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BIOC212 > MVU5 PROTEIN FOLDING IN THE CELL -4 > Flashcards

MVU5 PROTEIN FOLDING IN THE CELL -4 Flashcards

Ubiquitinylation

1
Q

why is degradation important?

A

key part of protein folding quality control
essential regulatory and homeostatic mechanism

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2
Q

what is the major route of degradation for proteins in the cytosol?

A

ubiquitin mediated degradation by proteasomes

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3
Q

what is the structure of Ubiquitin

A

small 8kDa protein (76 amino acids)

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4
Q

how does ubiquitin link to other molecules and to itself

A

covalently linked to lysine side chains

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5
Q

what is the addition of UB a type of?

A

type of post translational modification
covalent addition of a full protein to a protein

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6
Q

what enzymes attach chains of UB to substrate proteins?

A

ubiquitination enzymes

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7
Q

what enzyme selects the substrate to be UB?

A

E3 ligase

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8
Q

what recognises the polyUB chain?

A

receptors on the proteasomes

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9
Q

what does E1 do?

A

UB activating enzyme
activates ubiquitin and binds it to itself
transfers it to E2

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10
Q

what does E2 do?

A

conjugating enzyme
transfers it to the substrate

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11
Q

what does E3 do?

A

ligase recognises substrate (target protein) and the E2 bound to UB
E3 and E2 stay together to keep adding Ub to Ub

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12
Q

what happens to the PolyUb when the protein enters the proteasome?

A

cut out and recycled

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13
Q

what does the proteasome do?

A

large protein complex that unfolds and degraded substrates

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14
Q

what can remove Ub?

A

DUB, deubiquitinating

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15
Q

how many genes code for Ub and why?

A

a dozen genes
doesn’t require specificity

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16
Q

how many genes code for E2 and why?

A

around 50
not much specificity required

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17
Q

how many genes code for E3 and why?

A

around 600 genes code for E3
need lots of specificity, many different types of E3

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18
Q

how many genes code for DUBs?

A

90

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19
Q

how many genes code for UBPs? Ub binding protein

A

300

20
Q

what is the exact pathway of the Ubiquitination enzymes?

A
  1. E1 activating enzyme attaches Ub to itself in a chemically reactive state, on a Cys side chain (thioester bond)
  2. E2 conjugating enzyme transfers Ub to its own Cys
  3. E3 ligase selects the substrate to be modified
  4. E3 triggers Ub transfer from E2 to Lys side chain on substrate
  5. E2-E3 add more Ub to Lys on previous Ub, to make poly-Ub chain
21
Q

what are the two ways in which Ub can be transferred from E2 to the substrate?

A

most E3 enzymes are in the ring family, which does a direct transfer
some E3 enzymes belong to HECT family, which to indirect transfer:
E2 transfers UB to E3, E3 transfers Ub to substrate, all thioester bonds

22
Q

how is ubiquitin linked to the substrate lysine side chain? and what is that bond called

A

C terminus carboxyl is covalently linked to side chain amine
isopeptide bond

23
Q

can all lysines be a site of Ub?

A

no, depends on accessibility

24
Q

how many lysines does UB itself have?

A

7

25
Q

how many N and C termini does a ubiquitinylated protein have?

A

2 N termini
1 C terminus

26
Q

on which lysines of Ub can another Ub be attached, and which one signals degradation?

A

Lysine 63,48, or 11
Lysine 48 ubiquitination signals for degradation

27
Q

how many genes encode for most proteasome subunits?

A

1 gene
no specificity needed, only one type of proteasome
expressing different E3 enzymes is more efficient than expressing different types of proteasomes

28
Q

what are examples of substrate selection by E3 ligases? (situations where you would need it)

A
  1. quality control degradation of misfolded proteins
  2. constitutive degradation of native protein to control its level
  3. degradation of native protein in response to a signal (cyclins)
29
Q

what regulates the misfolded protein degradation system?

A

regulated by CHIP co chaperone

30
Q

what domains does CHIP co chaperone have and what do they do?

A

TPR domain binds HSC70 or HSP90
E3 ligase domain (U-box) binds E2 that is ubiquitinated

31
Q

what makes up the E3 ligase complex?

A

Chaperone, CHIP and E2

32
Q

what are CHIP interactions with chaperones like?

A

transient
fast binding and release

33
Q

how is balance between chaperone mediated folding and degradation established?

A

substrates bound by chaperones for a long time are more likely to form a complex with CHIP and be ubiquitinated
substrates bound for short times are likely to escape ubiquitination
sometimes several cycles of chaperones is needed, do everything to save the protein if you can

34
Q

how does constitutive degradation on native proteins work?

A

N-end rule
proteins are processed by cleavage within their sequences, so a different residue becomes the N terminus (not methionine)
certain N terminal residues are bound by N end rule E3 ligase which ubiquitinates the protein
N end rule degrades protein very rapidly, regardless or not if they are folded

35
Q

which amino acids on the N terminus will E3 recognise and mark for degradation?

A

Arg, Lys, His, Phe, Trp, Tyr, Leu, Ile
side chains that are basic or large hydrophobic

36
Q

how are some N terminal residues enzymatically modified too be recognised by the N end rule?

A

if Asp and Glu (acidic) are on the N terminus, Arginine is added, recognised by E3

if you have Asn or Gln (amides) as the N terminus AAs, convert them to Asp or Glu by removal of amine, and then add Arginine

37
Q

what are the different N end rule pathways (comp program)

A
  1. if N terminal N, convert to D, if Q, convert to E
  2. if N terminal D or E, add N terminal R
  3. if N terminal R,K,H,F,W,Y,L,I ubiquitinate the protein
  4. else, leave the protein alone
38
Q

how does regulated degradation (SCF E3) work?

A
  1. E3 ubiquitin ligase complex is made up of Skp1 (adaptor protein) Cullin (scaffold) and F-box (substrate binding arm) (SCF)
  2. scaffold binds E2 and substrate binding F box protein
  3. F box protein binds phosphorylated substrate
  4. substrate is presented to the E2 for ubiquitination (bring it close to the E2)
39
Q

what do F box proteins recognise?

A

phosphorylated peptide sequences
phosphorylation by kinases is used as a signal for degradation
degrade native proteins to stop their functions

40
Q

what is the structure of the proteasome, and what do the different parts do?

A

large oligomeric complex
central 20S core particle: cylinder, cuts the polypeptide
two 19S regulatory particles (caps): recognise poly Ub, cut the chain so it can be recycled
core and two caps make up the 26S proteasome
around 2.5MDa

41
Q

what is the function of the proteasome?

A

responsible for protein degradation in the cytosol and nucleus, and proteins from the ER

42
Q

what is the structure of the 20S core of the proteasome?

A

2 outer rings of similar alpha subunits and 2 inner rings of 7 similar beta subunits
3 of the beta subunits have protease activity on the inside surface
19S cap attaches to the outer ring

43
Q

what is the structure of the 19S regulator cap of the proteasome?

A

base with 6AAA-family ATPase subunits:
protein “unfoldase”
lid with non ATPase subunits:
poly Ub receptors
deubiquitinases (DUBsP)

44
Q

what are AAA proteins?

A

family of ATP dependent proteins with many different functions

45
Q
A

BIOC212 (32 decks)

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