Intrinsic Pathway of Apoptosis (Mitochondrial Pathway)

The intrinsic pathway of apoptosis is triggered within the cell, in response to internal stress signals such as DNA damage, oxidative stress, ER stress, or oncogene activation. Several elements of (Reactive oxygen species) ROS-induced apoptosis are also included, such as the formation of highly toxic nitrogen reactive species (NRS) It is mitochondria-dependent and leads to cytochrome c release, apoptosome formation, and caspase activation.

Key Steps in the Intrinsic Pathway:

1. Cellular Stress Triggers

DNA damage (e.g., by UV radiation), Hypoxia, Oxidative stress, Growth factor deprivation, Oncogene activation (e.g., Myc) triggers apoptosis.

2. Mitochondrial Outer Membrane Permeabilization (MOMP)

MOMP is regarded as the “point of no return” during apoptosis. The increase in permeability release numerous apoptotic proteins that are present in the peri-mitochondrial space. MOMP cause a collapse of mitochondrial membrane potential (Δym). Depending on the stimuli, the loss of Δym can occur before, during or after MOMP. Inner mitochondrial membrane (IMM) can also induce the MOMP through permeability transition pore (PTP) formation. Permeability transition pore (PTP) is a complex composed of voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane (OMM), The adenine nucleotide translocator (ANT) in the IMM and cyclophilin D (cypD) in the matrix. The opening of permeability transition pore (PTP) leads to an influx of ions into the mitochondrial matrix causing the loss of Δym, and swelling of the matrix as water enters leading to rupture of OMM and MOMP.

Mitochondrial Outer Membrane Permeabilization (MOMP) Controlled by the Bcl-2 family proteins:

• • Pro-apoptotic: Bax, Bak → Promote membrane permeabilization

  • Anti-apoptotic: Bcl-2, Bcl-xL → Block Bax/Bak

Bax and Bak are constitutively expressed and induce MOMP following apoptotic stimuli. Bax is present in a monomeric form in the cytoplasm and sometimes associated with OMM. During apoptotic stimuli, it is inserted into Outer Mitochondrial Membrane (OMM).  Stress causes Bax/Bak to oligomerize, forming pores in the outer mitochondrial membrane.

3. Release of Cytochrome c

Mitochondria release cytochrome c into the cytosol. Mitochondrial proteins known as SMACs (small mitochondrial-derived activator of caspases) are released in cytosol following an increase in permeability. Mitochondria Also release DIABLO and AIF (Apoptosis-inducing factor). The activator of caspases (Smac/DIABLO ) is a mitochondrial protein that inhibits the inhibitor of apoptosis protein (IAP). Inhibitor of apoptosis protein blocks the processing of caspases 3 and Caspases-9. The HtrA2 and Omi also inhibit inhibitor of apoptosis protein (IAP).

4. Apoptosome Formation

The Cytochrome C binds Apaf-1(Apoptotic proteases Activating Factor), dATP, and procaspase -9 to create a complex known as the Apoptosome. The procaspase-9 cleaves into caspase-9 to generate caspase-3, which causes apoptosis. This complex recruits and activates procaspase-9, forming the apoptosome.  Apoptosome = Cytochrome c + Apaf-1 + dATP + procaspase-9

5. Caspase Activation Cascade

Caspase-9 activates executioner caspases (e.g., caspase-3, caspase-7). These caspases cleave cellular substrates → nuclear condensation, membrane blebbing, and apoptosis.

Regulators of the Intrinsic Pathway

Bcl-2 Family Proteins in Regulation of Mitochondrial Outer Membrane Permeabilization (MOMP)

MOMP (Mitochondrial Outer Membrane Permeabilization) is the central and irreversible step in the intrinsic pathway of apoptosis. MOMP is regarded as the “point of no return” during apoptosis. It is tightly regulated by the Bcl-2 family proteins, which control whether a cell lives or dies by influencing mitochondrial membrane integrity.

Classification of Bcl-2 Family Proteins

Bcl-2 family members are grouped based on their structure (number of BH domains) and function (pro- or anti-apoptotic). The Bcl-2 family of proteins is divided into three groups based on their function and the number of BCL-2 homology

1. Pro-apoptotic Effectors (Executioners)

Bax and Bak are pro-apoptotic effectors (executioners) that play a crucial role in the intrinsic pathway of apoptosis, primarily by mediating mitochondrial outer membrane permeabilization (MOMP). These proteins, members of the Bcl-2 family, are activated upon apoptotic stimuli and oligomerize at the outer mitochondrial membrane, causing the release of cytochrome c and other factors that trigger the caspase cascade and cell death. Bax and Bak  contain multiple BH domains (BH1–3) . They are Essential for MOMP

2. Pro-apoptotic BH3-only Proteins (Initiators)

Pro-apoptotic BH3-only proteins are essential initiators of apoptosis. It Includes Bid, Bim, Bad, Puma, Noxa, tBid. They achieve this by interacting with and neutralizing pro-survival Bcl-2 family members, ultimately leading to the release of cytochrome c from mitochondria, which triggers the apoptotic cascade.

3. Anti-apoptotic Members (Inhibitors)

They are Present in OMM and have four BH domains (BH1-BH4). eg Bcl-2, Bcl-XL, Bcl-w, Mcl- 1, A1 and Bcl-B. Bcl2 and Bcl-x prevent the pore formation on OMM. They Bind and inhibit Bax/Bak oligomerization. They Preserve mitochondrial integrity. Thy Block MOMP and apoptosome formation

Functional Interactions and Balance

Inhibitor of apoptosis proteins (IAPs)

The IAP family of proteins consists of eight human analogues, including cellular IAP1 (c-IAP1), cellular IAP2 (c-IAP2), Xlinked inhibitor of apoptosis (XIAP), survivin, Apollon (also known as Bruce), melanoma IAP (ML-IAP, also known as Livin) and IAP-like protein 2 (ILP-2). All IAP proteins contain BIR (baculoviral IAP repeat) domain which inhibits the caspase activity. In addition to the BIR domain, all IAPs except survivin contains a RING domain, which possesses E 3 ubiquitin ligase activity. The linker region that precedes the BIR-domain binds to the active site of Caspase-3 or 7. This binding prevents substrate binding.

Glycogen synthase kinase-3

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase which is ubiquitously expressed in mammalian cells. GSK-3 is a kinase that phosphorylates glycogen synthase and makes it inactive. Akt phosphorylates GSK-3 on these serine residues. Overexpression of GSK-3 promotes mitochondrial intrinsic apoptosis. GSK-3 is present in mitochondria and mitochondrial GSK-3 activity is increased during DNA damage or ER stress, that leads to apoptosis. GSK-3 inhibits extrinsic apoptosis by inhibiting the death-receptor-mediated apoptosis.

Stress Proteins and Molecules That Inhibit Apoptosis

Link Between Intrinsic and Extrinsic Pathways of Apoptosis via tBid

Apoptosis occurs through two main pathways—intrinsic (mitochondrial) and extrinsic (death receptor-mediated)—which are interconnected through a critical molecule: tBid (truncated Bid).

Bid is a BH3-only Bcl-2 family protein (normally inactive in cytosol). The Caspase-8 cleaves Bid and  produces tBid (truncated Bid). The tBid translocates to mitochondria and activates Bax/Bak. This causes MOMP thus links extrinsic to intrinsic pathway. Even if extrinsic signals are weak, tBid amplifies the apoptotic signal via mitochondria.