IV. Cell Biology | 68. Protein quality control in the endoplasmic reticulum; the fate of misfolded proteins; ERAD

Question 1

1. Levels of protein organization
   a/ List the levels of protein organization

Answer 1

1/ Primary structure
2/ Secondary structure
3/ Tertiary structure
4/ Quaternary structure

Question 2

1. Levels of protein organization
   b/ Characteristics of primary structure?

Answer 2

It is a sequence of a chain of amino acids

Question 3

1. Levels of protein organization
   c1/ Characteristics of secondary structure

Answer 3

It is a sequence of amino acids that have hydrogen bridges between carbonyl and amino groups of peptide bonds (α-helix, β-sheet)

Question 4

1. Levels of protein organization
   c2/ What are the 2 types of secondary structure

Answer 4

1) α helix: right hand spiral,
H-bonds between amino acids located in three residues distance
- time of formation: ~ 100 ns

2) β pleated sheet:
two or more segments of a polypeptide chain line up next to each other and are held together by hydrogen bonds
- time of formation: ~ 1000 ns

Question 5

1. Levels of protein organization
   d/ Characteristics of tertiary structure

Answer 5

when certain attractions are present between α- helices and β-pleated sheets (ex: disulfide bridges, hydrophobic interaction, ionic bonds, H-bonds)
=> forms the overall 3D shape of the protein

Question 6

1. Levels of protein organization
   e/ Characteristics of quarternary structure

Answer 6

protein consisting of more than one amino acid chain

Question 7

2. What is Anfinsen’s dogma?

Answer 7

a thermodynamic hypothesis that propose:
- native structure of proteins is determined only by their amino acid sequence
(primary structure)

Question 8

3. What does Levinthal’s paradox state?

Answer 8

There are enormous numbers of possible conformations for protein folding – would take endless amount of years to try all, yet folding is done on a millisecond scale = contradiction.
E.g, a protein composed of 100 amino acids:
- every amino acid has 3 possible conformations
-> 3^100
-> 10^-13 seconds for trying one conformation
-> 10^27 years to try all conformations

Question 9

4. What is Hydrophobic collapse?

Answer 9

Because the main driving force is to find the thermodynamically more favorable position, the cytosol is water base solution (polar) –> try to hide the AA which poses the hydrophobic side chains, the polar AA will face to the cytosol.
=> It is called Molten globule state: secondary strucure is formed but tertiary structure is not finalized, yet.

Question 10

5. Why are proteins driven to their free energy minimum via the folding funnel?

Answer 10

• the folded state of the protein is very stable
• the undesired amino acid interactions along the folding pathway are reduced
• by forming the contacts of key residues the overall topology of the protein is
  established

Question 11

5. Success rate of in vitro folding of eukaryotic proteins is only 20-30 %
   -> WHY?

Answer 11

1) Macromolecular crowding inside the cells

2) Protein aggregation competes with folding (oligomers, amyloid fibrils and amorphous aggregations)
-> Formation of amyloid fibrils can accompany disease like
Alzheimer’s disease

Question 12

6. How does protein fold in ribosomes?

Answer 12

• α-helices and small tertiary structure elements could be formed inside the exit tunnel
• Prokaryotic proteins are folded post-translationally, while eukaryotic proteins are mainly folded co-translationally

Question 13

7. Protein folding in the cytosol
   a/ Which additional proteins are involved in protein folding in cytosol?

Answer 13

Chaperone proteins

Question 14

7. Protein folding in the cytosol
   b/ What are chaperone proteins?

Answer 14

They are a functionally related group of proteins assisting protein folding in the cell under physiological and stress condition

Question 15

7. Protein folding in the cytosol
   d/ Why are chaperone proteins heat shock proteins? Give an example

Answer 15

1/ Chaperones are heat shock proteins because they are proteins induced in a living cell in response to a rise in temperature (above normal level)
2/ E.g, in the salivary gland chromosome of fly, if put them at high temperature, it presents a specific puffing pattern showing active transcription and translation

Question 16

7. Protein folding in the cytosol
   c/ What is the role of chaperone proteins?

Answer 16

• Fold newly made proteins into functional conformations
• Refold misfolded / unfolded proteins into functional conformation
• Disassemble potentially toxic protein aggregates that form due to protein misfolding
• Mediate transformations between active + inactive forms of proteins
• They are ATP binding + use hydrolysis to (1) enhance the binding of target proteins and (2) switch their own conformation

Question 17

7. Protein folding in the cytosol
   e/ How can chaperones facilitate proper folding of nascent proteins?

Answer 17

• Prevent aggregation by binding to target polypeptide
• Sequestering it from other unfolded proteins to give nascent protein time to fold

Question 18

7. Protein folding in the cytosol
   f/ What are the 2-general families of chaperons?

Answer 18

1) Molecular chaperons: bind to segment of protein substrate and stabilize unfolded or partly folded protein
-> Prevent aggregation/degradation

2) Chaperonins: form folding chambers, where all or parts of unfolded protein can enter
-> Give time to fold properly in appropriate environment

Question 19

7. Protein folding in the cytosol
   g/ What is the working principle of Hsp70 (chaperon)?

Answer 19

• ATP binding and hydrolysis drive large conformational
  changes
• Non-properly folded polypeptide substrate interacts first with Hsp40 and is delivered to ATP-bound Hsp70
• Interaction with Hsp40 triggers ATP hydrolysis on Hsp70 generating the closed state
• ADP-release catalyzed by nucleotide exchange factor (NEF) and rebinding of ATP triggers substrate release for folding or possible transfer to downstream chaperones

Question 20

7. Protein folding in the cytosol
   h/ What is the Working principle of GroEL/GroES (chaperonin) nanocage?

Answer 20

• Substrate protein binds as folding intermediate to
  the empty GroEL-ring and is encapsulated by GroES
  in an ATP-dependent step
• The protein is free to fold within the chaperonin
  nanocage for the time required to hydrolyze ATP on
  each subunit of the heptameric GroEL-ring
• ATP binding to the opposite ring then triggers the
  release of folded protein and GroES, completing the
  cycle
• Incompletely folded protein will rebind after release

Question 21

8. Protein folding in ER
   a/ How does protein fold in ER?

Answer 21

• The proteins in the ER have a hydrophobic signal
  sequence near their N-terminal
• A protein-RNA complex, the signal recognition
  particle (SRP), binds to the ribosome and stops
  translation
• The SRP receptor has GTPase activity (SRP as well)
  and is located in the ER membrane
• The receptor will bind to SRP and open a translocon
  channel and the translation of the peptide continues
  straight into the lumen of the ER
• A signal peptidase in the ER membrane cleaves the
  signal sequence and the folded protein will be released into the ER lumen -> Proteins targeted for the ER, are never in contact with the cytosol

Question 22

8. Protein folding in ER
   a/ How does protein fold in ER?

Answer 22

• The proteins in the ER have a hydrophobic signal
  sequence near their N-terminal
• A protein-RNA complex, the signal recognition
  particle (SRP), binds to the ribosome and stops
  translation
• The SRP receptor has GTPase activity (SRP as well)
  and is located in the ER membrane
• The receptor will bind to SRP and open a translocon
  channel and the translation of the peptide continues
  straight into the lumen of the ER
• A signal peptidase in the ER membrane cleaves the
  signal sequence and the folded protein will be released into the ER lumen -> Proteins targeted for the ER, are never in contact with the cytosol

Question 23

8. Protein folding in ER
   b/ How do enzymes fold in ER?

Answer 23

• Classical chaperons (Hsp70 and 90) (ex: BIP and GRP94)
• Protein disulfide isomerase (PDI) – produces disulfide bridges (only in ER, since no
  possibility of formation of the bridges in the cytosol!)
• Carbohydrate binding chaperons = lectins

Question 24

8. Protein folding in ER
   c/ Characteristics of lectins

Answer 24

• Carbohydrate binding chaperons
• 2 types of lectin in the ER, both bind to Ca2+ = calnexin + calreticulin

Question 25

9. Quality control
   a/ 3 ways that quality control can occur?

Answer 25

1. Proteasomal degradation in cytoplasm
2. N-glycosylation in ER

Question 26

9. Quality control
   b/ How are Inappropriately folded protein degraded in the proteasome?

Answer 26

1/ HSP40 detects hydrophobic patches and it will interact with the misfolded protein.

2/ HSP40 take the protein to the aTP bound HSP70. And the interaction of HSP40 with the protein will hydrolyze ATP on HSP70.

3/ HSP70 will trap the protein in a closed state.

4/ NEF will convert ATP to ADP which will trigger the substrate release of protein

5/ The protein will be sent to other chaperons or will simply be folded into its native state

Question 27

9. Quality control
   c/ How is N-glycosylation in ER participate in quality control?

Answer 27

Folded protein after N-glycosylation in the ER having carbohydrate tree which all the 3 glucose from the precurser tree has removed.
* when there is a misfolded protein, glycosyl transferase can recognise it and add glucose to the end of the oligosacchride,
the protein won’t be able to leave to the next step and will be bound to the calnexin (chaperon) help with its folding.
* Glucosidase responsible for removing the glucose. The cycle will go one an long as the protein is misfolded.

Question 28

10. ERAD
    a/ What is ERAD?

Answer 28

ER-associated degradation (ERAD) is an endoplasmic-reticulum associated protein degradation (ERAD).

Question 29

10. ERAD
    b/ What are the 3 main steps of ERAD?

Answer 29

1) Recognition of misfiled proteins in ER
2) Retro-translocation into the ER (retro-translocation = reverse of translocation)
3) Ubiquitin-dependent degradation by the proteasome - enzymatic reactions

Question 30

10. ERAD
    c/ What happen during step 1: Recognition of misfolded proteins in ER?

Answer 30

• Misfolded secretory proteins are recognized by specific ER-membrane proteins and are targeted for transport from the ER lumen into the cytosol by a process known as dislocation
• Accumulation of misfolded proteins is referred to as ER stress

Question 31

10. ERAD
    d/ What happen during stEP 2 - Retro-translocation into the ER (retro-translocation = reverse of translocation)?

Answer 31

• Ubiquitin–proteasome system (UPS) is located in the cytosol, terminally misfolded
  proteins have to be transported from the ER back into cytosol
• a complex of at least 4 integral membrane proteins, known as ERAD-complex,
  enables dislocation of misfolded proteins across the ER membrane

Question 32

10. ERAD
    e/ What happen during step 3: Ubiquitin-dependent degradation by the proteasome - enzymatic reactions?

Answer 32

Ubiquitination takes place during the retro-translocation
1. E1 (Ubiquitin-activating enzyme) hydrolyses ATP and forms a high-energy thioester linkage between: (1) active site cysteine residue and (2) ubiquitin c- terminus
2. E2 (ubiquitin-conjugating enzyme) gets the activated ubiquitin from E1
3. E3 (ubiquitin-ligase enzyme) bind to the misfolded protein. E3 align the protein
and E2, facilitating the attachment of ubiquitin to lysine residues of the
misfolded protein
4. Polyubiquitin chain is formed after successive addition of ubiquitin molecules to lysine residues of the previously attached ubiquitin on the protein.