AbuArish_CPPS325_March19_2025_Lecture 6_Upload (1)

Question 1

What is the Integrated Stress Response (ISR) pathway?

Answer 1

A cellular response mechanism to stress that involves stress granules (SGs) formation and regulates gene transcription for cytoprotective proteins.

The ISR pathway involves various signaling molecules such as eIF2α, PERK, and PKR.

Question 2

What are stress granules (SGs)?

Answer 2

Large membrane-less cytoplasmic complexes of mRNA and RNA-binding proteins that form in response to stress.

SGs inhibit global protein synthesis and focus on the transcription of protective genes.

Question 3

Which proteins are involved in SGs formation in response to stress?

Answer 3

• eIF2α
• HRI
• PKR
• PERK
• GCN2

These proteins play crucial roles in the signaling pathways that lead to SGs assembly.

Question 4

What is the role of ATF4 in the ISR?

Answer 4

ATF4 is upregulated and translocated to the nucleus, promoting cytoprotective gene transcription.

This process is essential for cellular adaptation to stress.

Question 5

How do stress granules assemble in human airway epithelial cells (HAECs) after exposure to cigarette smoke extract (CSE)?

Answer 5

SGs assemble within 1 hour of CSE exposure and continue to form for up to 2 hours.

This timeline indicates the dynamic response of cells to stress.

Question 6

When do SGs begin to disassemble in HAECs after CSE exposure?

Answer 6

SGs begin to disassemble after 4 hours of CSE exposure.

This disassembly suggests a potential stress relief in the cells.

Question 7

What is the effect of the ΔF508-CFTR mutation on SGs formation in HAECs?

Answer 7

HAECs expressing ΔF508-CFTR form fewer and smaller SGs compared to those expressing WT-CFTR.

The mutation negatively impacts the cellular stress response.

Question 8

What does CSE upregulate in HAECs, indicating ISR activation?

Answer 8

CSE upregulates eIF2α phosphorylation.

This suggests the activation of the ISR pathway.

Question 9

What is the function of ISRIB in relation to SGs formation?

Answer 9

ISRIB inhibits the downstream function of p-eIF2α, abrogating SGs formation in HAECs.

ISRIB does not reduce eIF2α phosphorylation but affects its functional role.

Question 10

What is the role of p-PERK in SGs formation?

Answer 10

p-PERK induces SGs formation by phosphorylating eIF2α.

Inhibition of p-PERK halts SGs formation.

Question 11

Does CSE upregulate PKR phosphorylation in HAECs?

Answer 11

No, CSE does not upregulate PKR phosphorylation in HAECs expressing either WT-CFTR or ΔF508-CFTR.

This indicates PKR is not involved in the stress response to CSE.

Question 12

What are the major components of CSE that activate the PERK/eIF2α pathway?

Answer 12

• Superoxide radicals
• Hydrogen peroxide (H2O2)

These reactive oxygen species (ROS) are responsible for ISR activation.

Question 13

What is the effect of NAC administration prior to CSE exposure?

Answer 13

NAC administration abrogates SGs formation and p-eIF2α upregulation.

NAC acts as a ROS scavenger, preventing stress response activation.

Question 14

Can superoxide radicals alone induce SGs formation?

Answer 14

Yes, superoxide radicals can induce SGs formation in the absence of other CSE components.

This was demonstrated using DMNQ, which generates superoxide radicals.

Question 15

What is the significance of the findings from the lecture regarding CSE and CF?

Answer 15

The findings emphasize the role of the ISR pathway and stress granules in the context of cystic fibrosis and airway epithelial cell response to stress.

The implications suggest potential therapeutic targets for improving cellular stress responses in CF.