Cell Injury and Regulated Cell Death

Explain the causes and characteristics of accidental cell death.
Explain the structure and function of caspase enzymes in regulated cell death.
Explain the extrinsic pathway of apoptosis initiated by death receptors.
Explain the mitochondrial outer membrane permeabilization and the intrinsic pathway of apoptosis.

Etiological Factors:
- Ischemia and reperfusion injury.
- Toxicity from various sources including pollutants, heavy metals, drugs, household or agricultural chemicals.
- Cellular damage from toxins, cellular invasion, and immune responses.
- Changes in protein quantity or activity.
- Nutritional issues: inadequate vitamins or calories; excessive intake of calories, fats, sugars.
- Physical trauma, radiation exposure, temperature extremes, electric shock.

Ischemia: Inadequate blood supply to a tissue.
Hypoxia: Inadequate oxygen supply to cells.
Toxic insult: Damage from reactive chemicals.
Infectious pathogens: Includes bacteria, viruses, fungi, and parasitic protozoa.
Immune responses: Includes autoimmune disorders and chronic inflammation.
Genetic mutations: Can be either inborn or acquired.
Nutritional deficiencies: Lack of necessary nutrients, or overindulgence in them.
Physical events: Includes any harmful exposure or incident leading to injury.

Cell can:
- Adapt and Repair: Indicates a reversible injury.
- Die: Indicates an irreversible injury.
Decisions depend on:
- Specific cause of cell injury.
- Intensity and duration of the injurious assault.
- Type of cell affected.

Historically, two forms of cell death are:
- Necrosis: Uncontrolled cell death.
- Apoptosis: Controlled or programmed cell death.
Factors determining the type of cell death include:
- Specific cause of the cell injury.
- Severity and/or duration of the injurious assault.
- Type of cell affected.

Activation of specific proteases called caspases.
Fragmentation of DNA.
Orderly degradation of organelles and cytoskeleton.
Membrane blebbing.
Transfer of phosphatidylserine to the outer leaflet of the plasma membrane.
Formation of apoptotic bodies.
Inflammation: No inflammation occurs during apoptosis.
Apoptosis is also known as programmed cell death (regulated cell death), which is an organized and stepwise process requiring dedicated molecular machinery.

Caspases: These are proteolytic enzymes that contain cysteine in their active site and cleave substrates next to aspartate residues.
Caspases are synthesized as procaspases activated by proteolytic cleavage and rearrangement.
There are two categories of caspases:
- Initiator caspases:
- Examples: Caspase 8 and Caspase 9.
- Activation involves the binding of initiator procaspases to adaptor proteins, leading to dimerization, cross-cleavage, and rearrangement.
- Activated initiator caspases activate executioner caspases by cleavage.
- Executioner caspases:
- Example: Caspase 3.
- Cleaved by activated initiator caspases; cleaved executioner caspases activate other executioner caspases and cleave numerous cellular targets to produce the apoptotic phenotype.

Caspase-activated DNase (CAD): This enzyme is important for the fragmentation of DNA during apoptosis.

Flippases: These enzymes maintain phosphatidylserine (PS) localized to the cytoplasmic face of the plasma membrane.
Scramblases: These enzymes randomly distribute phospholipids between the two faces of the membrane.
Inactivation of flippases and activation of scramblases results in increased PS localization on the exoplasmic side of the membrane.

Extrinsic Pathway: Activated by extracellular signals.
- Ligands bind to death receptors.
- Examples of ligands: Tumor necrosis factor (TNF), Fas ligand (FasL).
Intrinsic Pathway: Activated by internal signals, such as DNA damage, cell stress, and loss of survival factors.
- Commonly referred to as the mitochondrial pathway due to the involvement of mitochondrial changes.

Extrinsic pathway: Involves binding of ligands such as TNF-α and Fas ligand to the death receptors, resulting in formation of homotrimers.
Intrinsic pathway: Involves the release of proteins such as cytochrome c from the mitochondrial intermembrane space, needed for activation of initiator procaspase 9.

MOMP occurs when proteins Bak and/or Bax assemble in the outer mitochondrial membrane to form pores.

Bax and Bak: Members of the Bcl-2 family sharing Bcl-2 homology (BH) domains.
- Anti-apoptotic Bcl-2 Family Members: Include proteins like Bcl-2 and Bcl-xL, possessing all 4 BH domains.
- Pro-apoptotic Bcl-2 Family Members:
- Proteins such as Bax and Bak with BH1, BH2, & BH3 domains.
- BH3-only proteins: Include Bad, Bim, Bid, Puma, Noxa, etc.
Binding Pocket Formation: Bcl-2 and Bcl-xL form a binding pocket using their BH1, BH2, and BH3 domains which binds pro-apoptotic proteins.

Sequestration/Inhibition: Bcl-2 and Bcl-xL bind tightly to Bax and Bak to maintain them in an inactive state, preventing MOMP.
MOMP is initiated when BH3-only proteins displace Bax and Bak, enabling their activation and the formation of pores in the mitochondrial membrane, which allow cytochrome c to be released.

Cytochrome c binds to Apaf-1 (apoptotic protease activating factor-1), inducing a conformational change that results in the exposure of CARDs (caspase recruitment domains).
Seven copies of Apaf-1 interact to form an apoptosome, which activates procaspase 9 leading to activation of the executioner procaspase 3.

Exposure to TNF or FasL induces disassembly of death receptors forming the DISC.
Caspase 8 Activation: Caspase 8 gets activated and cleaves the BH3-only protein Bid into the active form known as tBid.
tBid acts as a potent MOMP inducer, linking the extrinsic pathway with the intrinsic mitochondrial pathway.