MVU5 PROTEIN FOLDING IN THE CELL -4

Question 1

why is degradation important?

Answer 1

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

Question 2

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

Answer 2

ubiquitin mediated degradation by proteasomes

Question 3

what is the structure of Ubiquitin

Answer 3

small 8kDa protein (76 amino acids)

Question 4

how does ubiquitin link to other molecules and to itself

Answer 4

covalently linked to lysine side chains

Question 5

what is the addition of UB a type of?

Answer 5

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

Question 6

what enzymes attach chains of UB to substrate proteins?

Answer 6

ubiquitination enzymes

Question 7

what enzyme selects the substrate to be UB?

Answer 7

E3 ligase

Question 8

what recognises the polyUB chain?

Answer 8

receptors on the proteasomes

Question 9

what does E1 do?

Answer 9

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

Question 10

what does E2 do?

Answer 10

conjugating enzyme
transfers it to the substrate

Question 11

what does E3 do?

Answer 11

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

Question 12

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

Answer 12

cut out and recycled

Question 13

what does the proteasome do?

Answer 13

large protein complex that unfolds and degraded substrates

Question 14

what can remove Ub?

Answer 14

DUB, deubiquitinating

Question 15

how many genes code for Ub and why?

Answer 15

a dozen genes
doesn’t require specificity

Question 16

how many genes code for E2 and why?

Answer 16

around 50
not much specificity required

Question 17

how many genes code for E3 and why?

Answer 17

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

Question 18

how many genes code for DUBs?

Answer 18

90

Question 19

how many genes code for UBPs? Ub binding protein

Answer 19

300

Question 20

what is the exact pathway of the Ubiquitination enzymes?

Answer 20

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

Question 21

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

Answer 21

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

Question 22

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

Answer 22

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

Question 23

can all lysines be a site of Ub?

Answer 23

no, depends on accessibility

Question 24

how many lysines does UB itself have?

Answer 24

7

Question 25

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

Answer 25

2 N termini 1 C terminus

Question 26

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

Answer 26

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

Question 27

how many genes encode for most proteasome subunits?

Answer 27

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

Question 28

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

Answer 28

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)

Question 29

what regulates the misfolded protein degradation system?

Answer 29

regulated by CHIP co chaperone

Question 30

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

Answer 30

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

Question 31

what makes up the E3 ligase complex?

Answer 31

Chaperone, CHIP and E2

Question 32

what are CHIP interactions with chaperones like?

Answer 32

transient fast binding and release

Question 33

how is balance between chaperone mediated folding and degradation established?

Answer 33

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

Question 34

how does constitutive degradation on native proteins work?

Answer 34

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

Question 35

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

Answer 35

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

Question 36

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

Answer 36

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

Question 37

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

Answer 37

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

Question 38

how does regulated degradation (SCF E3) work?

Answer 38

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)

Question 39

what do F box proteins recognise?

Answer 39

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

Question 40

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

Answer 40

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

Question 41

what is the function of the proteasome?

Answer 41

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

Question 42

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

Answer 42

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

Question 43

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

Answer 43

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

Question 44

what are AAA proteins?

Answer 44

family of ATP dependent proteins with many different functions