Apoptosome
Encyclopedia
The apoptosome is a large quaternary protein
structure formed in the process of apoptosis
. Its formation is triggered by the release of cytochrome c
from the mitochondria in response to an internal (intrinsic) or external (extrinsic) cell death stimulus. Stimuli can vary from DNA damage and viral infection to developmental cues such as those leading to the degradation of a tadpole's
tail.
In mammalian cells, once cytochrome c
is released, it binds to the cytosolic protein Apaf-1 to facilitate the formation of apoptosome. An early biochemical study suggests a two-to-one ratio of cytochrome c to apaf-1 for apoptosome formation. However, recent structural studies suggest the cytochrome c to apaf-1 ratio is one-to-one. It has also been shown that the nucleotide dATP
as third component binds to apaf-1, however its exact role is still debated. The mammalian apoptosome had never been crystallized, but a human APAF-1/cytochrome-c apoptosome has been imaged at lower (2 nm) resolution by cryogenic transmission electron microscopy
10 years ago, revealing a wheel-like particle with 7-fold symmetry. Recently, a medium resolution (9.5 Ångström) structure of human apoptosome was also solved by cryo-electron microscopy
, which allows unambiguous inference for positions of all the APAF-1 domains (CARD, NBARC and WD40) and cytochrome c. Once formed, the apoptosome can then recruit and activate the inactive pro-caspase-9
. Once activated, this initiator caspase can then activate effector caspases and trigger a cascade of events leading to apoptosis.
in apoptosis
: apoptosomes or mitochondria?". However, the Apoptosome was known before this time as a ternary complex
. This complex involved caspase-9
and Bcl-XL
which each bound a specific Apaf-1
domain. The formation of this complex was then believed to play a regulatory role in mammalian
cell death. In December of the same year, a further article was released in The Journal of Biological Chemistry stating that Apaf-1 is the regulator of apoptosis, through activation of procaspase-9.
The criteria for an apoptosome were laid out in 1999. Firstly, it must be a large complex (greater than 1.3 million Daltons). Secondly its formation requires the hydrolysis
of a high energy bond of ATP
or dATP. And lastly it must activate procaspase-9 in its functional form. The formation of this complex is the point of no return, and apoptosis will occur. The stable APAF-1 and cytochrome mutimeric complex
fit this description, and is now called the apoptosome.
The apoptosome was thought to be a mutimeric complex
for two reasons. Firstly, to bring multiple procaspase-9 molecules close together for cleavage. And secondly, to raise the threshold for apoptosis, therefore nonspecific leakage of cytochrome c
would not result in apoptosis.
Once the apoptosome was established as the procaspase-9 activator, mutations within this pathway became an important research area. Some examples include human leukemia
cells, ovarian cancer
and viral infections. Current research areas for this pathway will be discussed in further detail. There are hidden routes for cell death as well, which are independent of APAF-1 and therefore the apoptosome. These routes are also independent of caspase-3 and 9. These hidden pathways for apoptosis are slower, but may prove useful with further research.
complex assembled around the adaptor protein Apaf1 (apoptotic protease activating factor 1)
upon mitochondria-mediated apoptosis
which must be stimulated by some type of stress signal T formation of the apoptosome requires the presence of ATP
/dATP and cytochrome c
in the cytosol
.
A stress stimulus can trigger the release of cytochrome c into the cytoplasm which will then bind to the C-terminus of Apaf-1 within a region containing multiple WD-40 repeats. The oligomerization
of Apaf-1 appears to be accompanied by synchronized recruitment of procaspase-9
to the CARD motif
at the Apaf-1 N-terminus. The apoptosome triggers the activation of caspases
in the intrinsic pathway
of apoptosis
.
The wheel-shaped heptameric
complex with sevenfold symmetry structure of the apoptosome was first revealed at 27 Å resolution by electron cryomicroscopy
techniques and has a calculated mass of about 1 MDa (Acehan et al. 2002).This wheel-like particle has seven spokes and a central hub. The distal region of the spoke has a pronounced Y shape. The hub domain is connected to the Y domain by a bent arm. Each Y domain comprises two lobes (a large one and a small one) between which cytochrome c binding sites. Because the resolution of the apoptosome structure was relatively low, two controversial models for apoptosome assembly were proposed. One model suggests NOD domains form the central hub and the CARD domains form a freer ring at the top of the NOD region. Another model proposes that Apaf-1 is organized in an extended fashion such that both the N-terminal CARD and the nucleotide binding region form the central hub of the apoptosome, whereas the 13 WD-40 repeats
constitute the two lobes. The large lobe is formed by seven repeats and the small lobe is formed by six repeats. Each caspase- 9 molecule binds a CARD domain at the central hub, forming a dome shaped structure. This controversy has been resolved by a recent high resolution structure of the human apoptosome-procaspase-9 CARD complex . This structure clearly demonstrated that only the NOD regions form the central hub of the apoptosome (see pictures), while CARD is flexibly linked to the platform of apoptosome and becomes disordered in the ground state apoptosome . Once apoptosome binds to procaspase-9, the Apaf-1 CARDs and procaspase-9 CARDs form a flexible disk-like structure sitting above the platform . The number of WD-40 repeats
has also been proved to be 15 instead of 13, and it is composed of a 7-bladed beta-propeller and a 8-bladed beta-propeller.
Evidence from Wang and colleagues indicates that the stoichiometric ratio of procaspase-9 to Apaf-1 within the complex is approximately 1:1 . This was further proved by quantitative mass spectrometry analysis . The stoichiometry of cytochrome c to Apaf-1 within the complex is proved to be 1:1 . There are some debates about whether stable incorporation of cytochrome c into the apoptosome is required following oligomerization, but recent structural data favor the idea that cytochrome c stabilizes the oligomeric human apoptosome. However, cytochrome c may be not required for the assembly of apoptosome in non-mammalian species, such as worms and fruit flies . In addition, several other molecules, most notably caspase-3, have been reported to co-purify with the apoptosome and caspase-3 has been proved to be able to bind the apoptosome-procaspase-9 complex .
Apaf-1 forms the backbone of the apoptosome. It has three distinct regions: the N-terminal caspase-recruitment domain (CARD, residues 1–90), a central nucleotide-binding and oligomerization region (NB-ARC/NOD, 128–586) and a C-terminal WD40 region (613–1248) making up a protein about 140 KDa.
A short linker and nucleotide binding a/b domains (NBD) that contain conserved Walker boxes A (p-loop 155-161) and B (239-243) follow the N-terminal CARD domain. The Walker boxes A/B are critical for dATP/ATP and Mg2+ binding. Following the NBD is a small helical domain (HD1), a second linker and a conserved winged helix domain (WHD). The NOD region comprises NBD, HD1 and WHD, creating an ATPase domain that is part of the AAA+ family of ATPases. There is a super helical domain (HD2) present in the junction between the NOD and the WD-40 repeats. The WD40 repeats are in groups of eight and seven with linkers connecting them.
. In animals, apoptosis can be catalyzed
in one of two ways; the extrinsic pathway involves biding of extracellular ligands to transmembrane receptors, while the intrinsic pathway
take place in the mitochondria. This intrinsic pathway involves the release of cytochrome C
from the mitochondria and subsequent binding to the cytosolic protein Apaf-
. Cytochrome C release is thus necessary for the initiation of apoptosome action; this release is regulated in a number of ways, most importantly by detection of calcium
ion
levels.
(PTP) when the mitochondria receives a death inducing signal, and releases intermembrane space proteins
(12). The PTP is composed of the voltage-dependent anion channel
(VDAC), the inner membrane
protein adenine nucleotide translocator
(AdNT) and the matrix protein cyclophilin D
(CyD) (12). This pore causes the mitochondria to swell and the outer mitochondrial membrane
to rupture (Diamond & McCabe, 2007). With this change in permeability
, proteins
such as cytochrome C are released into the cytosol
(12). This change likely causes the mitochondrial permeability transition
(MPT), where the mitochondrial transmembrane potential
collapses, and ATP production ceases (12). The inhibition of this method by the pharmaceutical agent cyclosporine A
(CsA), lead to the discovery of the second pathway (13). The second method of cytochrome c release is independent of the PTP and involves only the VDAC. Members of the Bcl-2 family of pro-apoptotic proteins
can induce the opening of the VDAC (12). This will cause the same release of intermembrane space proteins, including cytochrome C, and the subsequent MPT to occur (12).
form; it is thought that the WD-40 domain
remain folded back onto the protein, keeping Apaf-1 in an auto inhibited
state. In addition, several areas are so tightly wound that the protein is unable to bind to anything else. It has been determined through mass spectrometry
that in the autoinhibited, or “locked” state, ADP is bound to the ATPase domain of Apaf-1. In this state, this protein is singular, and incapable of activating any caspases
.
and NB-ARC domains remain in autoinhibited state. The CARD domain will only be released from this lock when Apaf-1 is bound to (d) ATP/ATP; when ATP binds, the CARD domain will then be allowed to bind to Caspase-9. When ADP is in the ATPase domain, oligomerization is inhibited. Thus, the binding of ATP also allows for the oligomerization of Apaf-1 into the heptagonal
structure necessary for downstream caspase activation. Mutations in the ATPase
domain render the protein inactive; however, the method of controlling this ADP-ATP exchange is unclear.
Oligomerization can thus only occur in the presence of 7 cytochrome C molecules, 7 Apaf-1 proteins and sufficient (d) ATP/ATP . The ATPase domain belongs to the AAA+ family of ATPases; this family is known for its ability to link to other ATPase domains and form hexa- or heptamers. The apoptosome is then considered active when there are seven Apaf-1 molecules arranged in a wheel structure, oriented such that the NB-ARC domains rest in the centre.
This functional apoptosome then can provide a platform activation of caspase 9.
Caspase 9 exists as a zymogen
in the cytosol and is thought to be found at 20 nM in cells. Though it is known that the zymogen does not need to be cleaved in order to become active, the activity of procaspase-9 may increase significantly once cleaved . The first hypothesis is that the apoptosome provides a location for the dimerization
of two caspase 9 molecules before cleavage; this hypothesis was favoured by Reidl & Salvasen in 2007. The second is that cleavage takes place while caspase 9 is still in its monomeric form. In each case, caspase 9 activation leads to the activation of a full caspase cascade and subsequent cell death. It has been suggested that the evolutionary reason for the multimeric protein complex activating the caspase cascade is to ensure trace amounts of cytochrome c do not accidentally cause apoptosis.
functions as a tumor suppressor
that is involved in preventing cancer and occurs naturally in apoptotic pathways. P53 causes cells to enter apoptosis and disrupt further cell division therefore preventing that cell from becoming cancerous (16). In the majority of cancers it is the p53 pathway that has become mutated resulting in lack of ability to terminate dysfunctional cells. P53 function can also be responsible for a limited life span where mutations of the p53 gene causes expression of dominant-negative forms producing long lived animals. For example in an experiment using C. elegans
, the increased life span of p53 mutants was found to depend on increased autophagy. In another experiment using Drosophilia the p53 mutation had both positive and negative effects on the adult life span, which concluded a link between sexual differentiation, PCD and aging. Determining how p53 are affecting life span will be an important area for future research.
In general the up regulation of anti-apoptotic proteins leads to the prevention of apoptosis which can be solved by inhibitors and the down regulation of anti-apoptotic proteins leads to the induction of apoptosis which is reversed by activators that are able to bind and modify their activity. An important target molecule in apoptosis based therapies is Bcl-2
for drug design. Bcl-2 was the first oncogene found to cause cancer-inhibiting apoptosis. It is over expressed in tumors and is resistant to chemotherapy. Scientists have found that binding depressors to Bcl-2 anti-apoptotic proteins inhibits them and leaves direct activators free to interact with Bax
and Bak
.
Another targeted molecule for cancer therapy involves the caspase family and their regulators. The inhibition of caspase activity blocks cell death in human disease including neurodegenerative disorders, stroke, heart attack and liver injury. Therefore, caspase inhibitors are a promising pharmacological tool providing treatments for stroke and other human diseases. There are several caspase inhibitors that are currently in the preclinical stage that have shown promising evidence of reversing effects of some neurodegenerative diseases. In a recent study researchers developed a reversible caspase-3 inhibitor called M-826 and tested it in a mouse model where it blocked brain tissue damage. Furthermore, it was tested on a mouse with Huntington’s disease and the inhibitor prevented striated neuron death revealing promising effects for further study of this caspase inhibitor.
The discovery of apoptosome inhibitors will provide a new therapeutical tool for the treatment of apoptosis mediated disease. Of particular importance are those new compounds able to inhibit apoptosome stability and activity, by acting on intracellular protein–protein interactions without altering the transcriptional levels of the apoptosome components.Recent structural studies of apoptosome may provide valuable tools for designing apoptosome-based therapies.
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
structure formed in the process of apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
. Its formation is triggered by the release of cytochrome c
Cytochrome c
The Cytochrome complex, or cyt c is a small heme protein found loosely associated with the inner membrane of the mitochondrion. It belongs to the cytochrome c family of proteins. Cytochrome c is a highly soluble protein, unlike other cytochromes, with a solubility of about 100 g/L and is an...
from the mitochondria in response to an internal (intrinsic) or external (extrinsic) cell death stimulus. Stimuli can vary from DNA damage and viral infection to developmental cues such as those leading to the degradation of a tadpole's
Tadpole
A tadpole or polliwog is the wholly aquatic larval stage in the life cycle of an amphibian, particularly that of a frog or toad.- Appellation :...
tail.
In mammalian cells, once cytochrome c
Cytochrome c
The Cytochrome complex, or cyt c is a small heme protein found loosely associated with the inner membrane of the mitochondrion. It belongs to the cytochrome c family of proteins. Cytochrome c is a highly soluble protein, unlike other cytochromes, with a solubility of about 100 g/L and is an...
is released, it binds to the cytosolic protein Apaf-1 to facilitate the formation of apoptosome. An early biochemical study suggests a two-to-one ratio of cytochrome c to apaf-1 for apoptosome formation. However, recent structural studies suggest the cytochrome c to apaf-1 ratio is one-to-one. It has also been shown that the nucleotide dATP
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
as third component binds to apaf-1, however its exact role is still debated. The mammalian apoptosome had never been crystallized, but a human APAF-1/cytochrome-c apoptosome has been imaged at lower (2 nm) resolution by cryogenic transmission electron microscopy
Transmission electron microscopy
Transmission electron microscopy is a microscopy technique whereby a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through...
10 years ago, revealing a wheel-like particle with 7-fold symmetry. Recently, a medium resolution (9.5 Ångström) structure of human apoptosome was also solved by cryo-electron microscopy
Cryo-electron microscopy
Cryo-electron microscopy , or electron cryomicroscopy, is a form of transmission electron microscopy where the sample is studied at cryogenic temperatures...
, which allows unambiguous inference for positions of all the APAF-1 domains (CARD, NBARC and WD40) and cytochrome c. Once formed, the apoptosome can then recruit and activate the inactive pro-caspase-9
Caspase
Caspases, or cysteine-aspartic proteases or cysteine-dependent aspartate-directed proteases are a family of cysteine proteases that play essential roles in apoptosis , necrosis, and inflammation....
. Once activated, this initiator caspase can then activate effector caspases and trigger a cascade of events leading to apoptosis.
History
The term Apoptosome was first introduced in Yoshihide Tsujimoto's 1998 paper "Role of Bcl-2 family proteinsBcl-2
Bcl-2 is the founding member of the Bcl-2 family of apoptosis regulator proteins encoded by the BCL2 gene. Bcl-2 derives its name from B-cell lymphoma 2, as it is the second member of a range of proteins initially described in chromosomal translocations involving chromosomes 14 and 18 in...
in apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
: apoptosomes or mitochondria?". However, the Apoptosome was known before this time as a ternary complex
Ternary complex
A Ternary complex refers to a protein complex containing three different molecules which are bound together. In structural biology ternary complex can be used to describe a crystal containing a protein with two small molecules bound, for example cofactor and substrate; or a complex formed between...
. This complex involved caspase-9
Caspase-9
Caspase-9 is an initiator caspase, encoded by the CASP9 gene.CASP9 orthologs have been identified in all mammals for which complete genome data are available. Unique orthologs are also present in lizards, lissamphibians, and teleosts....
and Bcl-XL
Bcl-xL
B-cell lymphoma-extra large is a transmembrane molecule in the mitochondria. It is involved in the signal transduction pathway of the FAS-L. It is one of several anti-apoptotic proteins which are members of the Bcl-2 family of proteins. It has been implicated in the survival of cancer cells. Other...
which each bound a specific Apaf-1
APAF1
Apoptotic protease activating factor 1, also known as APAF1, is a human homolog of C. elegans CED-4 gene. APAF-1 and CED-4 homologs have been found in all currently sequenced animal genomes.-Function:...
domain. The formation of this complex was then believed to play a regulatory role in mammalian
Mammal
Mammals are members of a class of air-breathing vertebrate animals characterised by the possession of endothermy, hair, three middle ear bones, and mammary glands functional in mothers with young...
cell death. In December of the same year, a further article was released in The Journal of Biological Chemistry stating that Apaf-1 is the regulator of apoptosis, through activation of procaspase-9.
The criteria for an apoptosome were laid out in 1999. Firstly, it must be a large complex (greater than 1.3 million Daltons). Secondly its formation requires the hydrolysis
Hydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...
of a high energy bond of ATP
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
or dATP. And lastly it must activate procaspase-9 in its functional form. The formation of this complex is the point of no return, and apoptosis will occur. The stable APAF-1 and cytochrome mutimeric complex
Oligomer
In chemistry, an oligomer is a molecule that consists of a few monomer units , in contrast to a polymer that, at least in principle, consists of an unlimited number of monomers. Dimers, trimers, and tetramers are oligomers. Many oils are oligomeric, such as liquid paraffin...
fit this description, and is now called the apoptosome.
The apoptosome was thought to be a mutimeric complex
Oligomer
In chemistry, an oligomer is a molecule that consists of a few monomer units , in contrast to a polymer that, at least in principle, consists of an unlimited number of monomers. Dimers, trimers, and tetramers are oligomers. Many oils are oligomeric, such as liquid paraffin...
for two reasons. Firstly, to bring multiple procaspase-9 molecules close together for cleavage. And secondly, to raise the threshold for apoptosis, therefore nonspecific leakage of cytochrome c
Cytochrome c
The Cytochrome complex, or cyt c is a small heme protein found loosely associated with the inner membrane of the mitochondrion. It belongs to the cytochrome c family of proteins. Cytochrome c is a highly soluble protein, unlike other cytochromes, with a solubility of about 100 g/L and is an...
would not result in apoptosis.
Once the apoptosome was established as the procaspase-9 activator, mutations within this pathway became an important research area. Some examples include human leukemia
Leukemia
Leukemia or leukaemia is a type of cancer of the blood or bone marrow characterized by an abnormal increase of immature white blood cells called "blasts". Leukemia is a broad term covering a spectrum of diseases...
cells, ovarian cancer
Ovarian cancer
Ovarian cancer is a cancerous growth arising from the ovary. Symptoms are frequently very subtle early on and may include: bloating, pelvic pain, difficulty eating and frequent urination, and are easily confused with other illnesses....
and viral infections. Current research areas for this pathway will be discussed in further detail. There are hidden routes for cell death as well, which are independent of APAF-1 and therefore the apoptosome. These routes are also independent of caspase-3 and 9. These hidden pathways for apoptosis are slower, but may prove useful with further research.
Structure
The apoptosome is a multimolecular holoenzymeEnzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
complex assembled around the adaptor protein Apaf1 (apoptotic protease activating factor 1)
APAF1
Apoptotic protease activating factor 1, also known as APAF1, is a human homolog of C. elegans CED-4 gene. APAF-1 and CED-4 homologs have been found in all currently sequenced animal genomes.-Function:...
upon mitochondria-mediated apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
which must be stimulated by some type of stress signal T formation of the apoptosome requires the presence of ATP
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
/dATP and cytochrome c
Cytochrome c
The Cytochrome complex, or cyt c is a small heme protein found loosely associated with the inner membrane of the mitochondrion. It belongs to the cytochrome c family of proteins. Cytochrome c is a highly soluble protein, unlike other cytochromes, with a solubility of about 100 g/L and is an...
in the cytosol
Cytosol
The cytosol or intracellular fluid is the liquid found inside cells, that is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondrion into compartments....
.
A stress stimulus can trigger the release of cytochrome c into the cytoplasm which will then bind to the C-terminus of Apaf-1 within a region containing multiple WD-40 repeats. The oligomerization
Oligomer
In chemistry, an oligomer is a molecule that consists of a few monomer units , in contrast to a polymer that, at least in principle, consists of an unlimited number of monomers. Dimers, trimers, and tetramers are oligomers. Many oils are oligomeric, such as liquid paraffin...
of Apaf-1 appears to be accompanied by synchronized recruitment of procaspase-9
Caspase-9
Caspase-9 is an initiator caspase, encoded by the CASP9 gene.CASP9 orthologs have been identified in all mammals for which complete genome data are available. Unique orthologs are also present in lizards, lissamphibians, and teleosts....
to the CARD motif
CARD domain
Caspase recruitment domains, or Caspase activation and recruitment domains , are interaction motifs found in a wide array of proteins, typically those involved in processes relating to inflammation and apoptosis. These domains mediate the formation of larger protein complexes via direct...
at the Apaf-1 N-terminus. The apoptosome triggers the activation of caspases
Caspase
Caspases, or cysteine-aspartic proteases or cysteine-dependent aspartate-directed proteases are a family of cysteine proteases that play essential roles in apoptosis , necrosis, and inflammation....
in the intrinsic pathway
Intrinsic apoptosis
Apoptosis is a programmed form of cell death involving the degradation of cellular constituents by a group of cysteine proteases called caspases. The caspases can be activated through either the intrinsic or extrinsic apoptotic pathways.The intrinsic apoptotic pathway is characterized by...
of apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
.
The wheel-shaped heptameric
Heptamer
-See also:* Heptameria, a genus of fungi in the class Dothideomycetes* Heptaméron, a collection of 72 short stories written in French by Marguerite of Navarre and published in 1558* Hepta, a prefix* -mer, an affix...
complex with sevenfold symmetry structure of the apoptosome was first revealed at 27 Å resolution by electron cryomicroscopy
Cryo-electron microscopy
Cryo-electron microscopy , or electron cryomicroscopy, is a form of transmission electron microscopy where the sample is studied at cryogenic temperatures...
techniques and has a calculated mass of about 1 MDa (Acehan et al. 2002).This wheel-like particle has seven spokes and a central hub. The distal region of the spoke has a pronounced Y shape. The hub domain is connected to the Y domain by a bent arm. Each Y domain comprises two lobes (a large one and a small one) between which cytochrome c binding sites. Because the resolution of the apoptosome structure was relatively low, two controversial models for apoptosome assembly were proposed. One model suggests NOD domains form the central hub and the CARD domains form a freer ring at the top of the NOD region. Another model proposes that Apaf-1 is organized in an extended fashion such that both the N-terminal CARD and the nucleotide binding region form the central hub of the apoptosome, whereas the 13 WD-40 repeats
WD40 repeat
The WD40 repeat is a short structural motif of approximately 40 amino acids , often terminating in a tryptophan-aspartic acid dipeptide...
constitute the two lobes. The large lobe is formed by seven repeats and the small lobe is formed by six repeats. Each caspase- 9 molecule binds a CARD domain at the central hub, forming a dome shaped structure. This controversy has been resolved by a recent high resolution structure of the human apoptosome-procaspase-9 CARD complex . This structure clearly demonstrated that only the NOD regions form the central hub of the apoptosome (see pictures), while CARD is flexibly linked to the platform of apoptosome and becomes disordered in the ground state apoptosome . Once apoptosome binds to procaspase-9, the Apaf-1 CARDs and procaspase-9 CARDs form a flexible disk-like structure sitting above the platform . The number of WD-40 repeats
WD40 repeat
The WD40 repeat is a short structural motif of approximately 40 amino acids , often terminating in a tryptophan-aspartic acid dipeptide...
has also been proved to be 15 instead of 13, and it is composed of a 7-bladed beta-propeller and a 8-bladed beta-propeller.
Evidence from Wang and colleagues indicates that the stoichiometric ratio of procaspase-9 to Apaf-1 within the complex is approximately 1:1 . This was further proved by quantitative mass spectrometry analysis . The stoichiometry of cytochrome c to Apaf-1 within the complex is proved to be 1:1 . There are some debates about whether stable incorporation of cytochrome c into the apoptosome is required following oligomerization, but recent structural data favor the idea that cytochrome c stabilizes the oligomeric human apoptosome. However, cytochrome c may be not required for the assembly of apoptosome in non-mammalian species, such as worms and fruit flies . In addition, several other molecules, most notably caspase-3, have been reported to co-purify with the apoptosome and caspase-3 has been proved to be able to bind the apoptosome-procaspase-9 complex .
Apaf-1 forms the backbone of the apoptosome. It has three distinct regions: the N-terminal caspase-recruitment domain (CARD, residues 1–90), a central nucleotide-binding and oligomerization region (NB-ARC/NOD, 128–586) and a C-terminal WD40 region (613–1248) making up a protein about 140 KDa.
- The CARD domain of Apaf-1APAF1Apoptotic protease activating factor 1, also known as APAF1, is a human homolog of C. elegans CED-4 gene. APAF-1 and CED-4 homologs have been found in all currently sequenced animal genomes.-Function:...
interacts with procaspase-9 and involved with recruitment within the apoptosome. - The NB-ARC/NOD region exhibits significant sequence similarity to the C. elegans Ced-4 protein.
- The C-terminal WD40 region of Apaf1 contains 15 WD-40 repeatsWD40 repeatThe WD40 repeat is a short structural motif of approximately 40 amino acids , often terminating in a tryptophan-aspartic acid dipeptide...
structured into two b-propeller-shaped domains. WD-40 repeats are sequences around 40 amino acids long which end in Trp-Asp and are typically involved in protein–protein interaction.
A short linker and nucleotide binding a/b domains (NBD) that contain conserved Walker boxes A (p-loop 155-161) and B (239-243) follow the N-terminal CARD domain. The Walker boxes A/B are critical for dATP/ATP and Mg2+ binding. Following the NBD is a small helical domain (HD1), a second linker and a conserved winged helix domain (WHD). The NOD region comprises NBD, HD1 and WHD, creating an ATPase domain that is part of the AAA+ family of ATPases. There is a super helical domain (HD2) present in the junction between the NOD and the WD-40 repeats. The WD40 repeats are in groups of eight and seven with linkers connecting them.
Initiation
The initiation of apoptosome action corresponds with the first steps in the programmed cell death (PCD) pathwayApoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
. In animals, apoptosis can be catalyzed
Catalysis
Catalysis is the change in rate of a chemical reaction due to the participation of a substance called a catalyst. Unlike other reagents that participate in the chemical reaction, a catalyst is not consumed by the reaction itself. A catalyst may participate in multiple chemical transformations....
in one of two ways; the extrinsic pathway involves biding of extracellular ligands to transmembrane receptors, while the intrinsic pathway
Intrinsic apoptosis
Apoptosis is a programmed form of cell death involving the degradation of cellular constituents by a group of cysteine proteases called caspases. The caspases can be activated through either the intrinsic or extrinsic apoptotic pathways.The intrinsic apoptotic pathway is characterized by...
take place in the mitochondria. This intrinsic pathway involves the release of cytochrome C
Cytochrome c
The Cytochrome complex, or cyt c is a small heme protein found loosely associated with the inner membrane of the mitochondrion. It belongs to the cytochrome c family of proteins. Cytochrome c is a highly soluble protein, unlike other cytochromes, with a solubility of about 100 g/L and is an...
from the mitochondria and subsequent binding to the cytosolic protein Apaf-
APAF1
Apoptotic protease activating factor 1, also known as APAF1, is a human homolog of C. elegans CED-4 gene. APAF-1 and CED-4 homologs have been found in all currently sequenced animal genomes.-Function:...
. Cytochrome C release is thus necessary for the initiation of apoptosome action; this release is regulated in a number of ways, most importantly by detection of calcium
Calcium
Calcium is the chemical element with the symbol Ca and atomic number 20. It has an atomic mass of 40.078 amu. Calcium is a soft gray alkaline earth metal, and is the fifth-most-abundant element by mass in the Earth's crust...
ion
Ion
An ion is an atom or molecule in which the total number of electrons is not equal to the total number of protons, giving it a net positive or negative electrical charge. The name was given by physicist Michael Faraday for the substances that allow a current to pass between electrodes in a...
levels.
Cytochrome C Release
Cytochrome C release is proposed to take place in one of two ways. Firstly, the permeability transition poreMitochondrial permeability transition pore
The Mitochondrial Permeability Transition, or MPT, is defined as an increase in the permeability of the mitochondrial membranes to molecules of less than 1500 Daltons in molecular weight. MPT results from the opening of a mitochondrial permeability transition pore, also known as the MPT pore or MPTP...
(PTP) when the mitochondria receives a death inducing signal, and releases intermembrane space proteins
Intermembrane space
The intermembrane space also known as IMS is the region between the inner membrane and the outer membrane of a mitochondrion or a chloroplast. The main function of the intermembrane space is oxidative phosphorylation....
(12). The PTP is composed of the voltage-dependent anion channel
Voltage-dependent anion channel
Voltage-dependent anion channels are a class of porin ion channel located on the outer mitochondrial membrane.This major protein of the outer mitochondrial membrane of eukaryotes. It forms a voltage-dependent anion-selective channel that behaves as a general diffusion pore for small hydrophilic...
(VDAC), the inner membrane
Inner membrane
The inner membrane is the biological membrane of an organelle or Gram-negative bacteria that is within an outer membrane....
protein adenine nucleotide translocator
Adenine nucleotide translocator
Adenine nucleotide translocator also known as the ADP/ATP translocator is a mitochondrial protein.- Function :ANT has long been thought to function asymmetrically as a homodimer of subunits in the inner mitochondrial membrane. The dimer was thought to be a gated pore through which ADP and ATP were...
(AdNT) and the matrix protein cyclophilin D
Cyclophilin
Cyclophilins are a family of proteins from vertebrates and other organisms that bind to cyclosporine, an immunosuppressant which is usually used to suppress rejection after internal organ transplants...
(CyD) (12). This pore causes the mitochondria to swell and the outer mitochondrial membrane
Outer mitochondrial membrane
thumb|300px|Mitochondria structure :1) [[Inner membrane]]2) Outer membrane3) [[Crista]]4) [[Matrix |Matrix]]The outer mitochondrial membrane, which encloses the entire organelle, has a protein-to-phospholipid ratio similar to the eukaryotic plasma membrane...
to rupture (Diamond & McCabe, 2007). With this change in permeability
Semipermeable membrane
A semipermeable membrane, also termed a selectively permeable membrane, a partially permeable membrane or a differentially permeable membrane, is a membrane that will allow certain molecules or ions to pass through it by diffusion and occasionally specialized "facilitated diffusion".The rate of...
, proteins
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
such as cytochrome C are released into the cytosol
Cytosol
The cytosol or intracellular fluid is the liquid found inside cells, that is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondrion into compartments....
(12). This change likely causes the mitochondrial permeability transition
Mitochondrial permeability transition pore
The Mitochondrial Permeability Transition, or MPT, is defined as an increase in the permeability of the mitochondrial membranes to molecules of less than 1500 Daltons in molecular weight. MPT results from the opening of a mitochondrial permeability transition pore, also known as the MPT pore or MPTP...
(MPT), where the mitochondrial transmembrane potential
Membrane potential
Membrane potential is the difference in electrical potential between the interior and exterior of a biological cell. All animal cells are surrounded by a plasma membrane composed of a lipid bilayer with a variety of types of proteins embedded in it...
collapses, and ATP production ceases (12). The inhibition of this method by the pharmaceutical agent cyclosporine A
Ciclosporin
Ciclosporin , cyclosporine , cyclosporin , or cyclosporin A is an immunosuppressant drug widely used in post-allogeneic organ transplant to reduce the activity of the immune system, and therefore the risk of organ rejection...
(CsA), lead to the discovery of the second pathway (13). The second method of cytochrome c release is independent of the PTP and involves only the VDAC. Members of the Bcl-2 family of pro-apoptotic proteins
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
can induce the opening of the VDAC (12). This will cause the same release of intermembrane space proteins, including cytochrome C, and the subsequent MPT to occur (12).
a. Absence of Cytochrome C
In the absence of cytochrome C, Apaf-1 exists in its monomericMonomer
A monomer is an atom or a small molecule that may bind chemically to other monomers to form a polymer; the term "monomeric protein" may also be used to describe one of the proteins making up a multiprotein complex...
form; it is thought that the WD-40 domain
WD40 repeat
The WD40 repeat is a short structural motif of approximately 40 amino acids , often terminating in a tryptophan-aspartic acid dipeptide...
remain folded back onto the protein, keeping Apaf-1 in an auto inhibited
Enzyme inhibitor
An enzyme inhibitor is a molecule that binds to enzymes and decreases their activity. Since blocking an enzyme's activity can kill a pathogen or correct a metabolic imbalance, many drugs are enzyme inhibitors. They are also used as herbicides and pesticides...
state. In addition, several areas are so tightly wound that the protein is unable to bind to anything else. It has been determined through mass spectrometry
Mass spectrometry
Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...
that in the autoinhibited, or “locked” state, ADP is bound to the ATPase domain of Apaf-1. In this state, this protein is singular, and incapable of activating any caspases
Caspase
Caspases, or cysteine-aspartic proteases or cysteine-dependent aspartate-directed proteases are a family of cysteine proteases that play essential roles in apoptosis , necrosis, and inflammation....
.
b. Presence of Cytochrome C
Cytochrome C binds to the WD-40 domain of Apaf-1. This allows for the “lock” is released, meaning this domain is no longer autoinhibited. However, the CARDCARD domain
Caspase recruitment domains, or Caspase activation and recruitment domains , are interaction motifs found in a wide array of proteins, typically those involved in processes relating to inflammation and apoptosis. These domains mediate the formation of larger protein complexes via direct...
and NB-ARC domains remain in autoinhibited state. The CARD domain will only be released from this lock when Apaf-1 is bound to (d) ATP/ATP; when ATP binds, the CARD domain will then be allowed to bind to Caspase-9. When ADP is in the ATPase domain, oligomerization is inhibited. Thus, the binding of ATP also allows for the oligomerization of Apaf-1 into the heptagonal
Heptameter
Heptameter is one or more lines of verse containing seven metrical feet .An example from Lord Byron's Youth and Age:...
structure necessary for downstream caspase activation. Mutations in the ATPase
ATPase
ATPases are a class of enzymes that catalyze the decomposition of adenosine triphosphate into adenosine diphosphate and a free phosphate ion. This dephosphorylation reaction releases energy, which the enzyme harnesses to drive other chemical reactions that would not otherwise occur...
domain render the protein inactive; however, the method of controlling this ADP-ATP exchange is unclear.
Oligomerization can thus only occur in the presence of 7 cytochrome C molecules, 7 Apaf-1 proteins and sufficient (d) ATP/ATP . The ATPase domain belongs to the AAA+ family of ATPases; this family is known for its ability to link to other ATPase domains and form hexa- or heptamers. The apoptosome is then considered active when there are seven Apaf-1 molecules arranged in a wheel structure, oriented such that the NB-ARC domains rest in the centre.
Active Apoptosome Action
This functional apoptosome then can provide a platform activation of caspase 9.
Caspase 9 exists as a zymogen
Zymogen
A zymogen is an inactive enzyme precursor. A zymogen requires a biochemical change for it to become an active enzyme. The biochemical change usually occurs in a lysosome where a specific part of the precursor enzyme is cleaved in order to activate it...
in the cytosol and is thought to be found at 20 nM in cells. Though it is known that the zymogen does not need to be cleaved in order to become active, the activity of procaspase-9 may increase significantly once cleaved . The first hypothesis is that the apoptosome provides a location for the dimerization
Protein dimer
In biochemistry, a dimer is a macromolecular complex formed by two, usually non-covalently bound, macromolecules like proteins or nucleic acids...
of two caspase 9 molecules before cleavage; this hypothesis was favoured by Reidl & Salvasen in 2007. The second is that cleavage takes place while caspase 9 is still in its monomeric form. In each case, caspase 9 activation leads to the activation of a full caspase cascade and subsequent cell death. It has been suggested that the evolutionary reason for the multimeric protein complex activating the caspase cascade is to ensure trace amounts of cytochrome c do not accidentally cause apoptosis.
What happens when mutations occur?
While apoptosis is required for natural body function, mutations of the apoptosome pathway cause catastrophic effects and changes in the body. Mutations of the cell pathway can either promote cell death or disallow cell death creating a huge amount of disease in the body. Mutated apoptosis pathways causing disease are plentiful and have a wide range from cancer, due to lack of apoptosome activity, Alzheimer’s disease due to too much apoptosome activity, and many other neurodegenerative diseases such as Parkinson’s disease and Huntington’s disease. Neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s are all age-related diseases and involve increased apoptosis where cells die that are still able to function or that contribute to support function of tissue. Apaf-1-ALT is an Apaf-1 mutant found in prostate cancer, which does not have residues 339-1248. Recent structural studies of apoptosome prove that Apaf-1-ALT cannot form apoptosome as it misses key structural components for assembly.Repression of Apoptosis causing cancer
Genetic and biochemical abnormalities within a cell normally trigger programmed cell death to rid the body of irregular cell function and development; however, cancer cells have acquired mutations that allow them to repress apoptosis and survive. Chemotherapies like ionizing radiation have been developed to activate these repressed PCD pathways by hyper-stimulation to promote normal PCD.P53 mutations in Apoptosis
P53P53
p53 , is a tumor suppressor protein that in humans is encoded by the TP53 gene. p53 is crucial in multicellular organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor that is involved in preventing cancer...
functions as a tumor suppressor
Suppressor
A suppressor, sound suppressor, sound moderator, or silencer, is a device attached to or part of the barrel of a firearm which reduces the amount of noise and flash generated by firing the weapon....
that is involved in preventing cancer and occurs naturally in apoptotic pathways. P53 causes cells to enter apoptosis and disrupt further cell division therefore preventing that cell from becoming cancerous (16). In the majority of cancers it is the p53 pathway that has become mutated resulting in lack of ability to terminate dysfunctional cells. P53 function can also be responsible for a limited life span where mutations of the p53 gene causes expression of dominant-negative forms producing long lived animals. For example in an experiment using C. elegans
Caenorhabditis elegans
Caenorhabditis elegans is a free-living, transparent nematode , about 1 mm in length, which lives in temperate soil environments. Research into the molecular and developmental biology of C. elegans was begun in 1974 by Sydney Brenner and it has since been used extensively as a model...
, the increased life span of p53 mutants was found to depend on increased autophagy. In another experiment using Drosophilia the p53 mutation had both positive and negative effects on the adult life span, which concluded a link between sexual differentiation, PCD and aging. Determining how p53 are affecting life span will be an important area for future research.
Targeting the Apoptosome for Cancer therapy
The inhibition of apoptosis is one of the key features of cancer so finding ways to manipulate and overcome this inhibition to form the apoptosome and activate caspases are important in the development of new cancer treatments. The ability to directly cause apoptosome activation is valuable in cancer therapies because the infected cancerous genes are unable to be destroyed causing a continuation of the cancer to form. By activating the apoptosome by an outside stimulus apoptosis can occur and get rid of the mutated cells. Numerous approaches to achieve this are currently being pursued including recombinant biomolecules, antisense strategies, gene therapy and classic organic combinatorial chemistry to target specific apoptotic regulators in the approach to correct excessive or deficient cell death in human diseases.In general the up regulation of anti-apoptotic proteins leads to the prevention of apoptosis which can be solved by inhibitors and the down regulation of anti-apoptotic proteins leads to the induction of apoptosis which is reversed by activators that are able to bind and modify their activity. An important target molecule in apoptosis based therapies is Bcl-2
Bcl-2
Bcl-2 is the founding member of the Bcl-2 family of apoptosis regulator proteins encoded by the BCL2 gene. Bcl-2 derives its name from B-cell lymphoma 2, as it is the second member of a range of proteins initially described in chromosomal translocations involving chromosomes 14 and 18 in...
for drug design. Bcl-2 was the first oncogene found to cause cancer-inhibiting apoptosis. It is over expressed in tumors and is resistant to chemotherapy. Scientists have found that binding depressors to Bcl-2 anti-apoptotic proteins inhibits them and leaves direct activators free to interact with Bax
Bcl-2-associated X protein
The Bcl-2–associated X protein, or Bax is a protein of the Bcl-2 gene family. It promotes apoptosis by competing with Bcl-2 proper.The BAX gene was the first identified pro-apoptotic member of the Bcl-2 protein family....
and Bak
Bcl-2 homologous antagonist killer
Bcl-2 homologous antagonist/killer is a protein that in humans is encoded by the BAK1 gene. BAK1 orthologs have been identified in most mammals for which complete genome data are available....
.
Another targeted molecule for cancer therapy involves the caspase family and their regulators. The inhibition of caspase activity blocks cell death in human disease including neurodegenerative disorders, stroke, heart attack and liver injury. Therefore, caspase inhibitors are a promising pharmacological tool providing treatments for stroke and other human diseases. There are several caspase inhibitors that are currently in the preclinical stage that have shown promising evidence of reversing effects of some neurodegenerative diseases. In a recent study researchers developed a reversible caspase-3 inhibitor called M-826 and tested it in a mouse model where it blocked brain tissue damage. Furthermore, it was tested on a mouse with Huntington’s disease and the inhibitor prevented striated neuron death revealing promising effects for further study of this caspase inhibitor.
Apoptosome complex has revealed new potential targets for molecular therapy
The Apaf1/caspase-9 apoptosome formation is a crucial event in the apoptotic cascade. The identification of new potential drugs that prevent or stabilize the formation of active apoptosome complex is the ideal strategy for the treatment of disease characterized by excessive or insufficient apoptosis. Recently taurine has been found to prevent ischemia-induced apoptosis in cardiomyocytes through its ability to inhibit Apaf1/caspase-9 apoptosome formation without preventing mitochondrial dysfunction. The possible mechanism by which taurine inhibits the apoptosome formation was identified as being capable of reducing the expression of caspase-9, a fundamental component of apoptosome. However, there are studies that show Aparf1 and caspase-9 have independent roles other than the apoptosome so altering their levels could alter cell function as well. So despite encouraging experimental data several problems remain unsolved and limit the use of experimental drugs in clinical practice.The discovery of apoptosome inhibitors will provide a new therapeutical tool for the treatment of apoptosis mediated disease. Of particular importance are those new compounds able to inhibit apoptosome stability and activity, by acting on intracellular protein–protein interactions without altering the transcriptional levels of the apoptosome components.Recent structural studies of apoptosome may provide valuable tools for designing apoptosome-based therapies.