IV. Cell Biology | 67. Endoplasmic reticulum stress, connection between the Unfolded Protein Response (UPR) and apoptosis

Question 1

I. Basics
1. What is Organelle stress?

Answer 1

Organelle stress is when the functional capacity of an organelle becomes insufficient compared with the cellular demand.

Question 2

I. Basics
2. What is the role of ER?

Answer 2

• ER plays a central role in both lipid and protein biosynthesis.
• Several quality control mechanisms function to ensure the proper processing and folding of proteins.
• Unfolded or misfolded proteins are retained in the ER and degraded by ER-associated degradation – ERAD (next topic).
• If unfolded protein build up in the ER, eukaryotic cells upregulate the expression of ER chaperones and components of the ERAD machinery to enhance the capacity of folding and degradation of unfolded proteins => UPR

Question 3

I. Basics
3A. What are the 3 mechanisms of ER stress?

Answer 3

1) Biotransformation process
2) Disulfide bond formation
3) Misfolded proteins

Question 4

I. Basics
3B. One of the mechanism of ER stress is Biotransformation process
=> Explain

Answer 4

Biotransformation process: cytochrome p450 in phase 1 can induce formation of ROS O2- (superoxide anion) or intermediate reactive substance

Question 5

I. Basics
3C. One of the mechanism of ER stress is Disulfide bond formation
=> Explain

Answer 5

Disulfide bond formation: in the process of ER chaperon-mediated protein folding, it causes formation of H2O2

Question 6

I. Basics
3D. One of the mechanism of ER stress is “Misfolded proteins”
=> Explain

Answer 6

Misfolded proteins: many disturbances in the ER can cause accumulation of misfolded proteins or quality control problems (calreticulin chaperon bind misfolded proteins and prevent them from moving to the Golgi)

Question 7

II. Unfolded protein response (UPR)
1. What is Unfolded protein response (UPR)?

Answer 7

Disturbances in normal functions of the ER lead to a cell stress response, the UPR
=> This is aimed initially at compensating for damage, but can eventually trigger cell death if the ER dysfunction is severe or prolonged.

Question 8

II. Unfolded protein response (UPR)
2. What are the 2 phases of UPR?

Answer 8

UPR does therefore have 2 phases:
1) Tries to increase the protein folding capacity to remove the ER stress and decrease the amount of accumulated misfolded proteins
2) If stress cannot be removed, UPR promotes ER stress-induced apoptosis

Question 9

II. Unfolded protein response (UPR)
3. How many pathways executing UPR are there?

Answer 9

There are 3 parallel pathways that execute the UPR, involving the proteins IRE1, PERK and ATF6.

Question 10

II. Unfolded protein response (UPR)
4. What is the mechanism of The chaperone BiP (binding immunoglobulin protein)?

Answer 10

• There are 3 parallel pathways that execute the UPR, involving the proteins IRE1, PERK and ATF6.
• The chaperone BiP (binding immunoglobulin protein) binds to the proteins mentioned above, when they are not needed.
• But if unfolded proteins accumulate in ER, then the BiP dissociates because it needs to bind to the unfolded proteins instead (to retain them in the ER), allowing them to be activated (dimerize, autophosphorylation)

Question 11

II. Unfolded protein response (UPR)
5A. What are the features of IRE1 (inositol requiring enzyme 1)?

Answer 11

A TM protein kinase which oligomerize and phosphorylates itself
=> causes activation of an endoribonuclease domain in the cytosolic surface of IRE1
=> cleaves specific cytosolic mRNA molecules at 2 positions

Question 12

II. Unfolded protein response (UPR)
5B. What is the mechanism of IRE1 (inositol requiring enzyme 1)?

Answer 12

• Cleavage of mRNA will remove an intron, allowing the two exons to be joined by an RNA ligase, creating a new spliced mRNA which is translated into an active transcription regulatory protein called XBP1
• XBP1 is translocated into the nucleus and activates genes encoding proteins that help mediate the UPR

Question 13

II. Unfolded protein response (UPR)
6A. What are the features of PERK (Protein kinase R [PKR]-like ER kinase)?

Answer 13

• Also a TM kinase in the ER

Question 14

II. Unfolded protein response (UPR)
6B. What is the mechanism of PERK (Protein kinase R [PKR]-like ER kinase)?

Answer 14

• When BiP is released, it dimerizes and auto-phosphorylates itself
• PERK then phosphorylates and inactivates eIF2 (eukaryotic translation initiation factor), which inhibits general translation of new proteins on the ribosome => reducing amount of proteins being fed and needed to be folded in the ER
• Some proteins are translated when the eIF2 is inhibited, one of them being the ATF4 (activation TF), which is a TF that induces transcription of genes involved in the UPR (CHOP)

Question 15

II. Unfolded protein response (UPR)
7A. What are the features of ATF6 (Activation TF 6)?

Answer 15

A transcription regulator, initially synthesized as a TM ER protein, is embedded in the ER membrane
=> cannot activate the transcription of genes in the nucleus

Question 16

II. Unfolded protein response (UPR)
7B. What is the mechanism of ATF6 (Activation TF 6)?

Answer 16

• A transcription regulator, initially synthesized as a TM ER protein, is embedded in the ER membrane => cannot activate the transcription of genes in the nucleus
• When misfolded proteins accumulate, the AFT6 protein is transported to the Golgi where it undergoes proteolysis => cytosolic domain of the ATF6 will be cleaved off
• AFT6 can now move to the nucleus and induce transcription of proteins needed for the UPR

Question 17

III. ER stress-induced apoptosis
1. What are the events that lead to ER stress-induced apoptosis?

Answer 17

• If the ER stress is not relieved, the UPR will result in cellular death => ER stress-induced apoptosis.
• The TF and proteins produced in this stage are meant to result in apoptosis, because the dysfunction of the ER is so severe that the cell cannot survive.

Question 18

III. ER stress-induced apoptosis
2. As mentioned, PERK activates ATF4 which transcriptionally activates the apoptotic TF called CHOP. It will have many effects
=> What are the 3 effects?

Answer 18

1) CHOP will decrease the expression of Bcl2 proteins that would normally prevent apoptosis, and induce transcription of BH3-only proteins to aid in formation of the pore in the mitochondria
=> release of cytochrome C (strong apoptotic signal)
2) CHOP also activates GADD34 which is a phosphatase that dephosphorylates eIF2 = translation is no longer inhibited
=> growth arrest and DNA damage inducible proteins
3) CHOP also increases the expression of D5R (death receptor) that interacts with pro- caspases, activating them to caspases (programmed cell-death)