards)

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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?

Study These Flashcards
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?

Study These Flashcards
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?

Study These Flashcards
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?

Study These Flashcards
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?

Study These Flashcards
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?

Study These Flashcards
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
What are the characteristics of p97 and its mechanism in ERAD?
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
What are HRD1 and gp78?
They are E3 ligases → homologous, multiple TM helices - Poly-Ub as soon as the protein gets in the cytosol
27
Why is disulfide breakage by Thioredoxin domain of ERdj5 important for degradation of ER proteins?
Proteins can be translocated if have disulfide bonds → too bulky
28
What are the characteristics and roles of the AAA protein family?
Large family of ATPase, usually hexameric rings Unfoldase subunit of the proteasome 19S regulator
29
What is the ERAD process in 7 steps?
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) 6. p97 ATPase helps extract substrates from membrane (AAA-family) 7. Re-Ubiquitinated by Cytosolic E3 mechanisms (CHIP) 8. Substrates are recognized by proteasome or shuttling receptor
30
How is the stress response specific to ER proteins called?
Unfolded protein response (UPR)
31
What factors activate the ER unfolded protein response?
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
What is upregulated in case of UPR?
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
What are the 3 UPR signaling pathways? What are they important for?
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
How does IRE1 work? What is its importance?
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
What is the process of IRE1 activation/prevention from activation?
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
How does PERK work? What is its importance?
- 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
What is ATF4?
*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
What is the integrated stress response? (ISR)
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
How does ATF6 work? what is its importance?
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
Why is AFT6 transported to the Golgi when BiP dissociates from it?
Because BiP was masking its Golgi export signal, which is now exposed
41
What proteins are transcribed in the presence of ATF4? (still transcribed after PERK is activated)
- XBP1 - CHOP - cell death
42
What is the most common disease mutation is CFTR that causes Cystic Fibrosis? By what process does it affect the final protein?
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
What is the structure of CFTR and its characteristics? Which characteristics are important for translocation of CFTR?
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
How does the CFTR ER associated degradation occur?
*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 4. Specialized co-chaperones also promoted ERAD 5. p97 helps to extract mutant CFTR from membrane
45
What is special about the C-term of Calreticulin?
Has KDEL-COO(-) → sequence for retention of proteins within the ER (ER retrieval signal)

BIOC 212 (15 decks)

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