4. Protein folding, misfiling and degradation

Question 1

sign of protein misfolding

Answer 1

hydrophobic patches exposed on protein surface

Question 2

spontaneous refolding

Answer 2

• protein synthesised from N-terminus to C-terminus
• N-terminal region starts to fold before C terminal region is synthesised

Question 3

chaperones

Answer 3

proteins that help guide protein folding along productive pathways by permitting misfolded proteins to return to a proper pathway

Question 4

what do chaperones recognise?

Answer 4

exposed hydrophobic patches

Question 5

condition for chaperone upregulation

Answer 5

when misfolded proteins accumulate

Question 6

chaperone functions (5)

Answer 6

• fold newly made proteins into functional conformations
• refiled misfolded or unfolded proteins into functional conformations
• disassemble potentially toxic aggregates
• assemble and dismantle large multiprotein complexes
• mediate transformations between inactive/active forms of proteins

Question 7

how do chaperones work?

Answer 7

through ATP-dependent cycles, blocking exposed hydrophobic patches

Question 8

2 major classes of chaperones

Answer 8

• molecular chaperones
• chaperonins

Question 9

molecular chaperones overview

Answer 9

• operate as single molecules
• i.e. Hsp70: Heat shock protein
• bind to exposed hydrophobic residues of nascent proteins
• protect from aggregation until properly folded
• cycle of client protein binding and conformational change associated with ATP binding and hydrolysis

Question 10

Hsp70 function

Answer 10

help newly-synthesised proteins follow correct folding pathways

Question 11

molecular chaperone cycle (Hsp70)

Answer 11

1. substrate binding site available on chaperone: unfolded protein binds to it
2. ATP bound to nucleotide-binding domain leads to hydrolysis of ATP, producing ADP
3. ADP bound: substrate binding site blocked –> misfolding blocked
4. ADP exchanged to ATP: open binding site
5. protein released and folds correctly

Question 12

chaperonins overview

Answer 12

• form enclosed multisubunit refolding chamber made of inward-facing protein binding subunits
• i.e. Hsp60
• undergo concerted ATP-binding/hydrolysis and conformational change

Question 13

2 types of chaperonins

Answer 13

• group I chaperonins
• group II chaperonins

Question 14

group I chaperonins features

Answer 14

• 2 rings (7 subunits) + co-chaperone lid (7 subunits)
• each ring is a folding chamber where an unfolded protein enters
• found in bacteria, mitochondria

Question 15

group II chaperonins features

Answer 15

• no separate lids: lid function is ATP dependent
• 2 rings (8/9 homomeric/heteromeric subunits)
• found in eukaryotic cytoplasm

Question 16

group I chaperonin process (Hsp60)

Answer 16

1. misfolded protein binds in chamber –> hydrolysis of ATP to ADP removes bottom cap (GroES)
2. addition of ATP: GroES cap binds to top, sealing the protein
3. addition of ATP: GroES cap binds to bottom –> top GroES cap falls off
4. protein released properly

Question 17

what happens to irretrievably misfolded proteins?

Answer 17

they are destroyed by proteolytic cleavage into small fragments

Question 18

what shows chaperones are essential for life?

Answer 18

highly preserved in amino acid sequence, very ancient

Question 19

E1

Answer 19

ubquitin-activating enzyme

Question 20

E2

Answer 20

ubiquitin-conjugating enzyme

Question 21

E3

Answer 21

ubiquitin ligase

Question 22

E1 function

Answer 22

activate ubiquitin using energy of ATP, and transfer activated ubiquitin to E2

Question 23

E2 function

Answer 23

transfers the activated ubiquitin to the target protein

Question 24

E3 function

Answer 24

• necessary for E2 to work: instructs E2 which proteins to ubiquitinate
• recognises misfolded/damaged proteins

Question 25

what is ubiquitin/proteasome used for

Answer 25

protein degradation

Question 26

polyubiquitination

Answer 26

tagging damaged/misfolded proteins for degradation on poly-ubiquitin tail

Question 27

degradation of protein once polyubiquitin tail formed

Answer 27

1. proteins in cap recognise and bind polyubiquitin
2. hydrolysis of ATP: remove targeting ubiquitin
3. ubiquitin-tagged proteins fed into multisubunit chamber
4. subunits form inward-facing proteases that degrade proteins
5. peptides released: protein destroyed

Question 28

amyloid

Answer 28

aggregates to well ordered fibrils that have a common cross-B sheet structure, assembling into plaques that can cause amyloidoses

Question 29

amyloid pathologies

Answer 29

deposits in brain tissue as plaque/tangles leads to neurodegenerative diseases
-> Parkinson’s
-> Alzheimer’s

Question 30

similarity between proteasome and chaperonins

Answer 30

similar quaternary structures (evolved independently)

Question 31

proteasome structure

Answer 31

20S catalytic core and 1-2 19S regularity particles complexes (caps)

Question 32

ubiquitin-proteasome system

Answer 32

removes potentially toxic proteins and maintains proteostasis

Question 33

ubiquitin (Ub)

Answer 33

6-residue protein covalently linked to lysine residues on target proteins

Question 34

ubiquitin-proteasome system full steps (6)

Answer 34

1. E1 activated by ATP-dependent attachment of Ub molecule and then transfers Ub to cysteine in E2
2. E3 transfers bound Ub on E2 to NH2 side chain of lysine residue on target protein
3. repeating steps 1 and 2 to create polyubiquitin chain recognised by 19S regulatory particle
4. conformational changes in 19S RP + deubiquitinization allow target protein to move to ATPase
5. ATP hydrolysis enables subunits to unfold the protein and transfer it to the 20S core
6. protein cleaved and released in fragments