Stepwise intrinsic mechanism of apoptotic induction

Stepwise Intrinsic Mechanism of Apoptotic Induction

The intrinsic pathway of apoptosis, also known as the mitochondrial pathway, is initiated by intracellular stresses such as DNA damage, oxidative stress, endoplasmic reticulum (ER) stress, or growth factor withdrawal. This pathway relies on the balance between pro-apoptotic and anti-apoptotic proteins within the Bcl-2 family.

1. Initiation Phase: Stress Signals and Bcl-2 Family Proteins

Intracellular stress signals activate pro-apoptotic members of the Bcl-2 family, including Bax and Bak. These proteins undergo a conformational change and oligomerize, inserting into the outer mitochondrial membrane (OMM). Anti-apoptotic proteins like Bcl-2, Bcl-xL, and Mcl-1 normally inhibit Bax and Bak, preventing their activation. However, under stress conditions, these anti-apoptotic proteins are neutralized or downregulated, allowing Bax and Bak to proceed with OMM permeabilization.

2. Mitochondrial Outer Membrane Permeabilization (MOMP)

The oligomerization of Bax and Bak creates pores in the OMM, leading to MOMP. This is a critical commitment step in apoptosis. MOMP results in the release of several pro-apoptotic factors from the intermembrane space of the mitochondria into the cytosol. Key factors released include:

• Cytochrome c: Essential for activating the apoptosome.
• Smac/DIABLO: Inhibits IAPs (Inhibitors of Apoptosis Proteins).
• Omi/HtrA2: Also inhibits IAPs.
• Apoptosis-inducing factor (AIF): Translocates to the nucleus and induces DNA fragmentation.

3. Apoptosome Formation and Caspase Activation

In the cytosol, cytochrome c binds to Apaf-1 (Apoptotic protease activating factor-1) and dATP, forming a multi-protein complex called the apoptosome. The apoptosome then recruits and activates pro-caspase-9. Activated caspase-9 is an initiator caspase.

4. Caspase Cascade: Execution Phase

Activated caspase-9 cleaves and activates downstream effector caspases, primarily caspase-3, -6, and -7. This initiates the caspase cascade, a proteolytic cascade that amplifies the apoptotic signal. Effector caspases cleave a variety of cellular substrates, leading to the characteristic morphological changes of apoptosis.

5. Morphological Changes and Cell Dismantling

Effector caspases trigger a series of events leading to:

• DNA fragmentation: Caspase-activated DNase (CAD) is activated, leading to internucleosomal DNA cleavage.
• Blebbing: The cell membrane bulges outwards, forming blebs.
• Cell shrinkage: The cell volume decreases.
• Formation of apoptotic bodies: The cell breaks down into small, membrane-bound vesicles called apoptotic bodies.
• Phagocytosis: Apoptotic bodies are rapidly engulfed by phagocytes, preventing inflammation.

6. Role of IAPs and Smac/DIABLO

IAPs (Inhibitor of Apoptosis Proteins) normally bind to and inhibit caspases, preventing apoptosis. However, Smac/DIABLO, released from the mitochondria during MOMP, binds to IAPs, neutralizing their inhibitory effect and allowing the caspase cascade to proceed. This is a crucial regulatory step ensuring the apoptotic signal is not prematurely terminated.

Phase	Key Events	Key Proteins Involved
Initiation	Intracellular stress, activation of pro-apoptotic Bcl-2 proteins	Bax, Bak, Bcl-2, Bcl-xL, Mcl-1
MOMP	Formation of pores in OMM, release of pro-apoptotic factors	Bax, Bak, Cytochrome c, Smac/DIABLO, AIF
Apoptosome Formation	Cytochrome c binding to Apaf-1, caspase-9 activation	Cytochrome c, Apaf-1, caspase-9, dATP
Caspase Cascade	Activation of effector caspases	Caspase-3, -6, -7
Execution	DNA fragmentation, blebbing, cell shrinkage, apoptotic body formation	CAD, caspases