28.14: Programmed Cell Death

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Learning Goals 

 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

Overview, Physiological Roles, and Comparison to Other Cell Death Pathways

• Distinguish apoptosis from autophagy and necroptosis, describe the four sequential stages of apoptosis (decision, cell suicide, engulfment, degradation), and explain the molecular hallmarks of apoptotic cell death—nuclear condensation and fragmentation, cytoplasmic condensation into membrane-bound apoptotic bodies, internucleosomal DNA ladder formation, and phosphatidylserine externalization that signals phagocyte recognition.
• Explain the physiological contexts in which apoptosis is essential (embryonic sculpting, immune cell homeostasis, elimination of virally infected and tumor cells) and describe how dysregulation in either direction—insufficient apoptosis (cancer, viral persistence) or excessive apoptosis (AIDS-related T helper cell depletion, neurodegeneration, ischemic stroke)—underlies human disease.

The Extrinsic and Intrinsic Apoptotic Pathways

• Trace the extrinsic apoptotic pathway from death ligand binding (FasL, TNF-α, TRAIL) to death receptor aggregation (Fas/CD95, TNFR1), recruitment of adaptor proteins (FADD, TRADD) through death domain interactions, assembly of the Death-Inducing Signaling Complex (DISC), and activation of initiator caspases 8 and 10 that cleave and activate effector caspases 3 and 7.
• Trace the intrinsic apoptotic pathway from intracellular stress signals (DNA damage, ROS, radiation, Ca²⁺ elevation) through BH3-only protein activation, neutralization of pro-survival Bcl-2 family members, Bax/Bak homo-oligomerization and outer mitochondrial membrane permeabilization, cytochrome c and Smac release into the cytoplasm, apoptosome assembly (cytochrome c:Apaf-1:procaspase-9 heptameric complex), and activation of caspase 9 leading to effector caspase activation.
• Explain how the extrinsic and intrinsic pathways are interconnected through caspase-8-mediated cleavage of BID to truncated tBID, which activates Bax/Bak at the mitochondrial membrane, and describe how IAP proteins inhibit caspase activity while Smac promotes apoptosis by neutralizing IAPs.
• Describe the structural basis of caspase activation—explaining that they are Cys-Asp proteases synthesized as inactive zymogens that undergo limited proteolysis for activation, and that initiator caspases (8, 9) are activated by proximity-induced dimerization at oligomeric platforms (DISC or apoptosome) while effector caspases (3, 7) are activated by initiator caspase cleavage—and explain why the cascade architecture provides both amplification and irreversible commitment.

Regulation of Apoptosis and Therapeutic Targeting

• Explain how pro-survival signaling through growth factor receptors→Ras→PI3K→Akt inhibits apoptosis by phosphorylating and inactivating the pro-apoptotic protein BAD and by phosphorylating procaspase to prevent its interaction with cytochrome c, and explain how p53 tumor suppressor activity promotes apoptosis in DNA-damaged cells through oxidative stress pathways—and why p53 loss-of-function mutations in ~50% of human tumors allow damaged cells to escape apoptosis.
• Describe the major therapeutic strategies targeting each pathway: TRAIL receptor agonist antibodies and recombinant TRAIL activating the extrinsic pathway; BH3 mimetics (such as venetoclax) blocking pro-survival Bcl-2 family proteins to restore intrinsic apoptosis in cancer cells; and Smac mimetics neutralizing IAPs—and explain why selectivity for cancer cells over normal cells is the central pharmacological challenge in each approach.

Introduction

We have often discussed how cell signaling might go awry and lead to cancer. However, there are signaling systems that lead to cell death. There are many ways in which cells can die. We'll discuss not "accidental" cell death but one, apoptosis, that is programmed into the genome and highly regulated. Figure \(\PageIndex{1}\) shows how normal cell proliferation and growth can be modulated by two classes of genes, oncogenes that cause proliferation, and tumor suppressor genes that inhibit it.

Figure \(\PageIndex{1}\): Overview of oncogenes, tumor suppressor genes, apoptotic genes, and antiapoptotic genes. Adapted from https://www.researchgate.net/figure/...fig1_332774933. Creative Commons Attribution 3.0 Unported

Apoptosis involves chromatin aggregation and cleavage, the concentration of cell material, and the formation of the apoptotic body. Mutations to aberrantly activate oncogenes or inhibit the expression of tumor suppressor genes lead to cancer. These cells would ideally undergo programmed cell death or apoptosis. As with proliferation control, some genes promote apoptosis, while anti-apoptotic genes inhibit programmed cell death. Dysregulation of these can also cause cancer. Apoptosis is an important mechanism for killing virus-infected cells. However, this can go too far. For example, T helper cells (TH) infected with HIV die. However, the collapse of these cells' population is partly attributed to apoptosis.

As we learn more about programmed cell death, it is clear that apoptosis is not the only way the genome is programmed to cause cell death. These other ways include:

autophagy - This is a catabolic pathway in which intracellular proteins, protein complexes, and organelles are collected into large autophagosomes, which are incorporated into lysosomes, and their degradative enzymes reprocess damaged or unneeded cell material. It is a highly programmed process, which, if dysregulated, could lead to cell death.

necroptosis: Infections and toxins are known to cause necrosis, which is a "passive" form of cell death. In contrast, programmed necrosis is called necroptosis.

Overview of apoptosis.

Apoptosis consists of 4 steps:

• the decision to activate the pathway;
• the actual "suicide" of the cell;
• engulfment of the cell remains by specialized immune cells called phagocytes;
• degradation of the engulfed cell.

The actual steps in cell death require:

• condensing the cell nucleus and breaking it into pieces
• condensing and fragmenting of cytoplasm into membrane-bound apoptotic bodies; and
• breaking chromosomes into fragments containing multiple numbers of nucleosomes (a nucleosome ladder)

To commit suicide must be an extremely important cellular decision. Hence, you would expect this process to be regulated and highly complicated. When would it be advantageous to the organism to want a cell to kill itself (or be told to kill itself)? Cell death would be used to:

• "sculpt" an organism during development, such as during embryo development, metamorphosis, and tissue atrophy
• regulate the total number of cells.
• defend and remove unwanted or dangerous cells, such as tumor cells, virally infected cells, or immune cells that recognize themselves (which could lead to autoimmune disease).

Unregulated apoptosis could exacerbate or cause diseases such as:

• AIDS, in which T helper cell numbers plummet. Part of the dramatic decline in these cells might be caused by healthy T helper cells being tricked into committing suicide;
• neurodegenerative diseases like Alzheimer's;
• ischemic stroke, when restricted blood flow to some areas of the brain, can lead to neural death through increased apoptosis;
• cancer, in which tumor cells lose their ability to undergo apoptosis;
• autoimmune disease, in which self-reactive immune cells trick normal body cells into killing themselves;
• viral disease;

Apoptosis does not require new transcription or translation, suggesting that the molecular machinery for cell death lies dormant and only requires appropriate activation. What "signals" induce apoptosis?

Signals can be extracellular:

• a hormone (such as thyroxine that causes apoptosis in tadpole tails
• a lack of a "survival" signal (which inhibits apoptosis), such as a growth factor
• a cell:cell contact from an adjacent cell

Signals can be intracellular:

• ionizing radiation
• virus infection
• oxidative damage from free radicals

Apoptosis

Much of this section was derived directly from the following reference, with modifications and additions.

Fox, J., MacFarlane, M. Targeting cell death signaling in cancer: minimizing ‘Collateral damage’. Br J Cancer 115, 5–11 (2016). https://doi.org/10.1038/bjc.2016.111. Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

There are two apoptotic pathways in cells:

• The extrinsic pathway: Extracellular apoptotic ligands bind to membrane death receptors, leading to the assembly of the death-inducing signaling complex (DISC). Similar to the inflammatory response we discussed in Chapter 5, two specific cysteine-aspartic proteases (caspases), caspases 8 and 10, are activated. These activate other caspases, amplifying the process.
• The intrinsic pathway: Intracellular signals, such as damaged DNA or proteins, are sensed by Bcl-2 proteins on the outer membrane of mitochondria. The Bcl-2 (B-cell lymphoma-2) family of proteins all have Bcl homology domains. Their functions are carried out at the outer mitochondrial membrane. Some members of this family are antiapoptotic (Bcl-2, Bcl-xL, Mcl-1, Bcl-w, A1/Bfl-1, and Bcl-B/Bcl2L10), while others are proapoptotic (Bid, Bim, Puma, Noxa, Bad, Bmf, Hrk, Bik Bax, Bak, and Bok/Mtd). Apoptosis leads to activation of Bax/Bak, which initiates mitochondrial degradation, starting with damage to the outer membrane and the release of pro-apoptotic proteins, such as the inner membrane space protein cytochrome C, into the cytoplasm. This leads to the assembly of the apoptosome and the activation of caspases 9 and 13. Again, this is very similar to the formation and activity of the inflammasome, which we saw in Chapter 5.

Figure \(\PageIndex{2}\) shows an overview of the extrinsic and intrinsic apoptotic pathways. We will explore some of the proteins involved in the section below.

Figure \(\PageIndex{2}\): Overview of the extrinsic and intrinsic apoptotic pathways

Death receptor ligands, including FasL, TNF-α, DR3, DR4, DR5, and TRAIL, activate the extrinsic death receptor pathway. FasL is an integral membrane protein found in cells. In addition, there are soluble versions of it. The binding of FasL to Fas, an integral membrane protein, initiates the recruitment of FADD, TRADD, and caspase-8 to form the DISC complex, which activates caspase-8 and downstream caspases. The binding of tissue necrosis factor alpha (TNF-α) to its receptor, TNFR1 (a Fas protein), initiates the recruitment of TRADD, RIP, TRAF2/5, and cIAP1/2 to form complex I, which activates NF-κB and JNK pathways and increases the transcription of pro-survival genes. However, RIP modification or degradation of cIAP1/2 can lead to dissociation of complex I. TRADD and RIP then associate with FADD and caspase-8 to form complex II, the so-called death complex.

The intrinsic death receptor pathway is initiated by the BH3-only protein BCL-2 homology 3 (BH3-only) in response to intracellular stress, such as DNA damage. The BH3-only proteins activate apoptosis by binding and neutralizing pro-survival proteins, thereby allowing Bax/Bak to homo-oligomerize and permeabilize mitochondria. For example, the BH3-only protein can inactivate Bcl-2, preventing it from effectively neutralizing Bax and Bak and thereby leading to their activation. The activated Bax and Bak on the mitochondrial membrane alter its permeability, depolarize the membrane, and lead to the release of cytochrome c and Smac, normally found in the inner membrane space, from mitochondria. Figure \(\PageIndex{3}\) shows how monomeric BAK can form an altered dimeric form in the presence of detergent.

Figure \(\PageIndex{3}\): Monomeric and membrane associated dimeric BAK

The extended left-hand helix on the right-hand side is color-coded to show nonpolar residue (orange) and the polar/charged amino acids in gray. That same protein section is shown in cyan in the monomeric protein to the left. One can easily imagine how the apparent amphiphilic helices of the BAK dimer could bind to the outer mitochondrial membrane and alter its structure.

Cytoplasmic cytochrome c associates with Apaf-1 and caspase-9 to form the apoptosome, which activates caspase-9 and downstream executing caspases. Smac can regulate apoptosis by inhibiting the inhibitor of apoptosis proteins (IAPs)." Zhou and Li. Chapter 9, Apoptosis in Polycystic Kidney Disease: From Pathogenesis to Treatment. License: This open-access article is licensed under Creative Commons Attribution 4.0 International (CC BY 4.0)

Another diagram of the extrinsic and intrinsic apoptotic pathways that shows more detail on the domain structures of some key proteins and the "executioner" caspases is shown in Figure \(\PageIndex{4}\).

Figure \(\PageIndex{4}\): Extrinsic and intrinsic apoptotic pathways https://commons.wikimedia.org/wiki/File:Apop1.png

Extrinsic pathway: The first step is the association of death receptors with their cognate ligands, which recruits adaptor molecules, including FAS-associated death domain protein (FADD), and then caspase 8. Caspase 8 cleaves and activates caspase 3 and caspase 7, and can proteolytically activate BH3-only protein BH3-interacting domain death agonist (BID). Proteolytically activated BID (tBID) promotes mitochondrial membrane permeabilization by activating the assembly of BAX-BAK channels and representing the main link between the extrinsic and intrinsic pathways.

Now let's look more closely at the ligands that activate the extrinsic pathway, as shown in Figure \(\PageIndex{5}\).

Figure \(\PageIndex{5}\): Fas/FasL activation pathway

Soluble Fas and soluble FasL bind their respective ligands, thereby inhibiting activation of the pathway. FLIP inhibits caspase-8 activation and is thus a major anti-apoptotic protein. Volpe E et al. (2016) Fas–Fas Ligand: Checkpoint of T Cell Functions in Multiple Sclerosis. Front. Immunol. 7:382. doi: 10.3389/fimmu.2016.00382. Creative Commons Attribution License (CC BY).

Now we are in a position to examine the actual structure of some key components of the extrinsic pathway.

Active human apoptosome with procaspase-9 (5JUY)

Figure \(\PageIndex{6}\) shows an interactive iCn3D model of the active human apoptosome with procaspase-9 (5JUY)

Figure \(\PageIndex{6}\): Active human apoptosome with procaspase-9 (5JUY) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...ek6wzp36TGUpp6 (long load time)

Each of the seven subunits of the apoptotic protease-activating factor (Apaf-1) is shown in a different color. The seven yellow subunits are cytochrome Cs. The 4 red subunits beneath the disk plane of the other subunits are the zymogen procaspase-9s. The small spacefill CPK color ligands are 2'-deoxyadenosine 5'-triphosphate. The Apaf-1:pc9 pairs, interacting through their CARD domains, form a spiral underneath the disk.

Apaf-1 is an adaptor protein with an N-terminal caspase activation and recruitment domain (CARD), followed by a nucleotide-binding and oligomerization domain (NOD, also known as NB-ARC).

Figure \(\PageIndex{7}\) shows the domain structure of caspase 9 and Apaf-1.

Caspase 9 domain structure

Apaf-1

Figure \(\PageIndex{7}\): domain structure of caspase 9 and Apaf-1.

The presence of CARD domains in both allows their mutual binding and the assembly of the full apoptosome.

An AlphaFold model of the Cas 9 zymogen

Figure \(\PageIndex{8}\) shows an interactive iCn3D model of human Cas 9 AlphaFold model (P55211)

Figure \(\PageIndex{8}\): Human Cas 9 AlphaFold model (P55211) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...74TiVhNfiX4376

The green is the CARD domain and the salmon is the caspase (peptidase_C14) domain. Procaspase 9 is cleaved at Asp 315 (sticks, CPK colors, labeled) into two chains for activation. The activated Cas9 has two key active-site residues: His237 and the catalytic nucleophile C287 (sticks, CPK colors, labeled). Phosphorylation at Thr-125 by MAPK1/ERK2 blocks procaspase activation by proteolysis. to block caspase-9 processing

Apaf-1

Oligomeric Apaf-1 mediates the cytochrome c-dependent autocatalytic activation of pro-caspase-9 (Apaf-3), leading to the activation of caspase-3 and apoptosis

Figure \(\PageIndex{9}\) shows an interactive iCn3D model of Human Apaf-1 AlphaFold model (O14727)

Figure \(\PageIndex{9}\): Human Apaf-1 AlphaFold model (O14727) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...9ZrDFxEpdHR9K6

Domain colors:

• The green is the N-terminal CARD domain
• light red NB-ARC (nucleotide-binding and oligomerization domain - NOD)
• purple is Apaf
• yellow is WD40, gold the C-terminal WD40.

Again, the model above does not show the actual structure because the intrinsically disordered regions are unstructured.

The CARD domain of Cas9 inhibits the catalytic domain of Cas9. When the CARD domain of Cas9 interacts with the CARD domain of Apaf-1, the autoinhibition is removed. In addition, Apaf-1 stimulates the protease's catalytic activity.

Before assembly into the apoptosome, Apaf-1 is monomeric and in an inactive dATP or ATP conformation. When cytochrome C is released into the cytoplasm, it binds to the WD domains, facilitating a dATP/ATP-cleavage-associated conformation change in Apaf-1. In the presence of heat shock protein 70 (Hsp), it folds into a conformation that promotes assembly of the active apoptosome.

Fas - Tumor necrosis factor receptor superfamily member 6 - P25445

Figure \(\PageIndex{10}\) shows an interactive iCn3D model of Fas-Tumor necrosis factor receptor superfamily member 6 AlphaFold model (P25445)

Figure \(\PageIndex{10}\): Fas-Tumor necrosis factor receptor superfamily member 6 AlphaFold model (P25445) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...Dpm3neKspeCrP8

The green is the N-terminal TNFR/NGFR domain highly enriched in Cys (spacefill, color CPK) in disulfide bonds. The gray spheres are the transmembrane helix. The Red shows the Death Domain.

The death domains are common protein:protein binding domains that serve as adaptors or scaffolds. They can form homo- or heterodimers with other proteins that contain the domain, which is part of the CARD domain, DED (Death Effector Domain), and PYRIN.

Human FasL and a soluble Fas Receptor DcR

Figure \(\PageIndex{11}\) shows an interactive iCn3D model of the complex of Human FasL and Its Decoy Fas Receptor DcR (4MSV)

Figure \(\PageIndex{11}\): Complex of Human FasL and Its Decoy Fas Receptor DcR (4MSV). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...bHLh8fBNZ7E5Q9

The three gray subunits are soluble decoy receptors (DcR) versions of the Fas TNFR/NGFR domain, which is highly enriched in Cys. It is structurally very similar to its typical membrane receptor ligand, Fas (tumor necrosis factor receptor superfamily member 6 - P25445). The green spheres are Mg²⁺ ions.  The FasL trimer is shown in different colors.

DcR is a secreted member of the TNF family and disrupts apoptosis, allowing tumors to survive.

Fas and FADD death domain interactions

Figure \(\PageIndex{12}\) shows an interactive iCn3D model of two Fas death domains bound to two FADD death domains (3EZQ)

Figure \(\PageIndex{12}\): Two Fas death domains bound to two FADD death domains (3EZQ). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...S9Uj45N4mqT5G8

Two Fas death domains are shown in a different shade of gray, and the two FADD death domains are shown in a different shade of magenta. Each domain consists of six alpha-helical bundles. The interactions between the dark gray and light gray Fas death domains, and between the light gray Fas and light magenta FADD death domains, are shown with stick side chains in CPK colors.

The Fas-FADD-procaspase 8 complex is collectively referred to as the Death-Inducing Signaling Complex (DISC). The Fas-FADD interactions lead to the binding of caspase 8 and the completion of the DISC. The actual disc appears to contain 4 FADD death domains bound to 4 Fas death domains. Conformational changes allow both FADD:Fas and Fas:Fas interactions, some of which are weak, but when formed, switch on the activity of the building complex. The need for four monomers probably prevents accidental assembly, which would be deadly to the cell.

Mechanism and regulation of apoptosis

Caspases

Characterization of apoptotic mechanisms and cellular players began with the study of C. elegans, a roundworm. The mature worm has about 1000 cells. During development, 131 cells die. Two mutations were found in which the 131 cells did not die. These mutations were called ced3 and ced4 (ced stands for cell death). The sequence of ced 3 was highly homologous to a protein called interleukin-converting enzyme (ICE), which is required for proteolytic activation of the interleukin 1 precursor, a protein hormone released by certain immune cells during activation that can promote inflammation. This suggested that proteolysis was required for apoptosis. Subsequent studies show that a family of proteases (about 10 in humans), called caspases (ICE has been renamed caspase 1), is required for programmed cell death. These proteases are inactive in the cell and must undergo limited proteolysis to be activated. These caspases form a cascade of proteases that are activated in this process. They are endoproteases that have an active site Cys (C) and cleave at the C-terminal side of Asp residues (asp) and hence are known as caspases - cys containing-asp-specific proteases).

ICE is not normally involved in apoptosis, but its artificial activation in cultured mammalian cells can induce apoptosis. Each caspase had the same sequence as the substrates they are designed to cleave, so it became evident that they probably cleave each other in an activation cascade, similar to the coagulation protease cascade that activates precursors (zymogens) of serine proteases, which in turn activate the next in the series. Two series of caspases seem to be involved. One set initiates caspase activation. Just as in the clotting system, the question of what activates the first caspase appeared problematic until investigators found that the initiator caspase can be activated if it aggregates to a critical concentration. This could occur by binding a suicide signal molecule at the cell surface. Conformational changes in the receptor can lead to the aggregation of surface receptor molecules, concomitant with the aggregation of intracellular caspases that interact with the aggregated receptors.

Intracellular signals

How might intracellular activators of apoptosis (like radiation or reactive oxygen species) work? Research indicated the involvement of mitochondria in the apoptotic pathway. Believe it or not, cytochrome C, the heme protein which acts as a water-soluble mobile carrier of electrons in mitochondrial oxidative phosphorylation, shuttling electrons through cytochrome C oxidase or complex IV, leaks out of the intermembrane space and binds to a cytoplasmic protein called Apaf-1, for apoptotic protease activating factor-1. This then activates an initiator caspase-9 in the cytoplasm.

These proteins seem to leak out of mitochondria after a collapse of the electrochemical potential across the inner membrane. The potential collapses as a consequence of the opening of a channel called a nonspecific inner membrane permeability transition pore, composed of both an inner membrane protein (adenine nucleotide translocator - ant) and an outer membrane protein (porin, the voltage-gated anion channel - VDAC). These proteins act together, probably at sites where the inner and outer membranes are in contact. This channel passes anything with a molecular weight below 1500. Collapsing the proton gradient uncouples oxidation and phosphorylation in the mitochondria. Changes in ionic strength cause a swelling of the matrix. Since the inner membrane is highly convoluted and has a much greater surface area than the outer membrane, swelling of the matrix leads to a rupture of the outer membrane, spilling the inner membrane space proteins (cytochrome C and Apaf-1) into the cytoplasm.

What causes all these changes in the mitochondria? Several interrelated events appear to be involved:

1. disruption of ox-phos and electron transport, caused by irradiation and certain second messengers such as ceramide.
2. changes in cell redox potential and reactive oxygen species (ROS) generation.
3. DNA damage (caused by radiation, ROS, etc). A protein called p53 is often expressed in cells with DNA damage. Expression of this protein results in inhibition of cell division or apoptosis, both of which would keep the damaged cell from becoming a tumor cell. Hence, the p53 gene is a tumor suppressor gene. It is inactivated by mutation in approximately 50% of all human tumor cells studied. p53 can induce gene expression. Of the 14 genes whose expression is significantly altered by p53, many appear to be involved in generating or responding to oxidative stress. Cells undergo p53 apoptosis through oxidative damage.
4. increases in intracellular calcium ions through signal transduction.

Caspase targets:

Apoptosis involves:

1. condensing of the cell nucleus and breaking into pieces
2. condensing and fragmenting of cytoplasm into membrane-bound apoptotic bodies
3. breaking chromosomes into fragments containing multiple numbers of nucleosomes (a nucleosome ladder)

How does caspase activation lead to these events? A protein has been uncovered that, when cleaved by a caspase, leads to a nuclear breakup. The target protein is usually bound to another protein, a DNA endonuclease. When the target protein is cleaved, the DNase can migrate to the nucleus and begin the execution. Membrane changes during apoptosis occur when caspase-3 cleaves gelsolin, a protein that maintains cell morphology. The cleaved gelsolin cleaves actin filaments inside the cell. Another protein required to form apoptotic bodies is p21-activated kinase 2 (PAK-2). This kinase is activated by caspase-3 by limited proteolysis. Caspases also cleave beta-amyloid precursor protein, which might generate more beta-amyloid protein, causing neural cell death in Alzheimer's patients.

Controlling Apoptosis

It should be clear that cells keep tight control of the caspases. Two players that appear to inhibit apoptosis are the mitochondrial proteins Bcl-2 and Bcl-X, which can block the release of cytochrome C from the mitochondria. The Bcl family of proteins has a hydrophobic tail and binds to the outside surface of mitochondria and other organelles, like the nucleus and endoplasmic reticulum. These proteins appear to form ion channels in liposomes. So far, 15 members of this family (related to ced-9 of C. elegans) have been discovered in humans. Bcl-2 can also bind to Apaf-1 (mentioned above) and inhibit its activation of initiator caspase-9. Bcl-2 is regulated by changes in the expression of the Bcl-2 gene, by post-translational phosphorylation by kinases, or by cleavage by caspases. Overexpression of Bcl-2 can cause a cell to become a tumor cell. Other family members, BAX and BAD bind to mitochondria and facilitate apoptosis by stimulating cytochrome C release.

In addition, other proteins called IAPs (inhibitors of apoptosis) can inhibit caspase or other apoptotic proteins. Some viruses produce proteases to keep their host cells viable.

Cell Membrane Events

Cells can be induced to undergo apoptosis through cell-surface interactions with other cells, often immune cells. One of the jobs of immune cells is to destroy altered cells (for example, virally infected cells or tumor cells). Immune cells must also die after being activated in an immune response. Activated lymphocytes (such as cytotoxic T cells or natural killer cells) can target and kill cells in several ways, including apoptosis. In one, an activated lymphocyte binds to a target cell (like a virally infected cell) and secretes perforin. This protein assembles in the target cell membrane to form a transmembrane channel. Other proteins released by the activated lymphocyte can enter the target cell through the pore and initiate apoptosis. One such protein that enters is granzyme B, a protease that activates caspases in the target cell.

Target cells that express the membrane protein CD95 (also called Fas) are also targeted for apoptosis. This protein receptor, a member of the tumor necrosis factor receptor (TNFR) binds to a membrane protein-ligand on the surface of an activated lymphocyte called CD95 Ligand - CD95L- (also called the Fas ligand). Upon binding, CD95 (Fas) receptors on the target membrane aggregate following conformational changes. An adapter protein in the cell, FADD (Fas-associated death domain), binds to the aggregated cytoplasmic domain (the death domain) of CD95 (Fas). It recruits inactive caspase-8 to the site, where its concentration increases. This leads to the activation of the caspases.

This mechanism removes activated lymphocytes after they have finished their work. Activated immune cells start expressing Fas a few days after activation, targeting them for elimination. Some cells that have been stressed express both Fas and Fas ligands and kill themselves. Various cells express CD95 (Fas), but CD95L (Fas-Ligand) is expressed predominantly by activated T cells.

Cell surface events can also inhibit apoptosis. Binding "survival" factors (such as growth factors) to cell-surface receptors can shut off apoptotic pathways in cells. Some survival factor receptors are coupled to PI-3-kinase (phosphoinositol-3-kinase) through the G protein Ras (p21), which is targeted to the cell membrane by post-translational addition of a hydrophobic anchor. The activated kinase produces PI-3,4-P2 and PI-3,4,5-P3, which activate Akt, a Ser/Thr protein kinase. This activated kinase phosphorylates the proapoptotic-protein BAD, which then becomes inactive. In addition, active Akt phosphorylates procaspase, which in its phosphorylated form will not interact with cytochrome C, inhibiting apoptosis.

The endpoint of apoptosis is the engulfment of the fragmented cell by a phagocytic cell (such as a macrophage). In a recent article (Nature, 405, pg 85, 2000), it was shown that phagocyte activity could be inhibited stereospecifically by adding phosphatidyl serine (PS) to the mixture, but not by other negatively charged phospholipids. If you remember from our description of lipids, PS is found exclusively in the inner leaflet of red blood cells. The investigators cloned a gene from a phagocytic cell encoding a receptor that recognizes PS. These cells could also take up apoptotic cells when added to ordinary T and B lymphocytes (immune cells). The gene is homologous to genes in Drosophila (fruit fly) and C. elegans (roundworm), suggesting that it is conserved in nature. The message: when cells undergo apoptosis, PS, normally found only in the inner leaflet, is exposed to the outside. It can then bind to receptors on phagocytic cells, thereby completing apoptosis.

Therapeutics

Figure \(\PageIndex{13}\) shows points of therapeutic intervention in the intrinsic and extrinsic apoptotic signaling pathways.

Figure \(\PageIndex{13}\): Intrinsic and extrinsic apoptotic signaling pathways and points of therapeutic intervention. Fox, J., MacFarlane, M. Targeting cell death signaling in cancer: minimizing ‘Collateral damage’. Br J Cancer 115, 5–11 (2016). https://doi.org/10.1038/bjc.2016.111. Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

Intrinsic and extrinsic apoptotic signaling pathways and points of therapeutic intervention. Apoptosis can be initiated by signals originating from either the plasma membrane via death receptor ligation (extrinsic pathway) or at the mitochondria (intrinsic pathway). Stimulation of the extrinsic pathway by TRAIL results in TRAIL receptor (TRAIL-R) aggregation and formation of the DISC, in which pro-caspase 8 becomes activated. It initiates apoptosis by directly cleaving downstream effector caspases. The addition of either agonistic TRAIL-R1/R2 antibodies or recombinant human TRAIL (rhTRAIL) has been used to trigger the extrinsic pathway for therapy. The BCL-2 family of proteins regulates the intrinsic pathway, which regulates pore formation in the outer mitochondrial membrane and the release of apoptogenic factors such as cytochrome c or SMAC from the mitochondria. The release of cytochrome c into the cytosol triggers caspase 9 activation by forming the cytochrome c/Apaf-1/caspase 9-containing apoptosome complex. SMAC promotes caspase activation by neutralizing the inhibitory effect of IAPs. The intrinsic pathway has been targeted for therapy either by blocking the pro-survival BCL-2 family proteins' inhibitory action with BH3 mimetics or by inhibiting the anti-apoptotic action of IAPs with SMAC mimetics. The extrinsic and intrinsic pathways are interconnected, for example, by BID, a BH3-domain-containing protein of the BCL-2 family, which triggers intrinsic apoptosis upon cleavage by caspase-8, thereby further amplifying the signal from the extrinsic pathway.

The entire pathway

Now we can present detailed pathways that show apoptosis in all its complexity. Trace the interconnections in the different views.

Figure \(\PageIndex{14}\): View 1

Figure \(\PageIndex{14}\): View 1 from Cell Signaling Technologies (with permission) https://www.cellsignal.com/pathways/...ptor-signaling

Figure \(\PageIndex{15}\) presents a second view.

Pathway 2 https://www.sinobiological.com/pathw...ceptor-pathway

Figure \(\PageIndex{15}\): View 2 from Sinobiological https://www.sinobiological.com/pathw...ceptor-pathway

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

Apoptosis is a genetically encoded, energy-independent form of programmed cell death that is executed through the activation of a cascade of cysteine-aspartate proteases (caspases) without requiring new gene transcription or translation, indicating that the molecular machinery lies dormant in all cells awaiting an appropriate death signal. Unlike necrosis (passive, inflammatory cell death), apoptosis proceeds through an ordered sequence: a decision phase, caspase-mediated cell suicide producing chromatin condensation, internucleosomal DNA fragmentation, and cytoplasmic condensation into membrane-bound apoptotic bodies, followed by phagocytic recognition and engulfment mediated by phosphatidylserine externalized from the inner to the outer leaflet of the plasma membrane. The biological necessity of apoptosis is illustrated by its roles in embryonic morphogenesis, immune cell homeostasis, and the elimination of genotoxically damaged, virally infected, or autoreactive cells; its dysregulation contributes to cancer, neurodegeneration, autoimmunity, and immune deficiency.

Two biochemically distinct but interconnected pathways converge on caspase activation. The extrinsic pathway is initiated when death ligands (FasL, TNF-α, TRAIL) bind their cognate trimeric death receptors (Fas/CD95, TNFR1, TRAIL-R), driving receptor aggregation and recruitment of the adaptor protein FADD through homotypic death domain interactions. FADD in turn recruits and concentrates procaspase 8, whose activation by proximity-induced dimerization initiates the caspase cascade. Activated caspase 8 directly cleaves and activates effector caspases 3 and 7, and also cleaves the BH3-only protein BID to truncated tBID, which bridges to the intrinsic pathway by activating Bax/Bak at the mitochondrial outer membrane.

The intrinsic pathway responds to internal stress signals—DNA damage, oxidative stress, radiation, and Ca²⁺ dysregulation—through a balance between pro-apoptotic BH3-only proteins (BID, BIM, PUMA, BAD) and anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-xL, Mcl-1). When BH3-only proteins neutralize the pro-survival proteins, BAX and BAK homo-oligomerize and permeabilize the outer mitochondrial membrane, releasing cytochrome c and Smac from the intermembrane space. Cytoplasmic cytochrome c binds the WD40 domains of Apaf-1, triggering a dATP-dependent conformational change and assembly of the heptameric apoptosome, which activates procaspase 9 through proximity-induced dimerization. Active caspase 9 cleaves and activates effector caspases 3 and 7, which then dismantle the cell by cleaving gelsolin (disrupting the actin cytoskeleton), activating DNases (producing the nucleosomal ladder), and processing beta-amyloid precursor protein.

Pro-survival signaling through the RTK→Ras→PI3K→Akt axis counteracts apoptosis by phosphorylating and inactivating BAD and blocking procaspase-cytochrome c interaction. The tumor suppressor p53, induced by DNA damage, promotes apoptosis through oxidative stress pathways; its mutation in roughly 50% of all human tumors represents perhaps the single most common mechanism by which cancer cells evade programmed death. Therapeutically, each node in the apoptotic cascade represents a potential drug target: TRAIL receptor agonists activate the extrinsic pathway selectively in cancer cells; BH3 mimetics such as venetoclax displace pro-apoptotic proteins from Bcl-2 to restore intrinsic apoptosis; and Smac mimetics neutralize IAPs to permit caspase activation—collectively representing a new generation of molecularly targeted cancer therapies designed to reactivate the cell's endogenous death program.