Caspases

Structure and Biochemistry

• All caspases contain an active-site cysteine residue and cleave substrate proteins at specific aspartate residues.
• They exist as inactive procaspases; activation requires dimerization or proteolytic cleavage.
• Over 600 substrate proteins have been identified as targets for apoptotic caspases (global proteomics analyses) - this "death by a thousand cuts" ensures cells cannot escape once caspases are activated.

Classification

Pathways of Apoptotic Caspase Activation

1. Intrinsic (Mitochondrial) Pathway

1. BH3-only proteins (BAD, BIM, BID, Puma, Noxa) are upregulated by stress sensors.
2. They activate BAX and BAK, which oligomerize in the outer mitochondrial membrane, forming channels that allow cytochrome c (and SMAC/DIABLO) to leak into the cytoplasm.
3. Anti-apoptotic proteins BCL2, BCL-XL, MCL1 normally suppress this by keeping the outer mitochondrial membrane impermeable.
4. Cytochrome c binds APAF-1 to form the apoptosome - a multimeric wheel-shaped complex.
5. The apoptosome recruits and autocatalytically activates caspase-9 (initiator caspase of this pathway).
6. Active caspase-9 activates executioner caspases-3, -6, and -7.

2. Extrinsic (Death Receptor) Pathway

1. FasL (on CTLs or self-reactive T cells) binds to Fas (CD95), clustering three or more Fas molecules.
2. Their cytoplasmic death domains recruit the adaptor FADD (Fas-associated death domain protein).
3. FADD recruits and brings together multiple molecules of procaspase-8 (or -10), leading to autocatalytic cleavage and generation of active caspase-8.
4. Active caspase-8 either:
   • Type I pathway: Directly activates downstream executioner caspases (caspase-3).
   • Type II pathway: Cleaves the BH3-only protein BID, which then feeds into the mitochondrial pathway (amplification loop). This cross-talk is especially important in hepatocytes and pancreatic β cells.
5. FLIP (a decoy protein produced by some viruses and normal cells) can bind procaspase-8 and block FADD binding, inhibiting this pathway.

Fas-ligand route to apoptosis. CTL-expressed FasL triggers Fas trimerization on the target cell → FADD recruitment → caspase-8 activation → Type I (direct caspase-3) or Type II (BID cleavage → mitochondria → apoptosome → caspase-9 → caspase-3) routes to cell death. (Roitt's Essential Immunology)

Execution Phase (Common Final Pathway)

• Cleaves ICAD (inhibitor of caspase-activated DNase), liberating CAD to degrade chromosomal DNA into oligonucleosomal fragments (the "DNA ladder").
• Proteolytically degrades nuclear lamins, causing nuclear fragmentation.
• Cleaves PARP (poly-ADP-ribose polymerase), disabling DNA repair.
• Triggers cytoskeletal reorganization and membrane blebbing.
• Ultimately produces apoptotic bodies - membrane-bounded fragments containing organelles and nuclear material, which are phagocytosed without triggering inflammation.

Clearance of Apoptotic Cells

• Phosphatidylserine flips from the inner to outer membrane leaflet ("eat me" signal for macrophage receptors).
• Apoptotic cells secrete soluble "find me" signals recruiting phagocytes.
• Complement C1q and natural antibodies coat apoptotic bodies for recognition.
• This efficient clearance means apoptotic cells rarely trigger inflammation and are rarely seen on routine H&E (apoptosis occurs >20x faster than mitosis).

Inflammatory Caspases - Caspase-1 and Pyroptosis

• Cleaves pro-IL-1β and pro-IL-18 into active, secreted cytokines.
• Cleaves gasdermin-D; the N-terminal fragment forms pores in the plasma membrane, causing pyroptosis - an inflammatory form of cell death characterized by cell swelling and membrane rupture (distinct from apoptosis).

BCL-2 Family - Regulators of the Intrinsic Pathway

Granzyme B Pathway (CTL Killing)

• Perforin forms pores in the target cell membrane.
• Granzyme B (a serine protease) enters the target cell and directly cleaves and activates caspases (including caspase-3), bypassing the upstream initiator steps.
• This secretory granule pathway is the dominant CTL killing mechanism (evidenced by perforin-knockout mice failing to clear viral infections).

Clinical Significance

• Robbins & Cotran Pathologic Basis of Disease, pp. 58-62 (Mechanisms of Apoptosis)
• Histology: A Text and Atlas (Ross), pp. 279-281 (Apoptosis, caspase cascades)
• Roitt's Essential Immunology, pp. 272-275 (Caspase activation, CTL killing)
• Goodman & Gilman's Pharmacological Basis of Therapeutics (Apoptosis section)
• Janeway's Immunobiology 10e (Inflammasome and caspase-1)
• Recent reviews: Apoptosis signaling pathways review (2024) (PMID: 39594587); Pyroptosis - mechanisms and clinical perspective (2024) (PMID: 39300122)