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Apoptosis Resource: The Beginning to the End
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Over the past three decades, the study of apoptosis (programmed cell death) has gained significant importance in human disease and its clinical management. Failure to regulate apoptosis is a common feature in several diseases including autoimmune disorders, neurodegenerative diseases, cancer, and AIDS. Hence, it is important to understand the apoptotic processes in cells in order to devise therapeutic means to intervene and reset the balance between cell survival and death. It is now well recognized that there are two main pathways for the induction of apoptosis, the extrinsic or receptor-mediated pathway, and the intrinsic or mitochondrial pathway. Induction of either pathway can result in the activation of caspases, a class of intracellular cysteine proteases that are responsible for the cleavage of a variety of cellular substrates and the morphological changes attributed to apoptosis. Other less well-defined caspase activation pathways, such as autophagy, have recently been described, but will not be discussed here.
A very large beneficiary of apoptosis research is oncology, since most cancer cells exhibit defects in their suicidal machinery. By better understanding caspase activation pathways, new therapeutic agents may be developed to induce death in cancerous cells. On the other hand, pharmacologic interference with the induction or completion of apoptosis holds promise for the treatment of several neurodegenerative disorders.
The extrinsic pathway is activated by the interaction of a specific death ligand with its cell surface death receptors (DR), which are members of the tumor-necrosis factor (TNF) superfamily. This pathway plays an important role in the regulation of apoptosis in cells involved in the immune system. Fas, TNFa, or TNF-related apoptosis-inducing ligand (TRAIL) interact with their cognate receptor to induce a conformational change. For example, following FasL binding to its receptor, an intracellular death-inducing signaling complex (DISC) is formed via the stepwise recruitment of cytosolic proteins, such as procaspase-8 and the Fas-associated death domain protein (FADD). FADD is an adapter protein that acts as a bridge to link the death receptor to death effector domains (DED) of caspases-8 and 10. Formation of the DISC leads to the dimerization and activation of caspase-8, which in turn can activate caspase-3 and other downstream events. The extrinsic pathway can also crosstalk with the intrinsic pathway via caspase-8 mediated cleavage of Bid, which can trigger the release of proapoptotic mitochondrial proteins.
The intrinsic pathway is the most common pathway for cell death in vertebrates and can be activated by a variety of stimuli, including growth factor withdrawal, heat shock, oncogene activation, DNA-damaging agents, reactive oxygen species, excessive cytosolic calcium, and other cellular stresses. These agents cause the permeabilization of the mitochondrial outer membrane (MOMP) and release of cytochrome c and other proteins. The permeabilization of MOMP can occur as a result of either a change in the mitochondrial permeability transititon pore (PTP) or by the action of pro-apoptotic members of the Bcl-2 family of proteins. The PTP complex is composed of the voltagedependent anion channel (VDAC) in the outer membrane, the adenosine nuclear transporter (ANT) channel in the inner mitochondrial membrane, and the soluble matrix protein cyclophilin D. Opening of the PTP, triggered by higher levels of cytosolic calcium, allows water and solutes of 1.5 kDa to freely diffuse from the cytosol to the mitochondrial matrix leading to mitochondrial swelling and collapse in the transmembrane potential. The second mechanism of MOMP permeabilization involves pro-apoptotic members of the Bcl-2 family, whereby Bax and Bak oligomerize and insert into the outer mitochondrial membrane. BH3-only proteins, such as Bim and Bid contribute to the oligomerization of these proteins. In contrast, the Bcl-2 antiapoptotic members Bcl-2, and Bcl-XL inhibit protein release. While significant progress has been made in our understanding of the regulation and interaction of Bcl-2 member proteins, the exact details remain to be elucidated.
Mitochondrial outer membrane permeabilization is the key event leading to caspase activation in the intrinsic pathway. Cytochrome c released into the cytosol from the intermembrane space binds to the apoptosis proteaseactivating factor (Apaf-1), which then oligomerizes in the presence of ATP. Pro-caspase-9 molecules can then bind to each of the Apaf-1 monomers via the caspase recruitment domain (CARD) forming a caspase-activating complex, the apoptosome. Active caspase-9 participates in activation of downstream caspases-3 and -7.
The extrinsic and intrinsic pathways for caspase activation converge on downstream effector caspases, which ultimately results in apoptotic cell death. Since caspases play a central role in regulation and execution of cell death, they must be tightly regulated. Regulation can occur by either inhibiting caspase activity, or by blocking its activation. The IAP family of proteins acts to inhibit caspase activity. Members of this family include XIAP, cIAP1, cIAP2, hILP-2, ML-IAP, NAIP, survivin, and apollon. A key domain present in all members is a baculovirus IAP repeat (BIR), a 65-residue domain rich in histidine and cysteine residues, which acts in concert with the flexible region preceeding the BIR domain to inhibit the activity of caspases. Also, present in some IAPs is a RING domain located at the carboxy terminus, which functions as a E3 ubiquitin ligase to provide specificity of transfer of ubiquitin moieties to the target protein.
The activity of IAPs can be regulated by the mitochondrial protein Smac/DIABLO, normally localized to the mitochondria. Upon its release from the mitochondria, Smac/DIABLO acts as an IAP antagonist to inhibit XIAP, thereby acting as a proapoptotic molecule. Omi/HtrA2 is another mitochondrial protein, that when released into the cytosol, can inhibit IAPs.
Caspases can also be regulated by blocking their activation. For example, the FLIP protein, a caspase-8 homolog lacking proteolytic activity, can block caspase-8 activation. FLIP possess DEDs at their N-termini and can be recruited to the DISC, and under conditions of overexpression, can prevent caspase-8 activation. However, when FLIP is present at lower concentrations in the DISC, it can aid in the cleavage of procaspase-8.
Cellular apoptosis is manifested by a number of distinctive biochemical and morphological changes to give the apoptotic phenotype. These changes provide measurable markers to indicate that apoptosis has occurred. At this point it is prudent to discuss the difference between apoptosis and necrosis. While necrosis and apoptosis can be distinguished in some situations, it is not so obvious in others because dying cells exhibit features of both apoptosis and necrosis. Since there are no clear biological markers to distinguish apoptosis from necrosis, morphological changes remain the most reliable method for differentiation. The following Table compares morphological features of apoptosis with necrosis.
Necrosis is characterized as a pathological or accidental cell death wherein the cell is rendered energetically incapable of surviving due to ATP depletion. On the other hand, apoptosis is a programmed form of cell death, which requires ATP.
| Type of cell death: | Apoptosis | Necrosis | | Feature | | | | Plasma Membrane | • | Phosphatidylserine translocated to surface | • | Blebbed |
| • | Ruptured, early lysis without formation of vesicles |
| | Mitochondria | • | Increased membrane permeability | • | Release of cytochrome c into cytocol |
| | | Cell Degradation | • | Phagocyosis | • | No inflammation |
| • | Release of proinflammatory molecules | • | Inflammation |
| | Cell Shape | • | Formation of apoptotic bodies |
| | | DNA Fragmentation | • | Internucleosomal DNA cleavage; free 3'-OH ends |
| | | Cytoplasm | • | Presence of apoptotic bodies |
| • | Vacuolation of cytoplasm |
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A number of experimental methods and techniques are available to study cell death that take advantage of the morphological and biochemical changes during apoptosis. This brochure is designed to provide an overview of the techniques available to study apoptosis. Due to the often complex machinery of cell death, it is advised to always use more than two (separate) techniques to validate apoptosis in the experimental system. This brochure will also act as a guide and provide researchers with the tools and tips to measure apoptosis-induced changes that occur at the plasma membrane, mitochondria, activation of caspases in the cytoplasm, and DNA fragmentation in the nucleus.
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