Tuesday, November 19, 2013



Proteolytic enzymes, includes the cell's degrading machine preoteasome, caplains, and integral membrane proteases (such as γ-secretase and rhomboid); participate in several intracellular signaling processes. Caspases are one of the protein families in the body which are the only constitute a formal multistep pathway able to transmit intracellular signals by proteolysis and the name of this family is an abbreviation of cysteine-dependent aspartate-directed proteases, with a cysteine in their active site, as catalytic nucleophile, that cleave on the C-terminal side of aspartate residues.

The first discovery by Robert Horvitz and his colleagues who found that the ced-3 gene was required for the cell to die that take place during the development of the nematode, which called c.elegans. They found in 1993 that the protein, which encoded by ced-3 gene, was a cystiene protease with similar properties to the mammalian interleukiene-1-beta converting enzyme (ICE, or IL-1β), in which at this time is the only known caspase, named caspase-1. Other caspases have been discovered and numbered in the order in which they have been identified.

The human body contains cells with different life expectancies. Some (white blood cells, skin) are programmed to rapidly die and be replaced. Others (nerve cells) are programmed to survive the lifetime of the individual and are seldom replaced. There is a central role of enzymatic pathways play in the life and death of cells. When death pathways slowing down in cells that are normally programmed to die, cancer results. Conversely, when death pathways become overactive in cells that are programmed to survive, a degenerative disease occurs. So there is must be a balance between cell survival and programmed cell death (apoptosis) and that balance is managed by a family of proteases called caspases.

Some of them are essential for apoptosis (which mean the falling of leaves in Greek), others are required in the immune system for the maturation of cytokines, and also they have a role in necrosis and inflammation.

Basic properties

Nowadays there are many caspases have been discovered in many species, currently there are 11 members from human, 4 from c.elegans and 7 from Drosophila with obvious conservation in evolution.
¨    The General structure
Caspases are regulated at post-translational level, ensuring that they can be activated rapidly. They are first synthesized as inactive single chain polypeptide precursor, called zymogens or pro-caspases, in each caspase zymogen an N-terminal peptide (prodomain) is followed by sequences comprising first a large and then a small subunit [Fig. 1 and 2 ]. Pro-caspases have an intrinsic proteolytic activity. This unusual property is essential for triggering the proteolytic pathways that lead to complete their functions as in apoptosis. 
Initiator caspases have a longer N-terminal prodomain                 (>90 amino acids) than the effector caspases, which their prodomain is very small (20-30 residues). The prodomain of the initiator caspases contain domains such as CARD or DED, which enables caspases to interact with other molecules that regulate their activation by responding to the stimuli that cause the clustering of the initiator caspases. This allowing them to be auto-activated via auto-catalytic intra-chain cleavage and then they can proceed to activate the effector caspases by intra-chain cleavage.

¨    The Specificity
Caspases recognizing at least 4 contiguous amino acids, named P4-P3-P2-P1, and cleaving after the C-terminal residue (P1), it is usually an Asp or D.  Although the P1 residue was thought to be exclusively Asp, recent studies indicate that some caspases can also cleave after Glu (Hawkins et al., 2000; Srinivasula et al., 2001).

Interestingly, the preferred P3 position is invariantly Glu or E for all mammalian caspases examined (Thornberry et al., 1997). Thus the preferred specificity can be described as X-Glu-X-Asp. All caspases contain a conserved QACXG (where X is R, Q or G) pentapeptide active-site motif [Table 1]. 

Table 1, shows the different human caspases with their different names and with their common and different active sites.

Figure 2, Proenzyme organization of the caspases: Caspases are synthesized as proenzymes, with an N-terminal peptide or prodomain (PRO), and two subunits sometimes separated by a linker peptide (black box). Based on caspase-1 and caspase-3, active enzymes are heterotetramers of two large (~20 KDa) & and two small (~ 10 KDa) subunits. The proenzymes are cleaved at specific Asp residues (Dn, where n is the position in the protein). The numbers at the right-hand side are the numbers of amino acids in the protein. a Exact cleavage site not known. b the cleavage site of caspase-3 may be at Asp-9 or Asp-28. ccaspase-9 is cleaved preferentially at Asp-330 by caspase-3 and at Asp-315 by granzyme B. Caspase-2 cleavage sites are based on equivalent sites being present in Nedd2.FADD (in the white box) represents the domains of caspase-8 and caspase-10 that are homologous to the DED of FADD/MORT1.

¨    The Activation of caspases
Conversion of each caspase zymogen [dormant state of the enzyme] to the mature enzyme requires a minimum of 2 cleavages, one separating the prodomain from the large subunit and another is separating the large and small subunits [Fig. 3]. All of these cleavages involve Asp-X bond. This process eventually yields a heterodimeric enzyme with both fragments contributing to the formation of the catalytic machinery.

It is believed now that the functional caspase unit is a homodimer [which referred to as "homodimer of heterodimer"], with each monomer comprising a large (20KDa, named p20.) and a small (10 KDa, named p10.) subunit, according to the structural characterization of the caspase-1 which reveals the homodimeric structure. In addition to caspase-1, structural information is available for caspase-3, -7, -8 and -9. The overall architecture of them is similar and consists of 2 heterodimers composed of large and small subunits. The subunits of each heterodimer are folded into a compact cylinder that is dominated by a central six-stranded β-sheet and five α-helices that are distributed on opposing sides of the plane that is formed by the β-sheet. This so-called caspase-fold is a unique quaternary structure among proteases and has only been described for caspases [Fig. 4].

Figure 3, Schematic diagram of the mammalian caspases: except caspase-11 and 12 (mouse), caspase-13 (bovine), all listed caspases are of human origin. Their phylogenetic relationship (left) appears to correlate with their function in apoptosis or inflammation. The initiator and effector caspases are labeled in purple and red, respectively.  The position of the cleavage (between the large and small subunits) is highlighted with a large arrow while additional sites of cleavage are represented by medium and small arrows. The four surface loops (L1-L4) that shape the catalytic groove are indicated.   The catalytic residue Cys is shown as a red line at the beginning of loop L2.

Homodimerization [Fig. 4a] is mediated by hydrophobic interactions, with 6 β-strands (5 are parallel and one anti-parallel) from each catalytic subunit forming a single contiguous 12-stranded β-sheets. Several α-helices (six helices) and short β-strands (βI, II, III, IV and V) are located on either side of the central β-sheet, giving rise to globular fold. The active sites, formed by 4 protruding loops form the scaffold, are located at 2 opposite ends of the β-sheet.
Crystallographic analysis has demonstrated that the active caspase is a tetramer composed of two such heterodimers. As first appreciated in the structure of caspase-1, this general rule is valid also for caspases, since the catalytic residue His-237 is located in the loop that connects β-strand (β3) to the "front" helix (α3), while the neighbouring  strand (β1) is followed by the "back" helix (α1) (‘back’ and ‘front’ refer to the standard caspase orientation shown in [Fig. 4a]). The β1-α1 loop has one of the residues that determine the characteristic P1 specificity, Arg-179.

The entire active center of caspases, consisting of the S4-S3-S2-S1 specificity sub-sites binding P4-P3-P2-P1 residues of the substrate, respectively, is formed by a flexible loops (L1– L4, Fig. 5).  Loop L1 and a portion of L2, which contains the catalytic Cys-285 residue, are a part of the large subunit, whereas L3 and L4 come from the small subunit. The activation-mediated cleavage of caspases occurs in the loop L2; liberating the C-terminus of the large (L2) and the N-terminus of the small subunits (L2\). [Fig. 6] is an example of caspases activation by proteolysis.

Figure 4, Structure of active caspases: (a) the crystal structure of human caspase-8 exemplifies the fundamental caspase fold, and is shown bound to the tetrapeptide aldehyde inhibitor acetyl-Ile-Glu-Thr-Asp-CHO (PDB entry 1QTN), which represents the highest-resolution structure of a caspase reported to date. Note the three-layer structure of a twisted, 12-stranded β-sheet that is sandwiched by α-helices. Most of the interdomain contact area is built by the central small subunits, with additional interactions (the characteristic ‘loop bundle’) tying together the C- and N-termini of large and small subunits from neighbouring domains. The bound inhibitor is represented with a ball-and-stick model, as are dithiane diol molecules trapped in the cleft between the two monomers (termed the central cavity, for obvious reasons).
(b) Simplified topological diagram of the caspase structure, following the CATCH definition of secondary structure elements for 1QTN. An additional N-terminal α-helix of variable length (α0; not shown) is present in caspases-1, -2 and -9, and closes the ‘bottom’ of the α/β barrel. Also not depicted is an additional α-helix found solely in the long 179-loop of caspase-8. The positions of catalytic dyad residues His-237 and Cys-285 (red), along with those of the specificity-determining arginine residues (Arg-179 and Arg-341), are indicated. The location of loops that contain important functional elements is indicated in blue text using the numbering convention designated throughout this review, along with an alternative designation.

Figure 5, The Generalized distribution of caspase catalytic center loops (L1– L4) on small and large subunits: Loops are shown in blue. Position of the activating cleavage processing is shown by arrows. Numbers represent the order of the activation cleavages. The active site Cys is shown by a red asterisk. Processing occurs in L2. The resulting large subunit portion of the L2 loop of one monomer and small subunit portion of the L2 loop of another monomer (L2\). On the right is Schematic diagram of the substrate-binding groove: L1 and L4 constitute two parallel sides of the groove while L3 serves as the base. L2, harboring the catalytic residue Cys, is positioned at one end of the groove, poised for catalysis. L2\ plays a critical role by stabilizing the conformation of the L2 and L4 loops.

Figure 6, Mechanism of activation for effector caspases as exemplified by caspase-7: A schematic diagram of procaspase-7 activation is shown here. The active-site loops before and after the proteolytic processing is shown in orange/cyan and magenta/green, respectively. The detailed conformational changes at the active site are depicted in the middle panel, in which the four surface loops, L1-L4, and the L2\ loop are labeled. 

Role of caspases in Apoptosis

Apoptosis is a physiological cell suicide program (one of the main types of PCD) that is critical for the development and maintenance of healthy tissues. Regulation of PCD allows the organism to control the cell number and the tissue size, and to protect itself from rogue cells that threaten homeostasis. The changed activity of numerous genes influences switching of cells to a self-destruction program. Apoptosis requires co-ordinate action and fine tuning of a set of proteins that are either regulators or executors of the process. Cancer, autoimmune diseases, immunodeficiency disease, reperfusion injury and neurodegenerative disorders are characterized by unregulated apoptosis. Modulation of the expression and activation of the key molecular components of the apoptotic process has emerged as an attractive therapeutic strategy for many diseases.
There are two distinct types of cell death, death by injury and death by suicide. Cells that are damaged by injury, such as mechanical damage or exposure to toxic chemicals, undergo a series of changes characterized by swelling of cells and their organelles, leakage of cell content and inflammation of the surrounding tissues. In other words, cells die by necrosis. In contrast, apoptosis is an organized, genetically directed process, which leads to cell death. Cells dying by apoptosis share unique morphological features, distinct from autolytic, degenerative cell changes observed during necrosis [Fig. 7].


Morphological changes of an apoptotic cell might be easily detected under the microscope. Some of these changes can be seen even by light microscopy using specific dyes, while other can be detected only by electron microscopy. The dying cell starts to show protrusion from the plasma membrane, referred to as bleb [Fig. 8]. Staining DNA with certain dyes allows observation of the condensation of the cell nucleus, which usually starts as a condensed ring along the nuclear envelope. The condensed nucleus can disassemble into several fragments. The entire cell condenses and is re-organized into apoptotic bodies [Fig. 8], which are membrane-bound vesicles varying in size and composition, containing the entire cell content in various combinations, such as cytosolic elements, organelles or parts of condensed nuclei. Additional changes have been described by electron microscopy. Condensation or swelling of mitochondria, dilatation of endoplasmic reticulum (ER), vacuolisation of cytoplasm and loss of plasma membrane microvilli have been observed. At a certain point, apoptosis affects all compartments and organelles in a dying cell.

Apoptosis includes cellular shrinking, chromatin condensation and margination at the nuclear periphery with the eventual formation of membrane-bound apoptotic bodies that contain organelles, cytosol and nuclear fragments and are phagocytosed without triggering inflammatory processes.The necrotic cell swells, becomes leaky and finally is disrupted and releases its contents into the surrounding tissue resulting in inflammation. Modified from [Van Cruchten, 2002].

Figure 7, Schematic diagram of differences between apoptosis and necrosis: (A) Apoptosis includes cellular shrinking, chromatin condensation and margination at the nuclear periphery with the eventual formation of membrane-bound apoptotic bodies that contain organelles, cytosol and nuclear fragments and are phagocytosed without triggering inflammatory processes (B). The necrotic cell swells, becomes leaky and finally is disrupted and releases its contents into the surrounding tissue resulting in inflammation.

Figure 8, Schematic diagram of morphological changes in the cell during apoptosis.

Molecular mechanism of apoptosis

Apoptosis can be triggered by various stimuli from outside or inside the cell, as by ligation of cell surface receptors,  DNA damage as a cause of defects in DNA repair mechanisms, treatment with cytotoxic drugs or irradiation, a lack of survival signals, contradictory cell cycle signaling or by developmental death signals. Death signals of such diverse origin nevertheless appear to eventually activate common cell death machinery leading to the characteristic features of apoptotic cell death [Fig. 9].The apoptosis process can be divided into at least three functionally distinct phases: initiation, effector and degradation. During the initiation phase, cells receive death-inducing signals: as lack of obligatory survival factors, shortage of metabolite supply, ligation of death-signal transmitting receptors, sub-necrotic damage by toxins, heat or irradiation.
During the effector phase, these signals are translated into metabolic reactions and the decision to die is taken. The ultimate fate of the cell is subject to regulatory events. Beyond this stage, during the degradation phase, an increase in the overall entropy, including activation of catabolic enzymes, precludes further regulatory effects. During the late phase, DNA fragmentation and massive protein degradation becomes apparent. Subsequently, fragments are encapsulated into ‘apoptotic bodies’.

Figure 9, Apoptosis may occur through 2 main signaling pathways: The intrinsic pathway may be triggered by DNA damage and other types of severe cell stress. It may involve the release of intracellular pro-apoptotic proteins that activate caspases, a network of proteases that ultimately destroy critical structural proteins in the cell and stimulate fragmentation of the chromosomal DNA, resulting in cell death. The extrinsic pathway may be triggered in response to external pro-apoptotic signals, such as endogenous Apo2L/TRAIL. Binding of this molecule to pro-apoptotic cell surface transmembrane receptors DR4 and DR5 may lead to apoptosis by initiating a signaling cascade, which results in the activation of the caspase system. The intrinsic and extrinsic apoptosis pathways converge via the activated caspases that ultimately trigger cell death.

Caspases in apoptosis
Caspases are the family which can be classified into 3 subgroups according to their function:
I-Initiator caspases [caspase-2, -8, -9 and -10]
II-Executioner caspases [caspase-3, -6 and -7]
Both (I) and (II) have either direct or indirect role in the processing, propagation and amplification of apoptotic signals, which result in the destruction of the cellular structure.
III-Inflammatory caspases, which involved in the maturation of pro-inflammatory cytokines [caspase-1, -4, -5, -11, -12, -13 and -14]
At the molecular level, two principal pathways of apoptotic cell death have been described (Twomey and McCarthy, 2005). The pathway of apoptosis often referred to as:
A) Extrinsic apoptosis (Receptor-mediated apoptosis or type 1 apoptosis).
B) Intrinsic apoptosis (mitochondria-independent apoptosis and or type 2 apoptosis) [Fig. 10].
Many of the proteins involved utilize at least two types of sequence motifs. One motif is known as DD and the other motif as DED [The instigators include the long prodomain, DED containing caspases 8 and 10, and the CARD containing caspase-9] 

Figure 10, Basic two principal pathways: the receptor and the mitochondria-mediated apoptosis.

A)    Extrinsic apoptosis:
Extrinsic apoptosis signalling is mediated by the activation of so called “death receptors” which are cell surface receptors that transmit apoptotic signals after ligation with specific ligands. DRs belong to (TNFR) gene superfamily, including TNFR-1, Fas/CD95, and the TRAIL receptors DR-4 and DR-5 [Fig. 11 B and 12 and table 2]. All members of the TNFR family consist of cysteine rich extracellular sub-domains which allow them to recognize their ligands with specificity, resulting in the trimerization and activation of the respective death receptor. Subsequent signalling is mediated by the cytoplasmic part of the DR which contains a conserved sequence termed the DD. Adapter molecules like FADD or TRADD themselves possess their own DDs by which they are recruited to the DDs of the activated DR, thereby forming the so-called DISC [Fig. 12 A]. In addition to its DD, the adaptor FADD also contains a DED which through homotypic DED-DED interaction sequesters procaspase-8 to the DISC [Fig. 11 B]. The local concentration of several procaspase-8 molecules at the DISC leads to their autocatalytic activation and release of active caspase-8. Active caspase-8 then processes downstream effector caspases which subsequently cleave specific substrates resulting in cell death. The signal needs to be amplified via mitochondria-dependent apoptotic pathways. The link between the caspase signalling cascade and the mitochondria is provided by the Bcl-2 family member Bid. Bid is cleaved by caspase-8 in to its truncated form (tBid), translocates to the mitochondria where it acts in concert with the proapoptotic Bcl-2 family members Bax and Bak to induce the release of cytochrome c (Apaf-2) and other mitochondrial proapoptotic factors into the cytosol [Fig. 9, 10 and (11 A)]. Cytosolic cytochrome c is binding to monomeric Apaf-1 which then, in a dATP-dependent conformational change, oligomerizes to assemble the apoptosome, a complex of wheel-like structure with 7-fold symmetry, which triggers the activation of the initiator procaspase-9. Activated caspase-9 subsequently initiates a caspase cascade involving downstream effector caspases such as caspase-3, caspase-7, and caspase-6, ultimately resulting in cell death. 

Figure 11, (A)

Table 2, Shows many different molecules and receptors involved in apoptosis.

Figure 11,(B)

B)    Intrinsic apoptosis

Besides amplifying and mediating extrinsic apoptotic pathways, mitochondria also play a central role in the integration and propagation of death signals originating from inside the cell such as DNA damage, oxidative stress, starvation, as well as those induced by chemotherapeutic drugs. Most apoptosis-inducing conditions involve the disruption of normal mitochondrial inner transmembrane potential (Δψ) [Fig. 13 A] as well as the so called permeability transition (PT), a sudden increase of the inner mitochondrial membrane permeability to solutes with a molecular mass below approximately 1.5 kDa. Concomitantly, osmotic mitochondrial swelling has been observed by influx of water into the matrix with eventual rupture of the outer mitochondrial membrane, resulting in the release of proapoptotic proteins from the mitochondrial intermembrane space into the cytoplasm. Released proteins include cytochrome c, which activates the apoptosome [Fig. 14] and therefore the caspase cascade [Fig. 11 A, B and Fig. 13 A], but also other factors such as the apoptosis-inducing factor (AIF), the endonuclease (endoG), Smac/Diablo, and Htr/Omi. In addition to the release of mitochondrial factors, the dissipation of Δψ and PT also cause a loss of the biochemical homeostasis of the cell: ATP synthesis is stopped, redox molecules such as NADH, NADPH, and glutathione are oxidized, and reactive oxygen species (ROS) are increasingly generated.

  Caspases and chromatin break down
 One of the hallmarks of apoptosis is the cleavage of chromosomal DNA into nucleosomal units. The caspases play an important role in this process by activating DNases, inhibiting DNA repair enzymes and breaking down structural proteins in the nucleus. The role of the caspases in the breakdown of chromatin is illustrated in [Fig. 15]. 
1) Inactivation of enzymes involved in DNA repair: The enzyme poly (ADP-ribose) polymerase, or PARP, is an important DNA repair enzyme and was one of the first proteins identified as a substrate for caspases. The ability of PARP to repair DNA damage is prevented following cleavage of PARP by caspase-3.
2) Breakdown of structural nuclear proteins: Lamins are intra-nuclear proteins that maintain the shape of the nucleus and mediate interactions between chromatin and the nuclear membrane. Degradation of lamins by caspase 6 results in the chromatin condensation and nuclear fragmentation.
3) Fragmentation of DNA: The fragmentation of DNA into nucleosomal units is caused by an enzyme known as CAD, or caspase activated DNase. Normally CAD exists as an inactive complex with ICAD (inhibitor of CAD). During apoptosis, ICAD is cleaved by caspases, such as caspase 3, to release CAD. Rapid fragmentation of the nuclear DNA follows.

Caspases in inflammation

In mammles, there are 5 inflammatory caspases that all have a CARD at their N-terminus. Human inflammatory caspases are clustered on chromosome 11q22 in following order from the telomere:  caspase-1, caspase-5, caspase-4, and finally the gene for caspase-12 [Fig. 16].
The role of caspase-1 in the maturation of IL-1β and IL- 18 renders it a key player in response to pathogenic infection as well as in inflammatory and autoimmune disorders. IL-1β and IL-18 are key cytokines in these conditions. Although they share certain proinflammatory activities, they also have very important individual functions. For example, IL-1β but not IL-18 is anorectic, pyrogenic, results in skin rashes and urticaria, induces hepatic-acute phase proteins, up-regulates prostanoid synthesis, and is involved in inflammatory pain hypersensitivity. IL-1β is also implicated in destructive joint and bone disease, tumor angiogenesis and invasiveness, and toxicity of insulin-producing pancreatic islets β-cells, and neurons in stroke and neurodegeneration. IL-18, in contrast, was originally known for its ability to stimulate IFN-γ production and to mediate T cell polarization. Now it is known that it possesses many other functions including modulation of the heart contractile force, upregulation of adhesion molecules and NO synthesis, and regulation of energy intake and insulin sensitivity. Therefore, inhibition of caspase-1 is an attractive therapeutic strategy aimed at blocking the effects of both cytokines in inflammatory and autoimmune diseases. Although we know of many biological effects of IL-1β and IL-18, we still lack information on the exact mechanisms by which caspase-1 is activated, and by which these cytokines are matured and secreted.

Our understanding of the inflammatory caspases and their regulatory mechanisms has recently improved substantially. Multiple new players have now been identified to modulate the function of these enzymes. The discovery of the caspase-12 polymorphism and the characterization of its function in sepsis and the host response to pathogenic infection have emphasized the essential role of these caspases in innate immunity. In parallel, the identification of the NLR family and its regulation of the inflammatory caspases has provided an important step forward in our knowledge of the tight and highly specific mechanisms of activation of these enzymes.

1. C.elegans: Caenorhabditis elegans, it is a type of nematodes.
2. Ced-3 gene: c.elegans death gene or Cell Death Defective-3, which is involved in the cell death.
3. CARD: caspase recruitment domains, they are interaction motifs found in a wide array in proteins, typically those involved in apoptosis and inflammation.
4. DED: death effector domain (DD is Death Domain), which is a protein domain found in procaspases and proteins that regulate caspase activation in apoptotic cascade such as FADD.
5. FADD: fas-associated with death domain protein.
6. Glu or E: Glutamic acid.
7. Asp or D: Aspartic acid.
8. X: any amino acid (aa\).
9. MORT-1: Mediator Of Receptor-induced Toxicity-1, which is also called FADD.
10. Nedd2: Neural precursor cell Expressed Developmentally Down-regulated 2, it is a developmental regulated mouse gene that encodes a protein (mouse homologue of ICH-1, ICE and Ced-3 Homolouge-1) similar to the ced-3 gene protein and mammalian ICE. It is renamed as caspase-2 (Alnemri et al Human ICE/CED-3 protease nomenclature, 1996).
11.  Cys or CCysteine aa\.
12. PDB: Protein Data Bank. (http://www.rcsb.org/pdb/home/home.do)
13. P: Pocket.
14. PCD: Programmed Cell Death.
15. TNFR1:  Tumor Necrosis Factor Receptor-1
16. TRAIL-R1 or 2: TNF-Related Apoptosis-Inducing Ligand Receptor-1 or -2
17. p75-NGFR: p75-Nerve Growth Factor Receptor.
18. TNF: Tumor Necrosis Factor.
19. Motif and domain appear to be used interchangeable.
20.   Initiator caspases (º activator or apical or upstream or instigator caspases).
21. Effector caspases (º Executioner or downstream or terminator caspases).
22.   DISC: Death Inducing Signaling Complex.
23.   L: Ligand.
24.   TRADD: TNF Receptor-Associated Death Domain.
25.    TRAF2: TNF Receptor-Associated Factor-2
26.   BH: Bcl-2 Homology (has 4 domains BH1, BH2, BH3 and BH4).
27.   DR: Death Receptor.
28.   APAF-1: Apoptotic Protease Activating Factor-1, it is a protein contains three functional regions: an N-terminal CARD domain that can bind to the zymogen form of caspase-9, a CED4 like region enabling self-oligomerization, and a regulatory C-terminus with WD-40 repeats masking the CARD and CED4 region. During apoptosis, cytochrome-C and dATP can relieve the inhibitory action of the WD-40 repeats and thus enable the oligomerization of APAF-1 and the subsequent recruitment and activation of caspase-9 from its zymogen form.
29.   Smac: Second Mitochondria-Derived Activator of Caspases.
30.   DIABLO: Direct Inhibitor of Apoptosis-Binding Protein with LOw pI.
31.    NLR: Nod-Like Receptor.

4-        Biochem. J. (2004) 384, 201–232, Review article: the protein structures that shape caspase activity, specificity, activation and inhibition.
5-        Acta Pharm. 53 (2003) 151–164, Review:  Biochemistry of apoptotic cell death.
6-        Oncogene (2003) 22, 8543–8567, 2003 Nature Publishing Group, A decade of caspases.
7-        Cell, Vol. 117, 855–858, June 25, 2004, Copyright 2004 by Cell Press, Mini-review: Caspases activation: Revisiting the Induced Proximity Model.
8-        Molecular Cell, Vol. 9, 459–470, March, 2002, Copyright 2002 by Cell Press, Mini-review: Mechanisms of Caspase Activation and Inhibition during Apoptosis.
14-    BioEssays 25:888–896, 2003. 2003 Wiley Periodicals, Inc. Review articles, Apoptosis—an introduction, Alfons Lawen.
15-    Current science, VOL. 80, NO. 3, 10 February 2001, Review article: Apoptosis: Molecular machinery.
17-    ApoReview - Introduction to Apoptosis: Page 1 of 26, this review was composed by Andreas Gewies in 2003.
18-    Cell, Vol. 91, 559–562, November 28, 1997, Copyright 1997 by Cell Press, Mini-review, Cytochrome c: Can’t Live with It—Can’t Live without It.
21-    Basic Medical Sciences, St.George’s, University of London, Apoptosis by Phil Dash.
22-    The journal of immunology, Copyright 2006 by The American Association of Immunologists, Inc. Brief reviews, the inflammatory caspases.

(***Note: The last update by me was in 2009. Therefore, if there any new updates, please do not hesitate to contact me.)