Apoptosis: 322 (Methods in Enzymology)


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Excessive apoptosis causes atrophy , whereas an insufficient amount results in uncontrolled cell proliferation, such as cancer. Some factors like Fas receptors and caspases promote apoptosis, while some members of the Bcl-2 family of proteins inhibit apoptosis. German scientist Karl Vogt was first to describe the principle of apoptosis in In , anatomist Walther Flemming delivered a more precise description of the process of programmed cell death. However, it was not until that the topic was resurrected.

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While studying tissues using electron microscopy, John Foxton Ross Kerr at the University of Queensland was able to distinguish apoptosis from traumatic cell death. Currie , as well as Andrew Wyllie , who was Currie's graduate student, [9] at University of Aberdeen. In , the trio published a seminal article in the British Journal of Cancer. Kerr, Wyllie and Currie credited James Cormack, a professor of Greek language at University of Aberdeen , with suggesting the term apoptosis. He shared the prize with Boston biologist H.


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Robert Horvitz. For many years, neither "apoptosis" nor "programmed cell death" was a highly cited term. Two discoveries brought cell death from obscurity to a major field of research: identification of components of the cell death control and effector mechanisms, and linkage of abnormalities in cell death to human disease, in particular cancer.

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Sulston for their work identifying genes that control apoptosis. The genes were identified by studies in the nematode C. In Greek, apoptosis translates to the "falling off" of leaves from a tree. Hippocrates used the term to mean "the falling off of the bones". Galen extended its meaning to "the dropping of the scabs". Cormack was no doubt aware of this usage when he suggested the name.

To show the derivation clearly, we propose that the stress should be on the penultimate syllable, the second half of the word being pronounced like "ptosis" with the "p" silent , which comes from the same root "to fall", and is already used to describe the drooping of the upper eyelid. The initiation of apoptosis is tightly regulated by activation mechanisms, because once apoptosis has begun, it inevitably leads to the death of the cell.

A cell initiates intracellular apoptotic signaling in response to a stress, which may bring about cell suicide. The binding of nuclear receptors by glucocorticoids , [21] heat, [21] radiation, [21] nutrient deprivation, [21] viral infection, [21] hypoxia , [21] increased intracellular concentration of free fatty acids [22] and increased intracellular calcium concentration, [23] [24] for example, by damage to the membrane, can all trigger the release of intracellular apoptotic signals by a damaged cell.

A number of cellular components, such as poly ADP ribose polymerase , may also help regulate apoptosis. Before the actual process of cell death is precipitated by enzymes, apoptotic signals must cause regulatory proteins to initiate the apoptosis pathway. This step allows those signals to cause cell death, or the process to be stopped, should the cell no longer need to die. Several proteins are involved, but two main methods of regulation have been identified: the targeting of mitochondria functionality, [28] or directly transducing the signal via adaptor proteins to the apoptotic mechanisms.

An extrinsic pathway for initiation identified in several toxin studies is an increase in calcium concentration within a cell caused by drug activity, which also can cause apoptosis via a calcium binding protease calpain. The mitochondria are essential to multicellular life. Without them, a cell ceases to respire aerobically and quickly dies.

This fact forms the basis for some apoptotic pathways. Apoptotic proteins that target mitochondria affect them in different ways. They may cause mitochondrial swelling through the formation of membrane pores, or they may increase the permeability of the mitochondrial membrane and cause apoptotic effectors to leak out.

During apoptosis, cytochrome c is released from mitochondria through the actions of the proteins Bax and Bak.

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The apoptosome cleaves the pro-caspase to its active form of caspase-9 , which in turn activates the effector caspase Mitochondria also release proteins known as SMACs second mitochondria-derived activator of caspases into the cell's cytosol following the increase in permeability of the mitochondria membranes. SMAC binds to proteins that inhibit apoptosis IAPs thereby deactivating them, and preventing the IAPs from arresting the process and therefore allowing apoptosis to proceed.

IAP also normally suppresses the activity of a group of cysteine proteases called caspases , [34] which carry out the degradation of the cell. Therefore, the actual degradation enzymes can be seen to be indirectly regulated by mitochondrial permeability. Two theories of the direct initiation of apoptotic mechanisms in mammals have been suggested: the TNF-induced tumor necrosis factor model and the Fas-Fas ligand -mediated model, both involving receptors of the TNF receptor TNFR family [35] coupled to extrinsic signals.

TNF-alpha is a cytokine produced mainly by activated macrophages , and is the major extrinsic mediator of apoptosis. FLIP inhibits the activation of caspase Apoptosis is known to be one of the primary mechanisms of targeted cancer therapy. In some types of cells type I , processed caspase-8 directly activates other members of the caspase family, and triggers the execution of apoptosis of the cell. In other types of cells type II , the Fas -DISC starts a feedback loop that spirals into increasing release of proapoptotic factors from mitochondria and the amplified activation of caspase This balance is the proportion of proapoptotic homodimers that form in the outer-membrane of the mitochondrion.

The proapoptotic homodimers are required to make the mitochondrial membrane permeable for the release of caspase activators such as cytochrome c and SMAC. Control of proapoptotic proteins under normal cell conditions of nonapoptotic cells is incompletely understood, but in general, Bax or Bak are activated by the activation of BH3-only proteins, part of the Bcl-2 family.

Caspases play the central role in the transduction of ER apoptotic signals. Caspases are proteins that are highly conserved, cysteine-dependent aspartate-specific proteases.

Recombinant Human PDGF R alpha Protein Summary

There are two types of caspases: initiator caspases, caspase 2,8,9,10,11,12, and effector caspases, caspase 3,6,7. The activation of initiator caspases requires binding to specific oligomeric activator protein. Effector caspases are then activated by these active initiator caspases through proteolytic cleavage. The active effector caspases then proteolytically degrade a host of intracellular proteins to carry out the cell death program. There also exists a caspase-independent apoptotic pathway that is mediated by AIF apoptosis-inducing factor.

Amphibian frog Xenopus laevis serves as an ideal model system for the study of the mechanisms of apoptosis. In fact, iodine and thyroxine also stimulate the spectacular apoptosis of the cells of the larval gills, tail and fins in amphibians metamorphosis, and stimulate the evolution of their nervous system transforming the aquatic, vegetarian tadpole into the terrestrial, carnivorous frog.

Negative regulation of apoptosis inhibits cell death signaling pathways, helping tumors to evade cell death and developing drug resistance. Many pathways and signals lead to apoptosis, but these converge on a single mechanism that actually causes the death of the cell. After a cell receives stimulus, it undergoes organized degradation of cellular organelles by activated proteolytic caspases. In addition to the destruction of cellular organelles, mRNA is rapidly and globally degraded by a mechanism that is not yet fully characterized.

A cell undergoing apoptosis shows a series of characteristic morphological changes. Early alterations include:. Apoptosis progresses quickly and its products are quickly removed, making it difficult to detect or visualize on classical histology sections. During karyorrhexis, endonuclease activation leaves short DNA fragments, regularly spaced in size.

These give a characteristic "laddered" appearance on agar gel after electrophoresis. Before the apoptotic cell is disposed of, there is a process of disassembly. There are three recognized steps in apoptotic cell disassembly: [57]. The removal of dead cells by neighboring phagocytic cells has been termed efferocytosis. Many knock-outs have been made in the apoptosis pathways to test the function of each of the proteins.

In order to create a tumor necrosis factor TNF knockout, an exon containing the nucleotides — was removed from the gene. This exon encodes a portion of the mature TNF domain, as well as the leader sequence, which is a highly conserved region necessary for proper intracellular processing.

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However, upon immunization with SRBC sheep red blood cells , these mice demonstrated a deficiency in the maturation of an antibody response; they were able to generate normal levels of IgM, but could not develop specific IgG levels. Apaf-1 is the protein that turns on caspase 9 by cleavage to begin the caspase cascade that leads to apoptosis. This assay is used to disrupt gene function by creating an intragenic gene fusion.

When an APAF-1 gene trap is introduced into cells, many morphological changes occur, such as spina bifida, the persistence of interdigital webs, and open brain. In addition, after embryonic day APAF-1 cells are protected from apoptosis stimuli such as irradiation. A BAX-1 knock-out mouse exhibits normal forebrain formation and a decreased programmed cell death in some neuronal populations and in the spinal cord, leading to an increase in motor neurons.

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Analysis of programmed cell death in the nematode Caenorhabditis elegans. - PubMed - NCBI

The caspase proteins are integral parts of the apoptosis pathway, so it follows that knock-outs made have varying damaging results. A caspase 9 knock-out leads to a severe brain malformation. A caspase 8 knock-out leads to cardiac failure and thus embryonic lethality. However, with the use of cre-lox technology, a caspase 8 knock-out has been created that exhibits an increase in peripheral T cells, an impaired T cell response, and a defect in neural tube closure.

Finally, a caspase 3 knock-out was characterized by ectopic cell masses in the brain and abnormal apoptotic features such as membrane blebbing or nuclear fragmentation. A remarkable feature of these KO mice is that they have a very restricted phenotype: Casp3, 9, APAF-1 KO mice have deformations of neural tissue and FADD and Casp 8 KO showed defective heart development, however in both types of KO other organs developed normally and some cell types were still sensitive to apoptotic stimuli suggesting that unknown proapoptotic pathways exist.

In order to perform analysis of apoptotic versus necrotic necroptotic cells, one can do analysis of morphology by time-lapse microscopy , flow fluorocytometry , and transmission electron microscopy. There are also various biochemical techniques for analysis of cell surface markers phosphatidylserine exposure versus cell permeability by flow cytometry , cellular markers such as DNA fragmentation [69] flow cytometry , [70] caspase activation, Bid cleavage, and cytochrome c release Western blotting. It is important to know how primary and secondary necrotic cells can be distinguished by analysis of supernatant for caspases, HMGB1, and release of cytokeratin However, no distinct surface or biochemical markers of necrotic cell death have been identified yet, and only negative markers are available.

These include absence of apoptotic markers caspase activation, cytochrome c release, and oligonucleosomal DNA fragmentation and differential kinetics of cell death markers phosphatidylserine exposure and cell membrane permeabilization. A selection of techniques that can be used to distinguish apoptosis from necroptotic cells could be found in these references. The many different types of apoptotic pathways contain a multitude of different biochemical components, many of them not yet understood.

In a living organism, this can have disastrous effects, often in the form of disease or disorder. A discussion of every disease caused by modification of the various apoptotic pathways would be impractical, but the concept overlying each one is the same: The normal functioning of the pathway has been disrupted in such a way as to impair the ability of the cell to undergo normal apoptosis.

This results in a cell that lives past its "use-by-date" and is able to replicate and pass on any faulty machinery to its progeny, increasing the likelihood of the cell's becoming cancerous or diseased. Major topics covered include measuring apoptosis and apoptosis-induced endonucleases, measuring apoptosis in lower organisms, proteases involved in apoptosis and their inhibitors, cell free systems for monitoring steps in apoptosis pathways, mitochondria and apoptosis, bCl-2 family proteins, and studying receptors and signal transduction events implicated in cell survival and cell death.

Analysis of programmed cell death and apoptosis in Drosophila.

The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since , each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike.


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Now with more than volumes all of them still in print , the series contains much material still relevant today--truly an essential publication for researchers in all fields of life sciences. Both polyclonal and monoclonal antibodies are available to both procaspases and active caspases.


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One method of caspase detection requires cell lysis in order to release the enzymes into the solution, coating of microwells with anti-caspases; followed by detection with a fluorescent labeled substrate.

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