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Caspases (cysteine-aspartic proteases, cysteine aspartases or cysteine-dependent aspartate-directed proteases) are a family of protease enzymes playing essential roles in programmed cell death (including apoptosis, pyroptosis and necroptosis) and inflammation. They are named Caspases due to their specific cysteine protease activity - a cysteine in its active site nucleophilically attacks and cleaves a target protein only at the C-terminal of an aspartic acid amino acid. As of 2009, there are 12 confirmed Caspases in humans and mice, carrying out a variety of cellular functions.

The role of these enzymes in programmed cell death was first identified in 1993, with their functions in apoptosis well characterised. This is a form of programmed cell death, occurring widely during development, and throughout life to maintain cell homeostasis. Activation of Caspases ensure that the cellular components are degraded in a controlled manner, carrying out cell death with minimal effect to surrounding tissues.

Caspases have other identified roles in programmed cell death such as Pyroptosis and Necroptosis. These forms of cell death are important for protecting an organism from stress signals and pathogenic attack. Caspases also have a role in inflammation, whereby it directly processes pro-inflammatory cytokines such as pro-IL1β. These are signalling molecules that allow recruitment of immune cells to an infected cell or tissue. There are other identified roles of caspases such as cell proliferation, tumour suppression, cell differentiation, neural development and axon guidance and ageing

Caspase deficiency has been identified as a cause of tumour development. Tumour growth can occur by a combination of factors, including a mutation in a cell cycle gene which removes the restrains of cell growth, combined with mutations in apoptopic proteins such as Caspases that would respond by inducing cell death in abnormal growing cells. On the contrary, over activation of some caspases such as caspase-3 can lead to excessive programmed cell death. This is seen in several neurodegenerative diseases where neural cells are lost, such as Alzheimers disease. Caspases involved with processing inflammatory signals are also implicated in disease. Insufficient activation of these caspases can increase the organisms susceptibility to infection as an appropriate immune response is not signalled for. The integral role caspases play in cell death and disease has led to research for using caspases as a drug target. For example, inflammatory caspase-1 has been implicated in causing autoimmune diseases; drugs blocking the activation of Caspase-1 have been used to improve the health of patients. Additonally, scientists have used caspases as cancer therapy to kill unwanted cells in tumours.

Functional Classification of Caspases

Most caspases play a role in programmed cell death. These are summarised in the table below. The enzymes are sub classified into three types: Intiator, Executioner and Inflammatory.

Programmed Cell Death	Type of Caspase	Enzyme	Organism
Apoptosis	Initiator	Caspase 2	human and mouse
		Caspase 8	human and mouse
		Caspase 9	human and mouse
		Caspase 10	human and mouse
	Executioner	Caspase 3	human and mouse
		Caspase 6	human and mouse
		Caspase 7	human and mouse
Pyroptosis	Inflammatory	Caspase 1	human and mouse
		Caspase 4	human
		Caspase 5	human
		Caspase 11	mouse
		Caspase 12	mouse
Other roles	Other	Caspase 14	human and mouse

Note that in addition to apoptosis, Caspase-8 is also required for the inhibition of another form of programmed cell death called Necroptosis. Caspase-14 plays a role in epithelial cell keratinocyte differentiation and can form an epidermal barrier that protects against dehydration and UVB radiation.

Activation of Caspases

Caspases are synthesised as inactive zymogens (pro-caspases) that are only activated following an appropriate stimulus. This post-translational level of control allows rapid and tight regulation of the enzyme.

Activation involves dimerization and often oligomerisation of pro-caspases, followed by cleavage into a small subunit and large subunit. The large and small subunit associate with each other to form an active heterodimer caspase. The active enzyme often exists as a heterotetramer in the biological environment, where a pro-caspase dimer is cleaved together to form a heterotetramer.

Dimerisation is facilitated by a particular domain called the pro-domain. This domain is larger in Initiator and Inflammatory Caspases, consisting of Caspase Recruitment Domains (CARD) and Death Effector Domains (DED). These domains are used to mediate protein-protein interactions between caspases. Adaptor proteins contain CARD or DED domains that allow it to bind to appropriate caspase domains (CARD-CARD interaction or DED-DED interactions) Multiprotein complexes often form during pro-caspase activation .

Some activating multiprotein complexes includes:

• Apoptosome and Death Induced Signalling Complex (DISC) during apoptosis
• Inflammasome during Pyroptosis

Cleavage:Once appropriately dimerised, the Caspases cleaves at inter domain linker regions, forming a large and small subunit. This cleavage allows the active-site loops to take up a conformation favourable for enzymatic activity.

Cleavage of Initiator and Executioner caspases occur by different methods outlined in the table below.

• Initiator caspases self proteolytically cleave whereas Executioner caspases are cleaved by intitiator caspases and other executioner caspases forming a hierarchy. This amplifying chain reaction or cascade originates from the activation of an ‘initiator caspase’ and leads to the activation of several ‘executioner caspases’ that degrade cellular components, during controlled cell death (see section below link).

Initiator Caspase Caspase-3	Inititator Caspases have a prodomain that allows recruitment and dimerisation. Both pro-caspase moleuceules undergo cleavage by autocatalysis. This leads to removal of the prodomain and cleavage of the linker region between the large and small subunit. A heterotetramer is formed Inititator Caspases have a prodomain that allows recruitment and dimerisation represented by the grey DED domains in (FADD). Both pro-caspase moleucules undergo cleavage by autocatalysis at the regions marked in red. This leads to removal of the prodomain and cleavage of the linker region between the large and small subunit. A heterotetramer is formed	PDB image of caspase 8 (3KJQ) in 'biological assembly'. Two shades of blue used to represent two small sunits, while two shades of purple represent two large subunits PDB image of caspase 8 (3KJQ) in 'biological assembly'. Two shades of blue used to represent two small sunits, while two shades of purple represent two large subunits
Executioner Caspase Caspase-8	Executioner caspase constitutively exist as homodimers. The red cuts represnt regions where intitiator caspases cleave the executioner caspases. The resulting small and large subunit of each Caspase-3 will associate,resulting in a heterotetramer. Executioner caspase constitutively exist as homodimers. The red cuts represnt regions where intitiator caspases cleave the executioner caspases. The resulting small and large subunit of each Caspase-3 will associate,resulting in a heterotetramer.	PDB image of Caspase 3 (4QTX) in 'biological assembly'. Two shades of blue used to represent two small sunits, while two shades of purple represent two large subunits PDB image of Caspase 3 (4QTX) in 'biological assembly'. Two shades of blue used to represent two small sunits, while two shades of purple represent two large subunits

Some roles of Caspases

Apoptosis

Initiator caspases are activated by intrinsic and extrinsic apoptopic pathways. This leads to the activation of other caspases including executioner caspases that carry out apoptosis by cleaving cellular components.

Apoptosis is a form of programmed cell death where the cell undergoes morphological changes, to minimises its effect on surrounding cells to avoid inducing an immune response. The cell shrinks and condenses - the cytoskeleton will collapse, the nucleur envelope disassembles and the DNA fragments up. This results in the cell forming self-enclosed bodies called 'blebs', to avoid release of cellular components into the extracellular medium. Additionally, the cell membrane phospholipid content is altered, which makes the dying cell more susceptible to phagocytic attack and removal.

Apoptopic caspases are subcategorised as:

1. Initiator Caspases (Caspase 2, Caspase 8, Caspase-9, Caspase 10)
2. Executioner Caspases (Caspase 3, Caspase 6 and Caspase 7)

Once initiator caspases are activated, they produce a chain reaction, activating several other executioner caspases. Executioner caspases degrade over 600 cellular components in order to induce the morphological changes for apoptosis. Some substrates that executioner caspases target and the morphological changes it contributes to are summarised in the table adjacent.

Examples of caspase cascade during apoptosis:

1. Intrinsic apoptopic pathway: During times of cellular stress, mitochondrial cytochrome c is released into the cytosol. This molecule binds an adaptor protein (APAF-1), which recruits initiator Caspase-9 (via CARD-CARD interactions). This leads to the formation of a Caspase activating multiprotein complex called the Apoptosome. Once activated, initiator caspases such as Caspase 9 will cleaving and activate other executioner caspases. This leads to degradation of cellular components for apoptosis.
2. Extrinsic apoptopic pathway:  The caspase cascade is also activated by extracellular ligands, via cell surface Death Receptors. This is done by the formation of a multiprotein Death Inducing Signalling Complex (DISC) that recruits and activates a pro-caspase. For example, the Fas Ligand binds the FasR receptor its the extracellular surface, which activates the death domains at the cytoplasmic tail of the receptor. This recruits an adaptor protein FADD that contains also contains a Death Domain and DED domain for pro-caspase recruitment. This FasR, FADD and pro-Caspase 8 form a DISC, where Caspase-8 is activated. This could lead to either downstream activation of the intrinsic pathway by inducing mitochondrial stress, or direct activation of Executioner Caspases (Caspase 3, Caspase 6 and Caspase 7) to degrade cellular components. (http://dx.doi.org/10.1016/j. it.2014.10.004

Pyroptosis

Pyroptosis is a form of programmed cell death that inherently induces an immune response. It is morphologically distinct from other types of cell death – cells swell up, rupture and release pro-inflammatory cellular contents. This is done in response to a range of stimuli including microbial infections as well as heart attacks (myocardial infarctions). Caspase-1, Caspase-4 and Caspase-5 in humans whereas Caspase-1 and Caspase-11 in mice play important roles in inducing cell death by Pyroptosis. This could lead to limiting the life and proliferation time of intracellular and extracellular pathogens.

Pyroptosis by Caspase-1

Caspase-1 activation is mediated by a repertoire of proteins, allowing detection of a range of pathogenic ligands. Some mediators of Caspase-1activation are: NOD-like Leucine Rich Repeats (NLRs), AIM2-Like Receptors (ALRs), Pyrin and IFI16.

These proteins allow Caspase-1 activation by forming a multiprotein activating complex called Inflammasomes. For example, a NOD Like Leucine Rich Repeat NLRP3 will sense an efflux of potassium ions from the cell. This cellular ion imbalance leads to oligomerisation of NLRP3 molecules to form a multiprotein complex called the NLRP3 Inflammasome. The pro-caspase-1 is brought into close proximity with other pro-caspase molecule in order to dimerise and undergo auto-proteolytic cleavage.

Some pathogenic signals that lead to Pyroptosis by Caspase-1 are listed below:

• DNA in the host cytosol binds to AIM2-Like Receptors inducing Pyroptosis
• Type III secretion system apparatus from bacteria bind NOD Like Leucine Rich Repeats receptors called NAIP’s (1 in humans and 4 in mice)

Pyroptosis by Caspase -4 and Caspase-5 in humans and Caspase-11 in mice

These caspases have the ability to induce direct pyroptosis when LPS molecules (found in the cell wall of gram negative bacteria) are found in the cytoplasm of the host cell. For example, Caspase 4 acts as a receptor and is proteolytically activated, without the need of an inflammasome complex or Caspase-1 activation.  

A crucial downstream substrate for all pyroptopic caspases is Gasdermin D (GSDMD)

Role in Inflammation

Inflammation involves an attempt to protect an organism and restore a homeostatic state, following disruption from a harmful stimulus, such as tissue damage or bacterial infection.

Caspase-1, Caspase-4, Caspase-5 and Caspase-11 are considered 'Inflammatory Caspases'.

• Caspase-1 is key in activating pro-inflammatory cytokines; these act as signals to immune cells and make the environment favourable for immune cell recruitment to the site of damage. Caspase-1 therefore plays has a fundamental role in the innate immune system. The enzyme is responsible for processing cytokines such as pro-ILβ and pro-IL18, as well as secreting them. Pro-ILa is also secreted, without needing processing. There is evidence of an inflammatory caspase, caspase-11 aiding cytokine secretion; this is done by inactivating a membrane channel that blocks IL-1β secretion

• Caspase-4 and -5 in humans, and Caspase-11 in mice have a unique role as a receptor, whereby it binds to LPS, a molecule abundant in gram negative bacteria. This leads to Caspase-1 (leading to processing and secretion of IL-1B and IL-18 cytokines) Caspase-1 and leads to direct pyroptosis and pro-IL1a secretion.

• Caspases can also induce an inflammatory response on a transcriptional level. It promotes transcription of nuclear factor-κB (NF-κB), a transcription factor that assists in transcribing inflammatory cytokines such as IFNs, TNF, IL-6 and IL-8. This role is exemplified by Caspase-1 activating Caspase-7, which in turn cleaves the poly (ADP) ribose – this activates transcription of NF-KB controlled genes.