The Human Caspases: Viral Inhibitor

Sequential Activation

Apoptosis, which is the controlled death of cells in an organism, is important in order to remove damaged and aged cells from the system. A group of cysteine aspartic acid-specific proteases (also referred to as caspases) is the one that regulates this death process. Caspases are activated within the cells that are designated for elimination, ensuring that the elimination process is well regulated. There are different types of caspases, each type designated to play a specific role in the elimination process. Some of them interface with signalling events, in the process initiating the proteolytic process. Other caspases are to be found within the cells designated for death, where they dismantle the cell from within. A class of executioner caspases come into the picture during the terminal phase of apoptosis. They terminate the viability of the cell by initiating a large scale proteolytic process. The dismantled cells are removed from the system by a group of scavenging phagocytes.

Structure and Typology of Caspases

Typology

Studies carried out in this field have identified fifteen caspases in mammals. These are divided into two broad categories depending on the role they perform during the apoptosis process.

Inflammatory Caspases

The first class is made up of inflammatory caspases, which are involved in macrophage activation. These are caspases -1, -4 and -5, which enable the maturation of cytokines produced by various macrophages in the body (Logue & Martin 2008). To date, only two substrates are known to react with inflammatory caspases, and these are interleukin-1β and -18.

There are several scenarios that are involved in the activation of the various caspases in this category. For example, the sensing of cytoplasmic DNA by the absent in melanoma 2 (AIM2) inflamasome is such one event that leads to the activation of caspase -1(Fernandes-Alnemri et al 2009). This activation leads to loss of cell viability, a necrosis-like process that is also referred to as pyroptosis.

Apoptotic Caspases

Whereas inflammatory caspases are involved in the activation of macrophage, apoptotic caspases are involved in programmed demise of cells (Logue & Martin 2008). This class is composed of caspases -3, -6, -7, -8 and -9, and according to Luthi & Martin (2007), they possess a more complete characterisation unlike the inflammatory ones.

Structure of Caspases

The basic structure of caspases involves a large and small subunit. This is preceded by an N-terminal pro-domain, whose length determines the function and position of the particular caspase in the caspase activation cascade. Initiator caspases have a longer N-terminal domain than the executioners.

There are two types of N-terminal domains in the initiator class, which enable the caspases to interface with signalling events and activate. These are caspase recruitment domain (also known as CARD) and death effector domain (also known as DED). These domains are absent in executioner caspases’ structure, and therefore, the activation of the latter to cell death is enabled by the initiator caspases.

Pathways Leading to the Activation of Caspase

Three pathways (namely extrinsic, intrinsic and granzyme B) have been identified as leading to activation of caspase and apoptotic demise of cell. Whichever the path followed in the activation process, it is noted that apical protease must first be introduced to activate the cascade. An analysis of each of these three paths follows below:

Extrinsic Pathway

When caspase activation and apoptotic cell death follows this pathway, it is as a result of death instructions provided by another cell in the body. This is for example cytotoxic T lymphocyte cell. This takes place through extracellular signalling that assumes the form of death receptor interfacing. Several processes lead to the recruitment of an activation launch pad referred to as the death receptor complex. The processes start with the binding of receptors expressed on the target cell to corresponding ligands that are expressed on cytotoxic cells.

According to Muzio et al (1996), the death receptor complex is made up of adapters such as FADD and caspase -8. When caspase -8 is activated, it acts on caspases -3 and -7, executioner caspases that dismantles the cell. Apart from activating the two executioners, caspase -8 can additionally cleave and activate BID, which, according to Luo et al (1998), is a form of pro-apoptotic protein.

Granzyme B Pathway

According to Cullen & Martin (2008), this pathway involves the killing of targeted cells through the delivery of lytic granules across the immunological synapse. This process is carried out by the cytotoxic natural killer cells. The granules contain perforin and granzyme B. According to Keckler (2007), the former permeabilises the target cells plasma membrane, creating a pathway for granzyme B to enter the cell and initiate apoptosis.

Intrinsic Pathway

This involves the release of cytochrome c from mitochondria of the cell, which activates the caspase cascade. Death of the cell is facilitated through intrinsic cellular stresses, such as the damage of the DNA and loss of growth factor (Youle & Strasser 2008).

Effectors of Cell Demolition

The end result of all the three pathways described above is the activation of the three caspases responsible for executing the cell. Caspase 3 is unique in the whole of this process. According to Walsh et al (2008), this enzyme is able to affect other caspases such as -8, -9, -7 and -6 with regard to its location within the pathway.

Slee et al (2001) are of the view that caspase -6 plays part in the cleavage of specific lamin proteins found within the nuclear. On the other hand, orthologues caspases -3 and -7 appears redundant at this stage (Thornberry et al 1997: Lakhani et al 2006). But a closer inspection reveals that the functionality of the two enzymes is different to some extent. For example, caspase -3 is responsible for many cleavage events than -7 (Slee et al 2001: Walsh et al 2008).

Demolition of the Cell during the Apoptosis Process

The demolition phase can be conceptualised through a number of events as follows:

Cell Detachment and Plasma Membrane Blebbing

After the cell has been demolished or killed, it means that it has to be removed from the tissue and this is done by the phagocytes. It has been noted that some proteins responsible for interconnection between the cells in the tissue are caspase substrates. This is for example β- and y-catenins, which mediate the link between cadherins and the cytoskeleton (Brancolini et al 1997). This leads to the break of the cell-cell connection, leading to cell detachment. The detachment of the cell from the extra cellular matrix is also realised through the cleavage of proteins such as focal adhesion kinase (Levkau et al 1998).

The plasma membrane of the apoptotic cells also goes through blebbing, which is the extrusion of the membrane. This leads to the release of apoptotic bodies, and this affects the microtubules, microfilaments and their associates.

Nuclear Condensation and Fragmentation

Apoptotic cells also undergo the breakdown of nucleus at the demolition stage. This involves chromatin condensation and degradation of the cell’s DNA. This leads to physical fragmentation of the nucleus.

Two separate processes takes place during nucleus fragmentation. The first is the weakening of the nuclear envelope, especially the lamina. This is followed by the generation of contractile forces within the actin network, completing the fragmentation process. According to Rao et al (1996), proteolysis of the lamins leads to disassembly of the nuclear membrane.

Cytoskeletal forces mediated by ROCK1 phosphorylation of myosin light chain then come in to physically tear apart the weakened nuclear membrane. This force leads to actin myosin based contraction, which further facilitates the disintegration.

Fragmentation of the nucleus is preceded by condensation and degradation of the internal chromatin/DNA. According to Cheung et al (2003), this condensation may be attributed to activation of Mst1 kinase by caspases. Chromatin condensation is subsequently followed by the degradation of the DNA by caspase activated dnase (CAD) nuclease.

Reducing Cell Physiology

Activation of caspases leads to the undermining of various cell physiological processes. Chiu et al (2002) are of the view that the Golgi and endoplasmic reticulum are some of the aspects of the cell that are affected. Other processes affected include proteosome, transcription/translation mechanism as well as the mitochondrial transport chain (Thiede et al 2001: Bushell et al 2000: Ricci et al 2003: Adrain et al 2004). The disruption of these processes at the same time leads to the failure of the cell, leading to death.

Apoptotic Cells and Immunity

The apoptosis process is concluded by the clearance of the cellular debris made up of apoptotic elements by the phagocytes. The immune system is able to differentiate between necrotic and apoptotic cells in the body. The release of cytochrome c by the mitochondria is enough to kill the target cell. As such, it is observed that a large portion of the caspase processing is aimed at preparing the apoptotic cell for its encounter with the immune system.

In order for the apoptotic cells to be removed from the system by the phagocytes, they must be identifiable to those phagocytes. This is achieved through labelling of these cells as targets for removal from the system. Labelling occurs through various changes on the membrane of the apoptotic cell. Martin et al (1996), Chang et al (1999) and Greenberg et al (2006) have identified phosphatidylserine and oxidized low density lipoprotein as some of the identifiers of the apoptotic cell.

Some chemicals such as lysophosphatidylcholine, which is dependent on caspase -3, help the phagocytes to home on the apoptotic cell (Lauber et al 2003). Antigenicity of the apoptotic cells also marks them for eventual removal from the system by the macrophages. Apoptotic cells maintain their plasma integrity, unlike their necrotic counterparts. As such, they do not release molecules such as HMGB1 which interact with the immune system. According to Birge & Ucker (2008), this maintenance of plasma integrity means that the apoptotic cells lack immunogenicity. As such, the immune system can tolerate the apoptotic cells within the body. The apoptotic cells, unlike necrotic cells, seems to be deactivated from the inside out (Patel 2006), indicating the fact that caspases are involved.

Caspases and Cell Death

Cysteinyl aspartate specific protease (caspases) is the cysteine proteases that are responsible for controlled death of cells within the organism. In humans, there are twelve such proteases that have been identified so far. Drosophila melanogaster has seven of them, while Caenorhabditis elegans has only one such protein. Structurally, all caspases- whether in humans or in animals- are made up of three structural domains. The first is a prodomain, followed by a large subunit and a small subunit.

The caspases are formed through two main processes. They may arise due to proteolytic cleavage of the subunits, or alternatively, by a proximity dependent activation process initiated by the prodomain. Positions P4, P3, P2 and P1 are indicative of the cleavage locality of substrates for these enzymes. Amino acids in P4, P3 and P2 are highly variable, but P1 is made up of aspartate. The death of the cell is brought about by the caspases’ cleavage of more than 1000 substrates within the cell. The activity of these enzymes can be controlled pharmacologically, and this has made them important in the treatment of various conditions.

Historical Background and Classification of Caspases

Historical Background

Caspases traces their roots back to the year 1989. This was the year that Sleath and Schmidt made the first description of caspase activity in a living organism. According to these two scholars, caspase activity was necessary for the activation of interleukin -1β.

Further observations were made in the year 1993 in this field. Horvitz and his colleagues discovered the cell death gene ced-3. According to their observation, the gene encodes a protease that is found within the same family. According to Wang et al (1999), the first human genetic condition emanating from caspase type 10 mutations was identified. This was by Michael Lenardo in the year 1999.

Categorisation of Human Caspases

The categorisation of human caspases into different classes can be carried out through the analysis of the caspases’ phylogenetic properties and their substrate specificity. This has led to three sub-groups of human caspases.

Group I

This is made up of caspases 1, 4, 5 and 12. The four of them are responsible for inflammation. According to Black et al (1989), Kostura et al (1989) and Yuan et al (1993), caspase 1 is the prototype for this subgroup. This caspase was earlier referred to as interleukin-1β, or ICE. Caspase 1’s enzymatic processes were mapped out in the year 1989. However, the caspases was purified and cloned three years later, in 1992. This followed the identification of the first caspase inhibitor by Miller in 1991. The inhibitor was the viral protein p35.

Caspase 1 regulates the maturation of interleukin-1β. This means that it is actively involved in the response to inflammation within the system. Hydrophobic residue usually occupies site P4 of the substrate. Caspase 12 is rare in humans, and it is observed that more than 98 percent of Africans lack this protein.

Group II

The group is made up of caspases 3, 6 and 7, with caspase 3 being the prototype for the subgroup. This caspase is the homologue of ced-3 in Caenorhabditis elegans, the organism that was earlier identified as having a single protein. The three caspases in this group are responsible for cell apoptosis at the terminal effector phases. All the three caspases possess a very small prodomain. This is a possible indication of minimal regulation of their enzymatic activation.

Group III

This group is made up of the remaining four caspases, which are 2, 8, 9 and 10. All of the four have a large prodomain. This point to the presence of an activation mechanism that is complex, requiring molecular adaptors. Two of the caspases in this group (8 and 10) are identified as components of the signal transduction mechanism. This is for receptors of apoptosis, for example CD95, which requires the adaptor molecule FADD. On the other hand, caspase 9 appears to be activated in the apoptosome. The adaptor molecule that is required for this process is apoptosis activating factor 1 (Apaf -1). Site P4 of the substrate required for this subgroup is occupied by aliphatic residue. The other protein in this group (caspase 2) is seen to be activated within the PIDDosome complex.

Despite the large prodomain of these proteins, they can also be cleaved and activated by other caspases, for example those from group 1. As such, group III caspases are regulators and at the same time effectors of apoptosis, with regard to the activation context.

There is one more caspase that is found in humans, but which falls in none of the above three classifications. This is caspase 14, which is involved in keratinocyte differentiation. Caspase 14 plays no role in either apoptosis or inflammation, and as such falls into none of the three groups above.

Caspases’ Structure and their Active Site

As earlier indicated in this paper, three structural domains makes up the structure of a caspase. These are the prodomain, p20 (large subunit) and p10 (small subunit). The two subunits (large and small), are the ones that make up the caspase. The large subunit contains the active cysteine, with four varying subsites. These are S4, S3, S2 and S1, which are dependent on the crystal structure data that is available.

The first subsite (S1) works on the carboxyl cluster of the polypeptide side chain in position one (P1). The subsite is made up of the large and small subunits, which also make up the site for recognition and location of the substrate in S4, S3, S2 and S1. But most of the residues involved in substrate specificity for S4 are to be found in the small subunit.

Recognition of Substrate and Mechanism for Caspase Activity

S4-S1 denotes caspases’ recognition site for the substrate, where the particular substrate is attached to residues P4-P1. The recognition of substrate is necessary for caspase specificity. The substrate recognition site varies from one caspase to the other, with S1 exhibiting constant and highly tight traits. S1 has a deep and basic pocket, leading to absolute specificity to bind Asp (D) as the substrate residue P1. This is especially significant given the fact that Asp is tightly held by a hydrogen bond. This means that the facets of S1 are maintained at a minimum, reducing tolerance to P1 substitutes. P2 and P3 for subsites S2 and S3 respectively are not as specific as S1 and P1. A significant determinant of specificity is the P4 and S4 site.

Caspases have a characteristic catalytic triad, with cysteine and vicinity of a histidine being the more constant components of the triad. The other component, which is usually a serine, is not constant as the other two, and it is easily substituted. A case in point is the activation of caspase 3. After binding to a substrate, catalysis can occur with Cys285 and His237 as two components of the triad. The other component is substituted by an oxyanion hole which is made up of amide nitrogens.

Caspases Action Mechanism

It is noted that it is the effector caspases (3, 6 and 7) that inform the demise of the target cell. The three caspases are diverse, and are activated in a sequential rather than a parallel fashion. This leads to an amplification cascade of proteolytic cleavage, which is not unlike that to be found in blood coagulation.

Caspase 9 is the one that is first activated in the apoptosome, and it in turn activates 3 and 7. However, it is noted that caspase 3 can also proteolytically activate 9, leading to a reciprocity relationship between the two. According to Inoue et al (2009), the activated caspases 3 and 7 the proteolytically activates 6 and 2. When this happens, the four caspases cleave their intracellular substrates informing the direction of cell apoptotic.

The molecular stages of cell apoptosis can be conceptualised schematically. The first stage is initiation, where death receptors are activated. This involves caspases 8 and 10. The second stage is determination, which is the activation of the apoptosome by caspase 9. The third stage is amplification, which increases the type and number of activated caspases. This involves caspases 3, 7, 6 and 2. The fourth is the demolition stage, which is the last stage that determines the death of the target cell. It involves cleavage of more than 700 substrates in the target cell.

Substrates Involved in the Apoptosis Process

More than 1000 different polypeptides have been found to result from the apoptosis process. Patterns in this field indicated that there are possibly more substrates that have not yet been identified. These substrates (the discovered ones and those not yet identified) are seminal in the apoptosis process. This is given the fact that the various caspases proteolytically cleavages them, affecting the physiology of the cell. This ultimately leads to the death of that particular cell.

Inhibitors of Caspases

Inhibitors of caspases can be categorised into two broad groups. These are the pharmacological and natural inhibitors, with the latter occurring naturally within the cell and the former introduced from without the organism. The pharmacological inhibitors were engineered through knowledge gained on the activities of molecular mechanisms in the P4-P1 sites. Aldehydes, nitriles and ketones are some of the pharmacological inhibitors available today. All of them acts by irreversibly interfacing with catalytic Cys in the system.

The discovery of the first natural inhibitor to caspases took place before the caspases themselves were discovered. This was in the year 1991, and it is credited to Lois Miller. The inhibitor was a p35 protein that was produced by baculovirus (Autographa californica). This inhibitor was both potent and selective, and it led to the identification of more natural inhibitors. Viruses are one of the major organisms that regulate death of cells within the organism, and this mechanism can be understood by the operations of natural inhibitors.

The Knocking Out of Caspase

Caspases are significant in the controlled and regulated death of cells in an organism. Several experiments have indicated that it is possible to manipulate the mechanism of the caspases by knocking them out. For example, the role that these enzymes play in the receptor mechanism can be identified by knocking out a particular caspase, such as 8.

Caspases, Cell Apoptosis and Pathological Implications

It is noted that caspases are significant in determining the pathologies of excessive apoptosis. A case in point is the critical role of these enzymes in the in vivo activation of demise which may occur in myocardial infarction and cerebral ischaemia. In the two conditions above, the death of cells results from both necrosis and apoptosis.

It is also noted that caspases also determine the vulnerability of cells to shocks and lethal insults. A case in point is the role of caspases in neurodegeneration conditions such as Alzheimer disease. In this disease, studies have shown that caspase 3 plays a very central role. It alters the normal processing of APP (amyloid β precursor protein), getting rid of the C-terminal. This leads to degradation of APP, leading to the formation of amyloid β peptide. The latter in turn leads to death of cells through apoptosis, but the exact mechanism of this is not yet fully understood.

Caspases and their use in Clinical Therapy

Various caspase inhibitors have been developed, although there are certain challenges riding on their clinical application. For example, they face difficulties entering the cell membrane and are less stable in solution form. This, together with their evident electrophilic promiscuity, increases their risk of attack by biological nucleophiles.

The limited clinical use of these molecules has not reduced their importance in the medical field. The research community has greatly benefited from the steps made in this field. This is evidenced by the current efforts by research organisations to come up with improved inhibitors with more therapeutic uses. This is an indication of the bright future of therapeutic application of caspases in the clinical community.