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ANAT 212 pre midterm > Professor Uglade Lecture 10 > Flashcards

Professor Uglade Lecture 10 Flashcards

1
Q

What types of bonds/linkages are only in the ER side of the lumen?
Where are proteins synthesized/folded?

A

disulfide bonds and N-linked glycosylation
in the ER

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2
Q

Where are misfolded secretory proteins degraded?

A

In the ER, the ubquitin proteasome system

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3
Q

What type of degradation occurs at lysosomes at the end of secretory pathway?

A

-digestion by proteases inside lysosome

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4
Q

Why is the ER so important for all organelles in the secretory pathway?

A

-ER is the protein quailty control checkpoint

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5
Q

What are the 4 quality control mechanisms in the ER protein homeostasis system

A
  1. ER chaperones
  2. ER N-linked glycosylation
    3.ER misfolded protein degradation
    4.ER stress response (UPR)
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6
Q

What does the ER chapersone section of protein homeostasis system include, chaperones+co-chaperones?

A

Bip (HSP70)
-ERdj Proteins (DNAJ (also called HSP40) co chaperones)
-NEF co chaperones
GRP94 (HSP90)-no co chaperone are needed
-thioredoxin family- PDI and ERp57

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7
Q

What does the ER N-linked glycosylation section of protein homeostasis system include?

A

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

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8
Q

What does the degradation take place in the ER misfolded protein degradation section of protein homeostasis system occur, and why is folding necessary?

A

-degradation takes place on cytosolic proteasomes
-folding is necessary to exit ER to the secretory pathway

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9
Q

WHat is the ER stress response?

A

it is the unfolded protein response (UPR)

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10
Q

what does DNAJ co chaperone do in BiP?

A

substrate binding DNAJ (ERdj3) assists folding

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11
Q

what does the translocon interact with in BiP?

A

interacts with proteins on the nascent polypeptides such as:
signal peptidase
-OST (oligsaccharyl transferase)

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12
Q

What does Sec 63 do in BiP?

A

it is a specialized TM DNAJ that recruits BiP to translocating polypeptides (does not bind directly to substrate

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13
Q

When do folding and modification occur at Bip?

A

folding and modification take place during translocation

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14
Q

What type of environment is the ER lumen, and how fast is disulfide formation?

A

It is an oxidizing environment, but spontaneous disulfide formation is inefficient and/or incorrect for folding (takes very long)

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15
Q

What is disulfide formation is catalyzed by?

A

-catalyzed by thioredoxins:
ex. PDI and ERp57

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16
Q

What is the structuer of PDI (protein disulfide isomerase)?

A

-have 2 reactive Cys residues close together, can oxidize substrate

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17
Q

What does oxidized PDI do?

A

it catalyzes formation of disulfide bonds in substrate, PDI becomes reduced

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18
Q

How does reduced PDI aid in rearrangement of disulfide bonds during folding?

A

-the first disulfides to form may not be correct for the native state
-native state disulfides are the most stable

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19
Q

What does PDI form in both reduction and oxidation?

A

PDI forms mixed disulfide intermediates with substrate

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20
Q

What regenerates PDI?

A

chemical cascade regenerates oxidized PDI

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21
Q

When does PDI become reduced?
What is PDI oxidized by?
3. What is Ero1 regenerated by?
4.What is FAD regenerated by?

A

it becomes reduced after oxidizng the substrate
-PDI is oxidized by Ero1 protein with cofactor FAD; Ero1 is reduced
-it is regenerated by FAD
-FAD is regenerated by O2

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22
Q

WHat is oxidation potential in ER controlled by in PDI regeneration?

A

-controlled by enzymes (PDI,Ero1)

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23
Q

What type of domain + anchor does calnexin (CNX) have?

A

It has a 50kDa lumenal domain and a TM helix anchor

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24
Q

What domain does calreticulin have (CRT)

A

Has a lumenal domain but no TM helix, has a signal for retention in the ER

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25
Q

What does CNX ans CRT lumenal domain do?

A

-recognizes got an pattern on polypeptides (N- linked glycosylation)
-binds to thioredoxin (ERp57)

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26
Q

what does the n-linked glycan contain in calnexin binding?
How are glucose and mannose trimmed off, what does this indicate?

A

-branched mannose polymer with 3 glucose residues at the end
-trimmed off step by step
-trimming mannose can indicate thst the protein needs to be degraded

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27
Q

what does calnexin binding specifically a glycan with 1 glucose do?

A

keeps the polypeptide in the ER
-single glucose is a signal for imcomplete folding, but is eventually removed

28
Q

what composes UGGT, and what does it bind?

A

UDP-glucose:glycoprotein glucosyltransferase
-binds non native polypeptides and reattaches a glucose to the glycan, and CNX can bind again

29
Q

what is not recognized by UGGT?

A

native folded polypeptides are not recognized?

30
Q

what does glucosidase remove in UGGT?

A

removes Glc (glucose) from native and non native polypeptides

31
Q

what does mannosidase do and how does this affect UGGT?

A

mannosidase trims sugars further, and UGGT does not recognize shortened glycosylation

32
Q

What does calnexin keep polypeptides in?
What does glucosidase remove in the calnexin and calreticulin cycle?

A

-1.CNX keeps polypeptide in ER
-2.glucosidase removed the last glucose

33
Q

what does UGGT restore in CNX and CRT cycle?
What do folded peptides contain/do?

A

-3.UGGT restores Glc on misfolded polypeptides-CNX binding
-4.folded polypeptides do not have Glc restored and exit to golgi

34
Q

What is the mannosidase process in the CNX and CRT cycle?
What does mannose bind?

A

-mannosidase trims glycans without Glc
-it is slow irreversible
-proteins retained in ER by chaperones likely to get trimmed
-mannose binding lectins (EDEM), select short glycans for degradation

35
Q

What does ERAD (ER associated degration) degrade?
-What does it do?

A

-both lumenal and transmembrane polypeptides
-it acts as quality control before proteins are sent to rest of secretory pathway, since many substrates are misfolded proteins

36
Q

What does ERAD regulate?

A

regulates metabolism in regulated degradation in response to signals

37
Q

1.Where are substrates brought in ERAD steps?
2.Where does the complex translocate?
3.How/where is substrate degraded?

A
  1. substrates are recognized and brought to E3 Ub ligase complexes
  2. E3 complex polyUb substrate, and retrotranslocates (dislocates) substrate into cytosol
    3.substrate is deglycosylated, and degraded by proteasome
38
Q

What prevents unfolded substrates from aggregating?

A

substrates which cannot fold are prevented from aggreagting by BiP

39
Q

What cochaperone/substrates does BiP use in binding?
What do these catalyze and what does ERdj5 contain?

A

BiP binds substrate in complex with specialized DNAJ (Erdj5) and lectin (EDEM)
-ERdj5: has J domain and thioredoxin domain
-catalyzes breakage of disulfide in substrate
-reverse of PDI (protein disulfide isomerase)

40
Q

what does BiP to ERAD complex target?

A

targets substrate to E3

41
Q

What substrate recognition adaptors does transmembrane E3 ligases (HRD1 and gp78) form?

A

-misfolded lumenal proteins (SEL1L)
-mannose-binding lectins (EDEM)
-misfolded TM proteins (erlin1/2, derlins)
-chaperones (BiP)
-also p97 interactions
-basically diff adaptors bring protein to retrotranslocon

42
Q

How is thee regulated opening and closing of retrotranslocon like?

A

-regulated opening and closing is pore-like
-but large enough to allow N-linked glycans

43
Q

1.Why is polyUb in the cytosol neccesary for retrotranslocation?
2. what complex perfoms all these functions?

A
  1. to mark that a protein needs to go through degradation
  2. large transmembrane E3 ligase complexes are thought to perform all of these functions
    -HRD1 and gp78 E3 ligases:homologous, multiple TM helices
44
Q

what is retrotranslocation assisted by?
What ligases are in the cytosol?

A

-assisted by the cytosolic protein p97/VCP, has pulling or unfolding activity
-N-end rule, CHIP, SCF E3 ligases are in cytosol

45
Q

what does the p97 mechanism work on?

A

-works on N-linked glycans and poly-Ub which are too large to fit through pore

46
Q

what proteins does p97 form a complex with?

A

-ub binding adaptors
-peptide: N-glycanase (PNGase) removes glycans
-DUBs which remove poly-Ub (but E3s re-Ub after extraction to complete targeting proteasomes

47
Q

what are AAA-proteins?
what do they contain?

A

they are large diverse superfamily of ATPases with many diff functions, usually hexameric rings
-unfoldase subunits of the proteasome 19s regulator

48
Q

what is p97 composed of and how does it extract proteins from membrane and where is it threaded?

A

-it is a homo-dimer of 97 kDa subunits
-uses ATPase energy to extract proteins from membrane
-substrate is also threaded through the central pore (hole)

49
Q

ERAD summary 7 steps (MDATESS)

A
50
Q

How do cells respond to stress that causes protein misfolding?

A

they increase the expression of chaperones, and other specialized proteins

51
Q

What does heat shock response do/for (HSR)

A

-its for cytosolic and nuclear protein
-protects against cell death

52
Q

what does Unfolded protein response (UPR) do/for?

A

-it is for ER proteins
-can promote cell death if stress is to severe

53
Q

how is the ER UPR (unfolded protein response) activated?
What does UPR do?

A

-activated by accumulation of unfolded proteins in ER
-it reduces stress-breaks disulfide bonds
-glycosylation inhibitors
-loss of calcium

54
Q

What happens to ER chaperones, ERAD components, and lipid synthesis during UPR?
What else does UPR do/limited to, where does signaling have to cross?

A

transcription of these factors is upregulated
-induces cell death (apoptosis) if response is insufficient
-it is limited to ER, no overlap with HSR (HSP was in cytosol)
-signaling has to cross the ER membrane

55
Q

what are the 3 parallel UPR signaling pathway?

A

IRE1
-PERK
-ATF6

56
Q

how are IRE1 and PERK related?

A

-they both rely on dimerization whereas ATF6 is not a dimer

57
Q

what domains does IRE1 have?
When does IRE1 dimerize?
WHat activates RNase activity?

A

it has lumenal domains, kinase and Rnase domains
-dimerizes in response to unfolded proteins
-autophosphorylation activates RNase activity

58
Q

When is XBP1u (unspliced) translated?
What does IRE1 splice to allow XBP1s (spliced) to be translated effciently?
what does XBP1 do?

A

-translated at very low levels
-IRE1 splices out the 26 base intron
-XBP1 is a transcription factor that upregulates UPR genes

59
Q

Why do we dimerize IRE1?
what causes dimerization?

A

-you dimerize IRE1 when you have misfolded proteins to allow unfolded proteins to bind
-direct binding of unfolded protein by 2 IRE1 proteins causes dimerization

60
Q

what prevents dimerization of IRE1?
What releases IRE1 to form dimers?

A

BiP (HSP70 binds inactive IRE1 to prevent dimerization
-BiP binding to unfolded proteins releases IRE1 to form dimers

61
Q

What type of domain does PERK have?
When does PERK dimerize?
What does phosphorylation of translation factor do?

A
  1. PERK has a lumenal and a kinase domain
  2. PERK dimerizes upon stress and autophosphorlyates (same activation as IRE1)
  3. phosphorlates translation factor to inhibit translation
62
Q

what mRNA is not inhibited in PERK?
what does this mRNA do?

A

-ATF4 transcription factor is not inhibited:
-expression of more XBP1 (transcription factor that upregulates UPR genes)
-CHOP-actviates apoptosis genes
-makes decision to recover from stress or commit to cell death

63
Q

What turns on general translation for integrated stress response (ISR), when is it inactive?

A

translation initation factor eIF2alpha turns on general transcription
-phosphorlyated eIF2alpha is inactive

64
Q

What does eIF2alpha do in response to stress?

A

-eIF2alpha kinases respond to diff stresses to inhibit translation
-decrease amount of unfolded new proteins
-special mRNA including ATF4 are still translated to promote cell death

65
Q

What is PERK and other kinases used for in integrated stress response (HSR)

A

PERK-ER stress
-other kinases-proteasome inhibition (HSR) starvation, viral infection, etc

66
Q

Where is ATF6 and what does it do?
2. What occurs under stress in ATF6

A

ATF6 is normally transmembrane at the ER- BiP binding sites mask ER exit signal
2. upon stress, BiP is competed away by unfolded proteins

67
Q

3.Where is ATF6 transported
4. What do the golgi proteases cleave
5.what does ATF6 upregulate?

A
  1. ATF6 transported to golgi
  2. Golgi proteases cleave off cytosolic domain ATF6(N) (n terminus of ATF6 is a transcription factor and is released)
    5.ATF6(N) is solible transcription that upregulates UPR gene

ANAT 212 pre midterm (9 decks)

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