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ANAT 212 pre midterm > Professor Uglade Lecture 10 > Flashcards

Professor Uglade Lecture 10 Flashcards

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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
What does CNX ans CRT lumenal domain do?
-recognizes got an pattern on polypeptides (N- linked glycosylation) -binds to thioredoxin (ERp57)
26
what does the n-linked glycan contain in calnexin binding? How are glucose and mannose trimmed off, what does this indicate?
-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
27
what does calnexin binding specifically a glycan with 1 glucose do?
keeps the polypeptide in the ER -single glucose is a signal for imcomplete folding, but is eventually removed
28
what composes UGGT, and what does it bind?
UDP-glucose:glycoprotein glucosyltransferase -binds non native polypeptides and reattaches a glucose to the glycan, and CNX can bind again
29
what is not recognized by UGGT?
native folded polypeptides are not recognized?
30
what does glucosidase remove in UGGT?
removes Glc (glucose) from native and non native polypeptides
31
what does mannosidase do and how does this affect UGGT?
mannosidase trims sugars further, and UGGT does not recognize shortened glycosylation
32
What does calnexin keep polypeptides in? What does glucosidase remove in the calnexin and calreticulin cycle?
-1.CNX keeps polypeptide in ER -2.glucosidase removed the last glucose
33
what does UGGT restore in CNX and CRT cycle? What do folded peptides contain/do?
-3.UGGT restores Glc on misfolded polypeptides-CNX binding -4.folded polypeptides do not have Glc restored and exit to golgi
34
What is the mannosidase process in the CNX and CRT cycle? What does mannose bind?
-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
What does ERAD (ER associated degration) degrade? -What does it do?
-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
What does ERAD regulate?
regulates metabolism in regulated degradation in response to signals
37
1.Where are substrates brought in ERAD steps? 2.Where does the complex translocate? 3.How/where is substrate degraded?
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
What prevents unfolded substrates from aggregating?
substrates which cannot fold are prevented from aggreagting by BiP
39
What cochaperone/substrates does BiP use in binding? What do these catalyze and what does ERdj5 contain?
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
what does BiP to ERAD complex target?
targets substrate to E3
41
What substrate recognition adaptors does transmembrane E3 ligases (HRD1 and gp78) form?
-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
How is thee regulated opening and closing of retrotranslocon like?
-regulated opening and closing is pore-like -but large enough to allow N-linked glycans
43
1.Why is polyUb in the cytosol neccesary for retrotranslocation? 2. what complex perfoms all these functions?
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
what is retrotranslocation assisted by? What ligases are in the cytosol?
-assisted by the cytosolic protein p97/VCP, has pulling or unfolding activity -N-end rule, CHIP, SCF E3 ligases are in cytosol
45
what does the p97 mechanism work on?
-works on N-linked glycans and poly-Ub which are too large to fit through pore
46
what proteins does p97 form a complex with?
-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
what are AAA-proteins? what do they contain?
they are large diverse superfamily of ATPases with many diff functions, usually hexameric rings -unfoldase subunits of the proteasome 19s regulator
48
what is p97 composed of and how does it extract proteins from membrane and where is it threaded?
-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
ERAD summary 7 steps (MDATESS)
50
How do cells respond to stress that causes protein misfolding?
they increase the expression of chaperones, and other specialized proteins
51
What does heat shock response do/for (HSR)
-its for cytosolic and nuclear protein -protects against cell death
52
what does Unfolded protein response (UPR) do/for?
-it is for ER proteins -can promote cell death if stress is to severe
53
how is the ER UPR (unfolded protein response) activated? What does UPR do?
-activated by accumulation of unfolded proteins in ER -it reduces stress-breaks disulfide bonds -glycosylation inhibitors -loss of calcium
54
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?
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
what are the 3 parallel UPR signaling pathway?
IRE1 -PERK -ATF6
56
how are IRE1 and PERK related?
-they both rely on dimerization whereas ATF6 is not a dimer
57
what domains does IRE1 have? When does IRE1 dimerize? WHat activates RNase activity?
it has lumenal domains, kinase and Rnase domains -dimerizes in response to unfolded proteins -autophosphorylation activates RNase activity
58
When is XBP1u (unspliced) translated? What does IRE1 splice to allow XBP1s (spliced) to be translated effciently? what does XBP1 do?
-translated at very low levels -IRE1 splices out the 26 base intron -XBP1 is a transcription factor that upregulates UPR genes
59
Why do we dimerize IRE1? what causes dimerization?
-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
what prevents dimerization of IRE1? What releases IRE1 to form dimers?
BiP (HSP70 binds inactive IRE1 to prevent dimerization -BiP binding to unfolded proteins releases IRE1 to form dimers
61
What type of domain does PERK have? When does PERK dimerize? What does phosphorylation of translation factor do?
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
what mRNA is not inhibited in PERK? what does this mRNA do?
-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
What turns on general translation for integrated stress response (ISR), when is it inactive?
translation initation factor eIF2alpha turns on general transcription -phosphorlyated eIF2alpha is inactive
64
What does eIF2alpha do in response to stress?
-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
What is PERK and other kinases used for in integrated stress response (HSR)
PERK-ER stress -other kinases-proteasome inhibition (HSR) starvation, viral infection, etc
66
Where is ATF6 and what does it do? 2. What occurs under stress in ATF6
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
3.Where is ATF6 transported 4. What do the golgi proteases cleave 5.what does ATF6 upregulate?
3. ATF6 transported to golgi 4. 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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