Questions and Answers: Role of Mitochondria in Apoptosis

1. Explain the role of mitochondria in intrinsic apoptosis.

Answer:
Mitochondria play a pivotal role in intrinsic apoptosis, which is triggered by internal cellular stress such as DNA damage, oxidative stress, or ER stress. The process involves the following steps:

• Mitochondrial Outer Membrane Permeabilization (MOMP): Pro-apoptotic proteins like Bax and Bak form pores in the outer mitochondrial membrane, allowing the release of apoptogenic factors.
• Release of Cytochrome c: Cytochrome c is released into the cytoplasm, where it binds with APAF-1 to form the apoptosome.
• Caspase Activation: The apoptosome activates procaspase-9, which in turn activates effector caspases like caspase-3, leading to apoptosis.

2. Describe the Bcl-2 protein family and its role in mitochondrial apoptosis.

Answer:
The Bcl-2 protein family regulates mitochondrial membrane permeability during apoptosis.

• Anti-apoptotic Proteins: Include Bcl-2 and Bcl-xL, which stabilize the mitochondrial membrane and prevent cytochrome c release.
• Pro-apoptotic Proteins: Include Bax and Bak, which promote MOMP and cytochrome c release.
• BH3-only Proteins: Such as Bid, Bad, and Puma, act as sensors of cellular stress and mediate the balance between anti- and pro-apoptotic proteins.

3. How does cytochrome c contribute to the apoptotic process?

Answer:
Cytochrome c is a critical player in apoptosis:

1. Release from Mitochondria: Upon MOMP, cytochrome c is released into the cytosol.
2. Apoptosome Formation: It binds APAF-1 and dATP to form the apoptosome complex.
3. Caspase Activation: The apoptosome recruits and activates procaspase-9, initiating the caspase cascade.
4. Execution of Apoptosis: Effector caspases like caspase-3 cleave key cellular proteins, leading to apoptosis.

4. What is the mitochondrial permeability transition pore (mPTP), and how does it regulate apoptosis?

Answer:
The mPTP is a multiprotein complex in the inner mitochondrial membrane.

• Role in Apoptosis: Its opening leads to loss of mitochondrial membrane potential, swelling, and release of pro-apoptotic factors like cytochrome c and AIF.
• Regulation: Pro-apoptotic signals, such as high calcium levels or oxidative stress, promote mPTP opening, while anti-apoptotic proteins inhibit it to maintain mitochondrial integrity.

5. Discuss the role of reactive oxygen species (ROS) in mitochondrial-mediated apoptosis.

Answer:
ROS are byproducts of mitochondrial metabolism and act as signaling molecules.

• Triggering Apoptosis: Excess ROS damages mitochondrial DNA, lipids, and proteins, leading to loss of membrane potential and MOMP.
• Cytochrome c Release: Oxidative damage facilitates the release of cytochrome c into the cytosol.
• Caspase Activation: ROS amplify the apoptotic cascade by promoting caspase activation.

6. What is the role of mitochondrial membrane potential in apoptosis?

Answer:
Mitochondrial membrane potential (ΔΨm\Delta \Psi_mΔΨm​) is crucial for mitochondrial function.

• Maintenance of Integrity: Anti-apoptotic proteins stabilize ΔΨm\Delta \Psi_mΔΨm​, preventing apoptosis.
• Loss of ΔΨm\Delta \Psi_mΔΨm​: Indicates mitochondrial dysfunction and triggers the release of apoptogenic factors, leading to apoptosis.

7. Explain the formation and function of the apoptosome.

Answer:

• Formation: The apoptosome is formed when cytochrome c binds APAF-1 in the cytoplasm, recruiting dATP and procaspase-9.
• Function: It activates procaspase-9, initiating a caspase cascade that leads to cell death.

8. How do Bax and Bak regulate apoptosis through mitochondria?

Answer:
Bax and Bak are pro-apoptotic proteins that promote apoptosis by:

1. Membrane Permeabilization: They oligomerize to form pores in the mitochondrial outer membrane.
2. Release of Cytochrome c: These pores facilitate the escape of cytochrome c and other factors.
3. Downstream Activation: They enable caspase activation, leading to apoptosis.

9. Describe the anti-apoptotic mechanisms of Bcl-2 and Bcl-xL.

Answer:
Bcl-2 and Bcl-xL inhibit apoptosis by:

• Stabilizing Mitochondria: Preventing MOMP and the release of apoptogenic factors.
• Inhibiting Bax/Bak: Directly binding and neutralizing pro-apoptotic proteins.

10. What is the significance of Smac/DIABLO in apoptosis?

Answer:
Smac/DIABLO is released from mitochondria during apoptosis.

• Inhibition of IAPs: It binds and inhibits inhibitors of apoptosis proteins (IAPs), ensuring caspase activation.
• Amplification of Apoptosis: Smac/DIABLO enhances the apoptotic response by promoting caspase activity.

11. Discuss the role of mitochondrial DNA (mtDNA) damage in apoptosis.

Answer:

• Triggering Apoptosis: mtDNA damage impairs mitochondrial function and promotes ROS generation.
• Caspase Activation: Damaged mtDNA signals the release of apoptogenic factors, activating the caspase cascade.

12. How does calcium overload in mitochondria initiate apoptosis?

Answer:
Excess calcium in mitochondria:

• Opens mPTP: Leads to loss of membrane potential and release of cytochrome c.
• Triggers ROS Generation: Further damages mitochondrial integrity, amplifying apoptosis.

13. Explain the extrinsic and intrinsic apoptosis pathway connection via mitochondria.

Answer:

• Bid Activation: In the extrinsic pathway, caspase-8 cleaves Bid to tBid, which activates Bax/Bak in the intrinsic pathway.
• MOMP: The mitochondria release cytochrome c, linking the two pathways.

14. What is the role of apoptosis-inducing factor (AIF) in apoptosis?

Answer:
AIF is a mitochondrial protein that mediates:

• Caspase-independent Apoptosis: Translocates to the nucleus to induce DNA fragmentation.
• Enhancing Apoptotic Signals: Amplifies the intrinsic pathway.

15. How does mitochondrial fragmentation occur during apoptosis?

Answer:

• Role of Drp1: Drp1 promotes mitochondrial fission.
• Facilitates Apoptosis: Fragmentation enhances the release of apoptogenic factors like cytochrome c.

16. What are the molecular events during mitochondrial-mediated apoptosis?

Answer:

1. Stress signals activate pro-apoptotic Bcl-2 family proteins.
2. Bax/Bak oligomerize to form pores.
3. Cytochrome c and Smac/DIABLO are released.
4. Apoptosome forms, activating caspase-9.
5. Executioner caspases cleave cellular proteins, causing apoptosis.

17. Describe the role of mitochondria-associated membranes (MAMs) in apoptosis.

Answer:
MAMs facilitate ER-mitochondrial communication:

• Calcium Transfer: High calcium influx into mitochondria triggers apoptosis.
• ROS Signaling: Enhances pro-apoptotic signaling.

18. What is the role of mitochondrial cardiolipin in apoptosis?

Answer:
Cardiolipin is a lipid found in the inner mitochondrial membrane:

• Cytochrome c Release: Oxidized cardiolipin promotes its release.
• ROS Production: Amplifies oxidative stress, facilitating apoptosis.

19. How does oxidative stress influence mitochondrial apoptosis?

Answer:
Oxidative stress damages mitochondrial components, leading to:

• Loss of ΔΨm\Delta \Psi_mΔΨm​.
• mPTP opening and cytochrome c release.
• Caspase cascade activation.

20. Summarize the dual role of mitochondria in cell survival and apoptosis.

Answer:

• Cell Survival: Mitochondria generate ATP and maintain redox balance.
• Apoptosis Regulation: Under stress, they release apoptogenic factors like cytochrome c, initiating cell death.