What are caspases?

Caspases: The Proteases of Apoptosis

Caspases are a family of cysteine proteases that play essential roles in programmed cell death (apoptosis), serving as the primary executioners of the cell death machinery. 1

Definition and Structure

• Caspases (cysteine-aspartate proteases) are characterized by their almost absolute specificity for cleaving proteins after aspartic acid residues and contain a conserved QACXG (where X is R, Q or G) pentapeptide active-site motif 1
• They are synthesized as inactive proenzymes (zymogens) comprising an N-terminal prodomain and one large and one small subunit 1
• The active enzyme forms a heterotetramer containing two small and two large subunits, as revealed by crystal structures of caspase-1 and caspase-3 1

Classification of Caspases

Caspases are grouped into two major subfamilies based on their primary functions:

1. Apoptotic Caspases: Further subdivided into:

   • Initiator Caspases (caspase-2, -8, -9, -10): Contain longer prodomains with protein-protein interaction motifs that facilitate their recruitment to signaling complexes for activation 2
   • Executioner Caspases (caspase-3, -6, -7): Responsible for the actual dismantling of cellular components during apoptosis 2

2. Inflammatory Caspases (caspase-1, -4, -5, -11, -12): Primarily involved in cytokine processing and inflammatory responses 2

Caspase Activation Pathways

Caspases can be activated through two main pathways:

1. Extrinsic Apoptosis Pathway:

   • Initiated by binding of death ligands (FASL/CD95L, TNFα, TRAIL) to cell surface death receptors (FAS/CD95, TNFR1, TRAILR1/2) 3
   • Formation of the Death-Inducing Signaling Complex (DISC) recruits and activates initiator caspase-8 (or -10) 3
   • In Type I cells: Caspase-8 directly activates executioner caspases (caspase-3, -6, -7) 3
   • In Type II cells: Caspase-8 cleaves BID to tBID, triggering mitochondrial outer membrane permeabilization (MOMP) and subsequent caspase-9 activation 3

2. Intrinsic Apoptosis Pathway:

   • Triggered by intracellular stress signals (DNA damage, oxidative stress, ER stress) 3
   • Results in MOMP and release of cytochrome c from mitochondria 3
   • Cytochrome c, APAF1, and dATP form the apoptosome, which activates caspase-9 3
   • Activated caspase-9 then cleaves and activates executioner caspases 3

Regulation of Caspase Activity

• Inhibitor of Apoptosis Proteins (IAPs): Directly bind to and inhibit active caspases 2
• c-FLIP proteins: Compete with caspase-8 for binding to FADD at the DISC 3
• Bcl-2 family proteins: Regulate the intrinsic pathway by controlling MOMP 4
• Viral inhibitors: Proteins like CrmA can suppress caspase activity 3
• Chemical inhibitors: Pan-caspase inhibitors such as Z-VAD-fmk can block caspase-dependent cell death 3

Caspase Substrates and Cellular Effects

• Caspases cleave a discrete and limited subset of cellular proteins during apoptosis 5
• Key substrates include:
  • Poly(ADP-ribose) polymerase (PARP) 1
  • Nuclear lamins 1
  • Cytoskeletal proteins 5
• Cleavage of these substrates leads to the characteristic morphological changes of apoptosis:
  • Cell shrinkage
  • Chromatin condensation
  • Nuclear fragmentation
  • Membrane blebbing
  • Formation of apoptotic bodies 3

Non-Apoptotic Functions of Caspases

• Caspases also participate in:
  • Inflammatory responses (particularly caspase-1 in pyroptosis) 3
  • Cell differentiation processes (e.g., megakaryocytes, granulocytes) 3
  • Immune cell activation 2
  • Cellular proliferation 2

Clinical Significance

• Dysregulation of caspase activity is implicated in various diseases:
  • Excessive activation: Neurodegenerative disorders, ischemia-reperfusion injury 4
  • Insufficient activation: Cancer, autoimmune disorders 4
• Caspase inhibitors have been investigated as potential therapeutic agents for diseases characterized by excessive apoptosis 4

Common Pitfalls in Caspase Research

• Caspase-dependent cell death is not synonymous with apoptosis; other forms of cell death can also involve caspases (e.g., pyroptosis) 3
• Caspase inhibition may not prevent cell death but rather shift the morphology from apoptotic to necrotic or autophagic 3
• Z-VAD-fmk, while commonly used as a pan-caspase inhibitor, does not inhibit all caspases equally and can also inhibit other proteases like calpains and cathepsins 3
• Morphological classification of cell death should be complemented with biochemical and molecular analyses for accurate characterization 3