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APOPTOSIS

THE EVOLUTION OF APOPTOSIS

     Life depends on death.  Living cells ranging from human cells to yeast and even bacteria contain genes which, when activated, cause programmed cell death or apoptosis.  Apoptosis is useful in multicellular organisms to degrade embryonic structures which are used by the opposite gender, neurons which do not form the proper connections, endometrial cells at the site of embryo implantation, in the remodeling of the skeletal system, and in the responses of prostate and breast cells to steroid hormones.  The cells which are killed by killer T cells undergo some steps of apoptosis.   Apoptosis is a defense against cancer and mutations in apoptotic genes can result in cancer cells which are unable to execute programmed cell death (Vaux, 1993). 

        In embryonic development, many cells must die.  In the developing cerebrum of a fetal pig (pictured below), normal development requires that many neurons die.

NEURAL
Neurons whose axons do not reach the proper targets must die.  (Axons extending from the spinal cord to the limb of an embryonic pig are depicted below.)
EMBRYO
Embryonic vertebrates develop a tail.  Many of them subsequently reduce the size of this tail as in pigs (an embryonic pig is depicted below) and humans.
TAIL
Jawed vertebrates develop 3 sets of embryonic kidneys: pronephros, mesonephros, and metanephros.  In tetrapods, only the metanephros is retained and the mesonephros (such as that of the pig depicted below) must degenerate.
MESONEPHROS

      Apoptosis eliminates cells without inducing the inflammatory response which is advantageous since if cells were to die in an uncontrolled fashion, the cellular contents disrupt the surrounding cells.  As a result, apoptosis is distinct from the necrosis which results from phenomena such as abrasion, chemicals, temperature change, anoxia, glucose shortage, and other phenomena.  Many of the genes which animals utilize in apoptosis have homologs in bacteria (Huettenbrenner, 2003; Fraser, 1998).

     Apopotosis may have originally been part of the ancestral immune response, rather than an aspect of the development of complex body plans (Kasahara, 2004).  Vertebrates possess two types of immunoglobulin which are thought to be similar to the ancestral immunoglobulin which gave rise to antibodies and TCR genes, the JAM/CTX and the nectin/poliovirus receptor (PVR) families.  JAM can activate apoptosis through the NF-κB pathway  (Du Pasquier, 2004).

    Programmed cell death occurs in unicellular eukaryotes, even those which lack mitochondria (Chose, 2003).  Some bacteria undergo death in culture in order to limit the spread of bacteriophage (Fraser, 1998). Homologs of apoptotic proteins are known in bacteria, such as homologs of caspases, TIR (Toll-interleukin-1 receptor),  AP-ATPases, HtrA2/Omi homolog (high temperature requirement protein A2), EndoG , and apoptosis-inducing factor (AIF)(Aravind, 2003 ; Chose, 2003).  Omi/HtrA2 is an apoptotic serine protease which is homologous to the bacterial endoprotease HtrA.  In bacteria, it functions in the folding and degradation of proteins.  In eukaryotic cells under normal conditions Omi/HtrA2 is contained in the mitochondria, but during apoptosis, it binds (and thus inactivates) the inhibitor of apoptosis proteins (IAPs).  The homology of mitochondrial HtrA-like proteases to bacterial enzymes supports the endosymbiotic origin of mitochondria (Lorenzo, 2004).  EndoG is a member of the magnesium-dependent nuclease enzymes.  It functions in the replication of mitochondrial DNA under normal conditions and in bacteria, it repairs DNA among other functions.  During apoptosis, it degrades nuclear DNA independent of caspases.  CAD (caspase-activated DNase) also performs this function as part of the caspase pathway (Lorenzo, 2004). Plants react to pathogens with apoptotic defenses utilizing proteins homologous to those used in animal defenses such as caspases, Bax, Ced/APaf, and Toll (del Pozo, 1998). The transport of materials in and out of mitochondria through voltage-dependent anion channels (VDACs, also known as mitochondrial porins) which represent the most common protein on the mitochondrial outer membrane. Not only can ADP and ATP pass through these channels in energy transactions, cytochrome c can pass through during apoptosis (Graham, 2005).

     Wild type yeast will die when exposed to a number of environmental stresses, but this cell death can be prevented in transgenic yeast which express nematode ced-9 proteins, human bcl-2 proteins, or chicken bcl-xl proteins.  Thus it seems that the cellular machinery utilized by anti-apoptotic proteins in higher eukaryotes also exist in unicellular eukaryotes although they seem to lack the signals themselves.  In response to stress, yeast cells condense their chromatin, break their DNA molecules into fragments, and expose phosphatidylserine on their cell membrane.  The same processes occur in apoptotic animal cells.  A number of the proteins which animal cells use to induce apoptosis (Bax, caspases, p53, and CED4/Apaf-1) also kill yeast cells (Chen, 2003; Skulachev, 2002; Maedo, 2004).   Yeast cells undergo programmed cell death in response to high levels of yeast pheromone.  Yeast pheromone not only causes some cells to mate, it causes apoptosis in those cells which have not mated (Skulachev, 2002).  In ciliates, the macronucleus is degraded through chromatin condensation and the DNA is cleaved into fragments when its micronucleus participates in conjugation (Fraser, 1998).  Caspases are required for apoptosis in plants and apoptosis functions in development (Maedo, 2004). Meiotic cells which do not undergo synapsis properly will begin apoptosis. The gene PCH2 functions at this checkpoint in both yeast and animals (Bhalla, 2005).

    Sponges utilize apoptosis in the formation of gemmules and in responses to environmental stresses.  Sponges also use caspases in the rejection of allografts.  These observations indicate that apoptosis evolved early in the history of multicellular animals (Wiens, 2003).  Sponge cells are depicted below.

OVA

Nematodes do not possess a homolog of TNF.  The Drosophila protein Eiger is a member of the TNF superfamily which can cause apoptosis (Moreno, 2002).  Apoptosis in flies and mammals is controlled by homologous genes (Cashio, 2005). Sea urchin embryonic cells can undergo apoptosis, perhaps as a way of eliminating cells which fail to develop properly (Vega, 2004).

     Most of the proteins involved in apoptotic pathways are ubiquitously present in cells and post-translational regulation determines whether their activity.  Nevertheless, there is transcriptional control over some of the processes as well.  For example, without Hoxa13, the loss of apoptosis of cells between developing digits leads to the fusion of these digits.  The signaling of members of the TNF family can induce the transcription of several components of apoptosis.  In frogs, the apoptosis which is required in metamorphosis is under the control of thyroid hormone (Kumar, 2004).  In vertebrates, red blood cells have a limited lifespan.  Those of a frog last an average 900 days, 700 days in turtles, 120 in humans, and 30 in chickens.  Tetraopd erythrocytes share a number of common apoptotic reactions (Brastosin, 2004).  The blood cells of a frog are depicted below.

BLOOD

     AIF seems to be an important protein in mitochondrial reactions under normal conditions.  Mitochondria release AIF from the intermembrane space during apoptosis which proceeds to the nucleus where it causes chromatin condensation and the degradation of the DNA into fragments of about 50 kb.  AIF is in the gene family which contains NADH oxidases, ascorbate reductases, ferredoxin reductases, and nitrite reductases and is particularly closely related to archaeal NADH oxidases (Lorenzo, 2004).  Flies possess Apaf-1 homolog, seven caspases and 2 Bcl-2 proteins in addition to other proteins which have homologs in mammals.  It appears that apoptotic mechanisms are conserved in coelomate animals given that flies possess cytochrome c, HtrA2/Omi, AIF, and EndoG (Igaki, 2004). 

 

BCL-2 FAMILY

     Yeast lack the Bcl-2/ced-9 proteins which regulate apoptosis in all animals (Fraser, 1998; Wiens, 2000).. The Drosophila protein Drob-1 was the first described member of the ced-9/Bcl-2 family (Ogaki, 2000). This gene family is divided into two main groups, the members of one group (the original) promote apoptosis and the members of the other opposes apoptosis.  Nematodes possess two Bcl-2 family members, one of which is pro-apoptotic, the other which is anti-apoptotic.  These Bcl-2 family members function upstream of the caspase enzymes which actually initiate cellular degradation.   

     In C.elegans, 131 of the 1090 cells undergo apoptosis (Lanaye, 2004).   C.elegans uses 3 genes to regulate apoptosis: ced-3 ( a homolog of mammalian caspases), ced-4 (homologous to Apaf-1 in mammals), and ced-9 of the Bcl-2 family.  The three of these form a complex known as the apoptosome.  Ced-9 travels to the mitochondria and Ced-4 to the nuclear membrane (Igaki, 2004).  Apoptotic pathways have been conserved in bilateran animals given that expression of the pro-apoptotic nematode protein CED-4 in the cells of flies causes apoptosis (Kanuka, 1999) and human Bcl-2 can prevent cell death in nematode cells (Vaux, 1993).

     Mitochondria are involved in most, if not all, apoptotic pathways.  The Bcl-2 family members Bax and Bak form clusters in the mitochondrial membrane after Bax travels there following induction by apoptotic signals (Frank, 2003).

 

ANKYRIN

     The ankyrin domain is a stable domain which possesses great versatility in its protein binding.  Ankyrins interact with a variety of proteins, such as spectrin in the cytoskeleton, ion channels, ion pumps, and cell adhesion molecules. Ankyrins allow the cytoskeleton (microtubules, intermediate filaments) to attach to the cell membrane (Bennett, 2001a; Rubstov, 2000).  Mutations in ankyrins can cause anemia and cerebellar disorders.    Ankyrins are only known to exist in animals, with one copy known in nematodes, two in flies, and three in humans (Bennett, 2001a; Rubstov, 2000).

Ankyrin-R (ANK1) is the major ankyrin in red blood cells and is also expressed in muscle, macrophages, endothelial cells, and neurons (including cerebellar Purkinje cells) (Bennett, 2001a; Rubstov, 2000).  A macrophage is depicted below.

MONOCYTE

Ankyrin-B (ANK2) is primarily expressed in the nervous system.

 

Ankyrin-G (ANK3) is expressed in a variety of tissues.  It seems to function in the localization osodium channels and L1 CAM proteins in neurons (Bennett, 2001a; Rubstov, 2000).

Ankyrin repeats are present in a diversity of proteins (including enzymes, transcription factors, and toxins), indicative of domain shuffling in ancestral genomes.  Horizontal transfer seems to be responsible fro the presence of ankyrin domains in bacteria and yeast (Bork, 1993).

     Some ankyrin family members function in the nucleus, some are attached to the cell membrane (such as notch) and others are secreted (Kohl, 2003).  One of the new domains which evolved in the first animals was a death domain, which was derived from ankyrin-like domains.  Death domains promote apoptosis and are known in sponges.  Death domains are part of the ankyrin family (Muller, 2001a; Wiens, 2000). 

     The death domain superfamily includes the proteins required to transmit the death signal to an apoptotic cell.  The family includes proteins with the death domain (DD), death effector domain (DED), and caspase recruitment domain (CARD) which have descended from a common ancestral protein.  These include Apaf-1, caspase 8, and caspase 9.  Caspases cleave a number of apoptotic proteins (such as polyADP-ribose polymerase and endonucleases), activating them (Weber, 2001). 

     A group of eukaryotic proteins called tankyrases are related to both ankyrins and poly(ADP-ribose) polymerases since they are composed of each of these domains.  They perform a variety of functions such as telomere maintenance and vesicle transport. A tankyrase homolog in nematodes induces apoptosis in response to DNA damage (Gravel, 2004).  

Tankyrase-1 interacts with telomeric repeat binding factor, which inhibits telomere extension and binds to an endosomic enzyme which, after insulin signaling, transports vesicles to the cell membrane where glucose can be transported (Gravel, 2004).

 

  Tankyrase-2 seems to perform functions similar to those of tankyrase-1 but is more highly expressed in the placenta and skeletal muscle while tankyrase-1 is more highly expressed in adipocytes (as those pictured below) and the testis.  Unlike tankyrase-1, tankyrase 2 can promote apoptosis.  (Gravel, 2004).

ADIPOSE

CASPASES

     Eubacteria, archebacteria, chloroplasts, and some mitochondria (such as the mitochondria of the algae Mallomonas splendens and Cyanidioschyzon), use the GTPase FtsZ in division.  Eubacteria are pictured below.

BACTERIA

The response of mitochondria to stress shares a number of characteristics with the formation of bacterial spores.  It appears that the early eukaryotes adapted the stress responses of endosymbiotic mitochondria to become the apopototic response to stress (Frank, 2003).  Programmed cell death might be a relic of the relationship between two separate genomes in the ancestral eukaryote (Ameisen, 2002).  Mitochondria release proteins which cause apoptosis through both caspase dependent and caspase-independent pathways.  Mitochondria store a variety of proteins which can be released to destroy the cell (Lorenzo, 2004).   

     During apoptosis, the outer mitochondrial membrane permits the release of mitochondrial proteins into the cytoplasm.  Such release is inhibited by anti-apoptotic members of the Bcl family and stimulated by the pro-apoptotic members of the family.  Cytochrome c is one of the proteins which is released and it can activate caspase 9 of the apoptosome complex. During apoptosis cysteine proteases (caspases) cleave proteins at aspartic acid residues. Caspases are consistently present in cells in their inactive forms which can be activated if the small subunit is cleaved from the large subunit.  An active caspase can then cleave the small subunits of similar or different caspases and thereby activate them.  Molecules such as Apaf-1 and FADD help to activate some of the caspases (caspases-2, -8, -9, and -10) which are called initiator caspases.  The initiator caspases then activate other caspases called effector caspases (caspase-3, -6, and -7) which subsequently cleave target proteins which induce apoptosis (Orrenius, 2004; Huettenbrenner, 2003).

     Caspases arose early in evolution and are known in bacteria. (Aravind, 2003). Two families of caspase-like proteins have been named metacaspases (found in plants, fungi, and protists) and paracaspases (known in animals and Dictyostelium) (Uren, 2000). Caspases are enzymes belonging to the interleukin 1beta converting enzyme family. Cnidarians use caspases similar to caspase 3 in apoptosis; the same is predicted in sponges based on their caspase sequence (Chowdhury, 2008). During oogenesis in Hydra, nurse cells utilize both caspase dependent and caspase independent pathways in apoptosis (Technau, 2003).   Cells of Hydra  are depicted below.

SPERM

  C. elegans possesses one caspase gene while multiple caspases are known in Drosophila.  The activation of caspases by CED-4 is a conserved apoptotic mechanism in animals (Kankura, 1999).  Although Ced-4 activates apoptosis through a caspase in animals, it can induce apoptosis in yeast despite the absence of caspases (Ameisen, 2002).  Primitive chordates evolved more complex apoptotic pathways.  Tunicates possess 11 caspase genes (compared to 14 in mammals and 1 in nematodes) and homologs of both the intrinsic and extrinsic pathways of caspase activation (including the Ced-4, Apaf-1 [intrinsic] and TNF receptors [extrinsic] which are absent in worms) (Dehal, 2002). The mammalian caspases (consisting of 15 genes) can be split into two subfamilies: one promotes apoptosis while the other promotes inflammation as a result of cytokine signaling (Chowdhury, 2008).

 

The TTL gene family (tubulin tyrosine ligase) can promote apoptosis after disruption of microtubules. One gene of this family is expressed in the testis and underwent positive selection in primates (Chen, 2006).

    Plants are also capable of apoptosis.  One plant pathway involved homologs of the Toll receptor family of coelomate animals.  Plants possess enzymes called metacaspases which are absent in mammals; seem to be members of the same caspase/paracaspase/metacaspase superfamily (Ameisen, 2002)  Mammalian pro-apoptotic and anti-apoptotic signals have been shown to induce and inhibit plant apoptosis, respectively (Ameisen, 2002)