2012 Group 3 Project
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- 1 Extrinsic Apoptosis
|1842||Carr Vogt is the first to make observations on the occurrence of cell death.|
|1965||John Kerr's work on ischaemic liver injury led him to distinguish between two types of cell death, namely classical necrosis, and the transformation of scattered cells into tiny globules of cytoplasm.|
|1972||In collaboration with Andrew Wyllie and Alastair Currie, John Kerr coins the term apoptosis. This process is characterized by the condensation of the nucleus and the cytoplasm, followed by the fragmentation of the cell into small masses. He observes that controlled cell death is involved in embryonic development, tumour growth, and cell turnover.|
|1975||E.A Carswell and colleagues find a substance, thereafter named Tumor Necrosis Factor (TNF), in a serum given to bacillus Calmette--Guerin (BCG)-infected mice that mimics the endotoxin that causes tumor necrosis. |
|1976||Cell death is found to occur in a specific pattern, as seen in the ventral cord and ganglia of the nematode Caenorhabditis elegans by J.E Sulston.|
|1986||Through their research on the nematode Caenorhabditis elegans, H.M Ellis and H.R Horvitz make the first discovery regarding the steps involved in the programmed cell death pathway, namely that ced-3 and ced-4 activate cell death.  A decade later, M.O Hengartner, who also conducts research on the nematode Caenorhabditis elegans and Drosophila finds that ced-3 and ced-4 genes kill cells, whereby the ced-9 gene inhibits cell death. |
|1990||DNA digestion is found to play a critical role in the early stages of apoptosis. Due to the increase in research in the pharmaceutical area at this time, this finding has important repercussions to the field, as it suggests that the effects of drugs are partially mediated by the apoptotic signaling pathway. |
|1997||Researchers find that the activation of caspase-3, which leads to apoptosis, may be prompted by the binding of cytochrome c to Apaf-1. J.P Medema and colleagues discover that the binding of CD95/APO-1/Fas to the death-inducing signaling complex (DISC) leads to the activation of FLICE. |
|2002||Given the increase in knowledge of the extrinsic apoptotic pathway, researchers are attempting to find various ways in which this signaling pathway can be used to treat cancer.   |
|2008||Researchers find that cerebral ischemia/reperfusion (I/R), may be elicited by the extrinsic apoptotic pathway. More specifically, they conclude that caspases, the BcI-2 family, as well as the inhibitors of apoptosis (IAPs) play a critical role in triggering I/R. Although there are promising treatments that have surfaced as a result of research in this area, there remains a lot to be understood about the molecular mechanisms of extrinsic apoptosis in relation to I/R. |
|Today||Although many components of the extrinsic apoptotic pathway have been established over the last few decades, there are many parts of this intricate process that remain unknown. Thus, researchers are currently attempting to simultaneously discover more about the molecular mechanisms involved in the extrinsic apoptosis signaling pathway, while attempting to find innovative ways of treating various diseases such as cancer  and Parkinson's disease. |
The following is a table that includes the various proteins involved in the extrinsic apoptosis pathway:
|TNF-α||Tumor necrosis factor alpha||A cytokine produced by lymphocytes and macrophages that is involved in the lysis of certain cells, and plays a crucial role in the immune system.|
|TNFR1||Tumor necrosis factor receptor 1||A transmembrane protein containing cysteine-rich domains (CRD). In response to the blocking of protein synthesis, it commences the regulation of inflammatory genes, thus signaling the activation of the apoptotic caspase cascade.|
|FasL||Fatty acid synthetase ligand||This is a type-II transmembrane protein that fits into the tumour necrosis factor (TNF) family. Its binding with its receptor is what induces apoptosis. The ligand/receptor relations play an important role in the regulation of the immune system and the progression of cancers.|
|FasR||Fatty acid synthetase receptor||The most commonly recognised member of the death receptor family. The gene is situated on chromosome 10 in humans and 19 in mice. Apoptosis-inducing Fas receptor is dubbed isoform 1 and is a type 1 transmembrane protein. It is made up of three cysteine-rich pseudorepeats, a transmembrane domain, and an intracellular death domain.|
|Apo3L||Apo3 ligand||This is the ligand for the death-domain-containing receptor Apo-3. The Apo-3 ligand is a 249 amino-acid, type II transmembrane protein that induces apoptosis.|
|DR3||Death Receptor 3||DR-3 is a member of the TNF receptor family that is also known as Apo-3.|
|Apo2L||Apo2 ligand||APO-2 ligand that is also known as TRAIL. APO-2L is a member of TNF family that induces apoptosis. It is a homotrimeric, type II transmembrane protein and just like FasL and TNF-a, it can be cleaved at the cell surface to form a soluble protein in the extracellular matrix.|
|DR4||Death receptor 4||One of the receptors for TRAIL. When activated, it mirrors FasR in the required aggregation and DISC formation.|
|DR5||Death receptor 5||Death receptor-5 is one of receptors for TRAIL that is also known as TRAILR2 and when activated, allows for aggregation and DISC formation.|
|FADD||Fas-associated death domain||This is an adaptor molecule that bridges the Fas-receptor, and other death receptors to the caspase-8 forming death-inducing-signalling complex during the process of apoptosis.|
|TRADD||TNF receptor-associated death domain||This is an adaptor protein that encodes through a death domain containing molecule that interacts and mediates apoptosis as well as signals for NF-kappa B activation.|
|RIP||Receptor-interacting protein||A Fas-binding protein containing death domain that is crucial for the mediation of TNF-R1 as well as the activation of NF-kB.|
|DED||Death effector domain||This is a protein interaction domain found to control a variety of cellular signalling pathways. The DED domain is found in inactive cysteine proteases and proteins that regulate caspase activation during the process of apoptosis.|
|caspase-8||Cysteinyl aspartic acid-protease 8||The CASP8 gene encodes a member of the cysteine-aspartic acid protease (caspase) family. Sequential activation of caspases allows for a successful process of apoptosis. Caspases exist as inactive proenzymes composed of large and small protease subunits. |
|caspase-3||Cysteinyl aspartic acid-protease 3||A caspase protein that interacts with caspase 8 and caspase 9. Its role and structure is similar to that of the caspase-8.|
|c-FLIP||FLICE-inhibitory protein||This is an inhibitory protein and regulates apoptosis. It also goes by the name FLICE and plays an important role in the pathogenesis and treatment of cancer.|
|Activation of the Fas pathway begins with the cleavage activation of FasL in neighboring cells to the target cell. The membrane bound, activated FasL’s trimerisation on the neighboring cells membrane then binds to and causes the formation of a FasR trimer complex. This alone is not enough to result in cell death. The aggregation of multiple activated FasR trimers are required to form the Death Domain (DD) or the DISC.   Once the DD is generated, Fas Associated Death Domain molecule (FADD) is enabled to bind. FADD, upon binding to the DD, undergoes a conformational change to reveal its Death Effector Domain (DED). Caspase-8, also known as FLICE, is activated when Pro-caspase-8 makes contact with the DED that induces a self-proteolytic cleavage into p10 and p18 subunits.  This cleavage results in the release of the subunits into the cytoplasm where they proceed to activate effector caspases leading to cell death.
|As in Fas-mediated apoptosis, membrane bound and soluble forms of the ligand TNF alpha (TNF-a) are found in tissue. TNF-a, like FasL, experiences trimerisation that, when bound to TNFR, results in a TNF-a/TNFR trimer complex. Unlike the Fas pathway, there is no aggregation or DISC formation for the complex to begin its function. The formation of the TNFR trimer causes a disjunction of SODD (a TNF inhibitory protein) from the cytoplasmic end of the complex revealing the Death Domain (DD) of TNFR. The appearance of the DD allows the adaptor protein TRADD to bind.  At this point the pathway diverges down three paths that proceed simultaneously.
One of these pathways mirrors the Fas pathway described earlier as the activation of TRADD after binding to the DD leads to the activation of FADD. The activation of FADD, as described earlier in Fas Pathway, has downstream effects on caspase-8. Activated Caspase-8 continues on to activate effector caspases as described in the Caspase Cascade. Unlike the Fas pathway, this road is quite weak and is often overwhelmed by one of the other effects that the TNF pathway takes.
Unexpectedly, activation of anti-apoptotic factors is also caused along the second path. As demonstrated in the image, intracellularly, TRAF2 and RIP form a complex with TRADD that reveals an activation domain on RIP. This domain is utilised by protein kinase IKK. The active IKK phosphorylates IkBa (an inhibitory protein) releasing the heterodimer transcription factor NF-kB. The release of NF-kB results in the transportation down cytoskeletal components into the nucleus where transcription of proteins required for cell survival occurs.
The third pathway is generally pro-apoptotic activating one of the MAPK cascades. This evokes downstream effects on the JNK pathway that is usually involved with cell differentiation and proliferation. 
The triad response of the TNF-pathway is intricate. The complexity establishes a solid groundwork for a response (by a vast diversity of cells) to inflammation inside a specific tissue. The activation of both death and survival proteins is to aid in minimal disruption to the surrounding tissue of an infection so as to activate death in lowest amount of cells required to return the tissue to normal function. 
|The TRAIL pathway is quite similar to the Fas pathway in almost all aspects. TRAIL, also known as APO2L, is also a homotrimeric, type II transmembrane protein and just like FasL and TNF-a, it can be cleaved at the cell surface to form a soluble protein in the extracellular matrix. TRAIL binds to a variety of receptors and this is its defining characteristic that sets it apart from Fas regulated apoptosis. It is able to bind and activate Death receptor 4 (DR4) and Death Receptor 5 (DR5) that, both, when activated mirror FasR in the required aggregation and DISC formation. TRAIL can also bind to DcR1, DcR2 and osteoprotegerin (OPG) that are considered to be decoy receptors. Activation of these receptors does not lead to apoptosis, as their intracellular death domain is non-existent before or after being activated by the ligand.   Activation of DR4 or DR5 and its aggregate formation leads to activation of FADD that continues down the same pathway as Fas towards cell death.
Diagram of Signaling Pathway
The Cell Executioners: Caspases
Caspases are a large family of proteases that play crucial roles in the activation and execution of apoptosis. They are expressed as proenzymes and require proteolysis to be activated. Their proteolytic cleavage is extremely specific and is in keeping with the dire consequences that would result if erroneous activation occurred (accidental apoptosis). As proenzymes, caspases have three domains; a NH2 terminal domain, a large subunit and a small subunit. On proteolysis, the small and large subunits join to form a heterodimeric activated caspase. 
Caspases function as both initiators and effectors in the apoptotic pathway. Initiator caspases are regulated by co-factors associated with the death receptors, which, as discussed above, are the start of the signalling pathway. The key intial cascade kick-starter of extrinsic apoptosis is caspase 8, which normally exists as a procaspase and is activated upon association with its cofactor FADD through the death effector domain.
So initiator caspases (8 and 10) are activated by death receptors and associated proteins and they then activate effector caspases (3,6 and 7). The next link in the chain is how do these effectors contribute to cellular disassembly? Well the process is not entirely lucid but some key examples demonstrate their input:
- caspase inactivation of proteins that protect cells from apoptosis. For example, the cleavage and inactivation of ICAD, the protein responsible for the inhibition of Caspase Activated DNase, a nuclease which fragments DNA. Bcl2 is another anti-apoptotic gene that is also inactivated by capsases.
- indirect reorganisation of cell structures by cleavage of proteins responsible for cytoskeleton regulation. Examples include gelsolin, focal adhesion kinase, and p21-activated kinase, all of which are deregulated when cleaved by caspase effectors.
- nuclear shrinking and budding caused by caspase cleavage of nuclear laminins
- inactivation of proteins involved in DNA repair (eg. DNA-PKC5)
- inactivation of proteins involved in mRNA splicing (eg. U1-70K)
- deregulation of proteins involved in DNA replication (eg. RF-C)
The relationship of these last three caspase functions to apoptosis is not well understood, but it is probable that the inactivation of crucial homeostatic and repair mechanisms aids with dismantling of the cell. All in all, the role of caspases is vast, varied, crucial and carried out in a speedy, efficient and deadly manner. 
Morphology of Apoptosis
|Changes in cell morphology due to triggering of extrinsic apoptosis are first evident under a light microscope as cellular and nuclear shrinkage as well as chromatin condensation around the inner margins of the nuclear envelope, forming crescent shaped aggregates. .
It seems that evolution has recognised, or in fact decided, that the most efficient and successful mechansim of cell death is to first disable the cell by dismantling and destruction of its programming and control centre, the nucleus. Without translatable DNA, the cell is doomed. Its almost as if apoptotic cell makes an emphatic statement and signs its own death warrant by targeting the nucleus as one of its initial steps.
Following chromatin condensation, nuclear apoptosis continues with the cleavage of double stranded DNA into fragments of sizes between 180 and 200 base pairs by caspase substrates such as DNA nuclear fragmentation factor 40 (DFF40). 
The nuclear lamina, which is reponsible for nuclear envelope strucure, is then dismantled due to caspase-mediated degradation of nuclear lamins A and B (possible caspase 2 involvement). The nucleus then disassembles into a few fragments which will be packaged into apoptotic bodies..
Despite surrounding protease activity, membranes and organelles (except the nucleus) such as the mitochondria are surprisingly unaffected by the cell remodelling that occurs in the intial stages of apoptosis. Effector caspase and protein kinase activity causes the separation of the dying cell from its neighbours by cleavage of proteins responsible for organisation of focal adhesins or by disassembly of microtubules.
Upon detachment from adjacent cells, protrusions begin to appear from the unusally round cell. These blister-like protrusions are called blebs and will eventually form membrane-bound apoptotic bodies filled with nuclear fragments, organelles and cytosol. The 'blowing' of these bubbly blebs is driven by increased hydrostatic pressure of the cell due to cytoskeleton contraction. After formation of the intial bleb, actin polymerisation (so some synthesis pathways are still capable of turning on) and myosin recrutiment allow for retraction of the bleb and separation from the dying cell. Prior to this breaking up of the cell, late stage aopotosis does demonstrate some changes in cell organelles as seen listed in the below table but there relevance and contribution to the process is still to be solved.
Phagocytosis of the apoptotic bodies is a key feature of apoptotis. The speedy engulfment of the remnants of the dead cell by macrophages and other phagocytic cells, not only prevents an inflammatory response in the surrounding tissue but also allows for the dead cells complete degradation by lysosmal enzymes. The phagocytes recognise the apoptosed cell fragments due to the externalisation of the phospholipid, phosphatidylserine, which is usually stored on the inside but now is displayed externally, chiefly as a recognition molecule for phagocytosis.
Abnormal Function and Diseases
This table, which describes diseases involved in apoptosis, aims at introducing interesting pathologies - each of which is caused by a different defect in the apoptotic pathway. It is impossible to discuss every disease which involves apotosis due to the fact that it is such a crucial cell function, abnormalities of which therefore involve countless diseases. What we hope to show are some interesting diseases which can result from as little as a single point mutation and truncated protein, up to infection with a virulent bacterium and sytemic effects.
|Disease||Aetiology/Pathogenesis||Signs and Symptoms||Treatment||Image|
|Autoimmune Lypmphoproliferative Syndrome (ALPS)||Fas germline or somatic mutation at the intracellular death domain results in defective lymphocyte apoptosis and consequent imbalance in lymphocyte homeostasis ||Enlargement of lymphoid organs such as slpeen and lymph nodes. Organ specific symptoms include dermatitis, thyroiditis and hepatitis||Splenectomy, immunosuppressants such as corticosteroids |
|Non-small cell lung cancer||Two mechanisms:1) downregulation of Fas receptor expression in tumor cells due to Fas gene polymorphisms (anti-apoptotic effect)
2)Upregulation of Fas ligand expression by tumor cells, triggering apoptosis of tumor-targeting immune cells (protective for tumor cells. Occurs due to FasL polymorphisms)
|Non-small cell lung cancers include squamous cell carcinomas, adenocarcinomas and large cell carcinomas. Often silent till late stage progression,but possible symptoms are persistent cough, pneumonia, haemoptysis and airway obstruction.||Chemotherapy and infrequently surgical resection |
|Parkinson's Disease (PD)||Apoptosis of neurons via extrinsic pathway due to increased production of TNF-alpha and TNF-alpha receptor (one of many implicated pathways in (PD))||4 cardinal symptoms: Tremor, postural instability, muscle rigidity, brady kinesia. Other possible symptoms include anxiety, constipation, depression, anosmia, fatigue ||There is no cure for PD but it can be managed. Prevention of dopamine metabolism (MAO type B inhibitors), dopamine replacement therapy (Levodopa). Surgery invlving area of the CNS is an option but only on a case by case basis, it is not appropriate for everyone. |
|Gastric Carcinomas||Gastric carcinomas have a complex, multifactorial aetiology and pathogenesis, and it has been shown that mutations in the gene coding for pro-caspase-8 play a central role in advanced stomach cancer. Defective caspase-8 → dysfunctional extrinsic apoptosis → tumour cell proliferation. ||Very similar to those of chronic gastritis- epigastric pain, heartburn, chest pain, nausea and vomiting. ||Surgical resection, chemotherapy, eradication therapy for H. Pylori |
|Leprosy (Hansen's Disease)||Infection with Mycobacterium lepra and mycobacterial agonist production leads to an acute inflammatory response accompanied with wide spread and accelerated cell death due primarily to TNF-alpha release by monocytes.  ||Numbing of hands and feet due to nerve damage which leads to infection due to inability to react to painful stimuli. These consequent infections can culminate in finger and toe loss or necessitate amputation. Paralysis may cause the fingers and toes to curl up permanently. ||Leprosy is curable but if untreated can lead to severe deformities. Although a known teratogen, thaliomide, was approved and recommended by the World Health Organisation for treatment of leprosy in 1998. Thaliomide inhibits production of TNF-alpha by accelerated degradation of the mRNA which codes for the pro-apoptotic ligand. Many other treatments exist. |
Many diseases are a result of the dysregulation of the apoptotic pathway. More specifically, extrinsic apoptosis plays a critical role in the development of cardiovascular disease - one of the most prevalent causes of death in today's society.  In addition, ischemia, autoimmune diseases such as AIDS, and various neurodegenerative conditions such as Parkinson's, Huntington's and Alzheimer's disease are caused by excessive apoptosis - an increase in the amount of programmed cell death. In other cases, such as cancer, there is insufficient apoptosis; thus, there is an increase in the number of cells, and/or a decrease in the programmed death of dysfunctional cells. Given the plethora of diseases that arise from malfunctions of this pathway, there has been a surge in current research in this particular field. Researchers are attempting to fully understand the molecular mechanisms of the critical proteins that underlie apoptosis. With this knowledge, there is hope that therapeutic interventions for the aforementioned diseases can be improved, and result in increased survival rates for afflicted patients.
One example of current research is Ovadje, Hamm, and Pandey's work on induced extrinsic apoptosis in human Chronic Myelomonocytic Leukemia (CMML) cells through the use of dandelion root extract (DRE). CMML is an aggressive type of leukemia that is, unfortunately, difficult to diagnose. To make matters worse, patients with this disease do not have a high survival rate. Those who do go through treatment, such as chemotherapy, experience severe side effects, and cancer cells quickly develop resistance during the course of treatment. However, this group of researchers studied the effects of DRE on CMML cells, and found that DRE's capacity to induce extrinsic apoptosis may lead to a more natural and safe way to treat this disease. They suggest that DRE speeds the activation of caspase-8, which in turn quickens the activation of caspase-3. In addition, the researchers found that certain death receptors, such as the Fas-Associated Death Domain (FADD) were required for DRE to efficiently induce extrinsic apoptosis in CMML cells. Furthermore, extrinsic cell death was not observed in non-cancerous peripheral blood mononuclear cells (ncPBMcs). DRE's ability to selectively induce extrinsic apoptosis in CMML cells and to not do so in ncPMBcs demonstrates that DRE may possibly be a more natural alternative to other forms of cancer treatment that exist today. 
The Reproductive and Cardiovascular Disease Research Group at St. George's at the University of London is investigating the role of extrinsic apoptosis in the stages of early pregnancy, particularly during the formation of the placenta, which is critical in determining the health of a pregnancy. Although many events at this stage of development can lead to disorders such as pre-eclampsia, little is known about the factors or processes that regulate this growth period. Reproductive and Cardiovascular Disease Research Group
Furthermore, scientists at the MRC Toxicology Unit in the United Kingdom are conducting research on the role of the extrinsic apoptotic pathway in the development of cancer. More specifically, they are investigating whether TNF-related apoptosis-inducing ligand (TRAIL) can potentially be utilized as a form of treatment for those with breast cancer or Chronic Lymphocytic Leukemia (CLL). The researchers claim that TRAIL has been shown to selectively activate the extrinsic death pathway in primary tumor cells, without causing high levels of toxicity in healthy cells. Although this was shown in breast cancer cells, it was not observed to be as effective in lymphoid tumor cells, which exhibit resistance to TRAIL. As a result, the MRC Toxicology Unit is looking further into the biological mechanisms involving TRAIL, in order to fully take advantage of its promising ability to treat cancer. MRC Toxicology Unit
Finally, researchers at Genentech BioOncology are also looking into the apoptotic pathway as a means of developing new forms of cancer therapy. Genentech BioOncology
- Activation: The transfer of a protein from a dormant state into a form that allows it to induce a response on another molecule.
- Aggregation: The localisation of multiple proteins to one area on a cell membrane.
- Apoptosis: A natural process of self-destruction in specific cells that are determined by the genes, it can be initiated through a stimulus or removed through a repressor agent. Another name for it is programmed cell death.
- Apo2L/TRAIL: An endogenous protein that triggers apoptosis by binding to pro-apoptotic receptors DR4 and DR5.
- Apoptosome: A multiprotein complex that consists of Apaf-1 and cytochrome c molecules and helps to initiate apoptosis by activating procaspase 9.
- Apoptotic bodies: Formed when blebs separate from the cell, taking a portion of cytoplasm with them.
- Bcl-2 superfamily: A group of proteins characterized by the presence of Bcl-2 homology (BH) domains. Bcl-2 superfamily proteins can either promote or inhibit apoptosis.
- Caspases: Are a large family of proteases that play crucial roles in the activation and execution of apoptosis. They are expressed as proenzymes and require proteolysis to be activated. This cleavage is extremely specific and is in keeping with the dire consequences that would result if erroneous activation occurred.
- Chromatin: The combination of DNA and proteins that make up the contents of the nucleus.
- Cleavage: A division of cells that does not increase total mass as the number of cells increase.
- Cytokine: A signaling protein that plays a major role in intercellular communication.
- Cytoskeleton: Provides structure of the cell.
- Endonuclease: Enzyme that cuts the DNA at phosphodiester bonds.
- Extracellular Signaling: Signalling process occurring outside of the cellular compartment.
- FADD: Fas-associated death domain. A protein that plays a key role in transmitting the apoptotic signal mediated by death receptors.
- Focal Adhesions: Large protein complex which connects with the extracellular matrix.
- Heterodimer: A macromolecule composed of two different types of proteins.
- Homotrimeric: A protein that is composed of three identical polypeptide units.
- Ischaemia: Occurs when cellular metabolism is blocked due to a lack of oxygen and glucose to that area of the body.
- IAP: Inhibitor of apoptosis.
- Fas ligand (FasL or CD95L): Is a type-II transmembrane protein that belongs to the tumour necrosis factor (TNF) family. Its binding with its receptor induces apoptosis.
- Lamina: The basal layer of the epithelial cells.
- Ligand: A molecule that binds specifically to its receptor and activates its signaling powers.
- Lymphocytes: A type of white blood cell found in the immune system of vertebrates.
- Lysis: The breaking apart of - as in the degradation of a cell.
- Lysosomal Enzymes: An enzyme which breaks down foreign bacteria or materials.
- Macrophages: White blood cells, which act as an defense mechanism in destroying foreign bacteria’s.
- Phagocytosis: The cellular process of engulfing solid particles by a cell membrane.
- Phosphorylation: The adding of a phosphate group onto a molecular structure.
- Protease: An enzyme that performs proteolysis.
- Proteolysis: The breakdown of proteins into smaller polypeptides or amino acids.
- Pseudorepeats: A protein's conserved secondary structure.
- TNF-α: A cytokine produced by lymphocytes and macrophages that is involved in the lysis of certain cells, and plays a crucial role in the immune system.
- TNFR1: A transmembrane protein containing cysteine-rich domains (CRD). In response to the blocking of protein synthesis, it commences the regulation of inflammatory genes, thus signalling the activation of the apoptotic caspase cascade.
- Transduction: The process by which a cell converts one kind of signal or stimulus into another.
- Trimerisation: The formation of one protein by the combination of three individual proteins.
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