Before discussing the mechanisms of apoptosis, the morphologic and biochemical characteristics of this process are described.

Mechanisms of Apoptosis

Apoptosis results from the activation of enzymes called caspases (so named because they are cysteine proteases that cleave proteins after aspartic residues). Like many proteases, caspases exist as inactive proenzymes, or zymogens, and must undergo enzymatic cleavage to become active. The presence of cleaved, active caspases is a marker for cells undergoing apoptosis (Fig. 1C). The process of apoptosis may be divided into an initiation phase, during which some caspases become catalytically active, and an execution phase, during which other caspases trigger the degradation of critical cellular components. The activation of caspases depends on a finely tuned balance between production of pro-apoptotic and anti-apoptotic proteins.

Fig1. Morphologic features of apoptosis. A, Apoptosis of an epidermal cell in an immune reaction. The cell is reduced in size and contains brightly eosinophilic cytoplasm and a condensed nucleus. B, This electron micrograph of cultured cells undergoing apoptosis shows some nuclei with peripheral crescents of compacted chromatin, and others that are uniformly dense or fragmented. C, These images of cultured cells undergoing apoptosis show blebbing and formation of apoptotic bodies (left panel, phase contrast micrograph), a stain for DNA showing nuclear fragmentation (middle panel), and activation of caspase-3 (right panel, immunofluorescence stain with an antibody specific for the active form of caspase-3, revealed as red color). (B, From Kerr JFR, Harmon BV: Definition and incidence of apoptosis: a historical perspective. In Tomei LD, Cope FO (eds): Apoptosis: The Molecular Basis of Cell Death. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, 1991, pp 5-29; C, Courtesy Dr. Zheng Dong, Medical College of Georgia, Augusta, Ga.)

Two distinct pathways converge on caspase activation: the mitochondrial pathway and the death receptor pathway (Fig 2). Although these pathways can intersect, they are generally induced under different conditions, involve different molecules, and serve distinct roles in physiology and disease.

Fig2. Mechanisms of apoptosis. The two pathways of apoptosis differ in their induction and regulation, and both culminate in the activation of caspases. In the mitochondrial pathway, proteins of the BCL2 family, which regulate mitochondrial permeability, become imbalanced and leakage of various substances from mitochondria leads to caspase activation. In death receptor pathway, signals from plasma membrane receptors lead to the assembly of adaptor proteins into a “death-including signaling complex,” which activates caspases, and the end result is the same.

The Intrinsic (Mitochondrial) Pathway of Apoptosis The mitochondrial pathway is the major mechanism of apoptosis in all mammalian cells. It results from increased permeability of the mitochondrial outer membrane with consequent release of death-inducing (pro-apoptotic) molecules from the mitochondrial intermembrane space into the cytoplasm (Fig. 3). Mitochondria are remarkable organelles in that they contain proteins such as cytochrome c that are essential for life, but some of the same proteins, in particular cytochrome c, when released into the cytoplasm (an indication that the cell is not healthy), initiate the suicide program of apoptosis. The release of mitochondrial pro-apoptotic proteins is tightly controlled by the BCL2 family of proteins This family is named after BCL2, which is frequently overexpressed due to chromosomal translocations and resulting rearrangements in certain B cell lymphomas (Chapter 13). There are more than 20 members of the BCL family, which can be divided into three groups based on their pro-apoptotic or antiapoptotic function and the BCL2 homology (BH) domains they possess.

• Anti-apoptotic. BCL2, BCL-XL, and MCL1 are the principal members of this group; they possess four BH domains (called BH1-4). These proteins reside in the outer mitochondrial membranes as well as the cytosol and ER membranes. By keeping the mitochondrial outer membrane impermeable they prevent leakage of cytochrome c and other death-inducing proteins into the cytosol (Fig. 3A).

• Pro-apoptotic. BAX and BAK are the two prototypic members of this group. Like their anti-apoptotic cousins they also have four BH domains. Upon activation, BAX and BAK oligomerize within the outer mitochondrial protein and promote mitochondrial outer membrane permeability. The precise mechanism by which Bax-Bak permeabilize membranes is not settled. According to one model illustrated in Fig 3B, they form a channel in the outer mitochondrial membrane, allowing leakage of cytochrome c from the intermembranous space.

• Sensors. Members of this group, including BAD, BIM, BID, Puma, and Noxa, contain only one BH domain, the third of the four BH domains, and hence are sometimes called BH3-only proteins. BH3-only proteins act as sensors of cellular stress and damage, and regulate the balance between the other two groups, thus acting as arbiters of apoptosis.

Fig3. The intrinsic (mitochondrial) pathway of apoptosis. A, Cell viability is maintained by the induction of anti-apoptotic proteins such as BCL2 by survival signals. These proteins maintain the integrity of mitochondrial membranes and prevent leakage of mitochondrial proteins. B, Loss of survival signals, DNA damage, and other insults activate sensors that antagonize the anti-apoptotic proteins and activate the pro-apoptotic proteins BAX and BAK, which form channels in the mitochondrial membrane. The subsequent leakage of cytochrome c (and other proteins, not shown) leads to caspase activation and apoptosis.

Growth factors and other survival signals stimulate the production of anti-apoptotic proteins such as BCL2, thus preventing the leakage of death-inducing proteins from the outer mitochondrial membrane. When cells are deprived of survival signals or their DNA is damaged, or misfolded proteins induce ER stress, the BH3-only proteins “sense” such damage and are activated. These sensors in turn activate the two critical (pro-apoptotic) effectors, BAX and BAK, which form oligomers that insert into the mitochondrial membrane and allow proteins from the inner mitochondrial membrane to leak out into the cytoplasm. BH3-only proteins may also bind to and block the function of BCL2 and BCL-XL. At the same time, the synthesis of BCL2 and BCL-XL may decline because of the relative deficiency of survival signals. The net result of BAX-BAK activation coupled with loss of the protective functions of the anti-apoptotic BCL2 family members is the release into the cytoplasm of several mitochondrial proteins that can activate the caspase cascade (Fig. 3). As already mentioned, one of these proteins is cytochrome c, well known for its role in mitochondrial respiration.

Once released into the cytosol, cytochrome c binds to a protein called APAF-1 (apoptosis-activating factor-1), which forms a wheel-like hexamer that has been called the apoptosome. This complex is able to bind caspase-9, the critical initiator caspase of the mitochondrial pathway, and the enzyme cleaves adjacent caspase-9 molecules, thus setting up an autoamplification process. Cleavage activates caspase-9, which triggers a cascade of caspase activation by cleaving and thereby activating other pro-caspases, and the active enzymes mediate the execution phase of apoptosis (discussed later). Other mitochondrial proteins, with arcane names like Smac/Diablo, enter the cytoplasm, where they bind to and neutralize cytoplasmic proteins that function as physiologic inhibitors of apoptosis (called IAPs). The normal function of the IAPs is to block the activation of caspases, including executioners like caspase-3, and keep cells alive. Thus, the neutralization of these IAPs permits the initiation of a caspase cascade.

The Extrinsic (Death Receptor-Initiated) Pathway of Apoptosis

This pathway is initiated by engagement of plasma membrane death receptors on a variety of cells. Death receptors are members of the TNF receptor family that contain a cytoplasmic domain involved in protein-protein interactions that is called the death domain because it is essential for delivering apoptotic signals. (Some TNF receptor family members do not contain cytoplasmic death domains; their function is to activate inflammatory cascades [Chapter 3], and their role in triggering apoptosis is much less established.) The best known death receptors are the type 1 TNF receptor (TNFR1) and a related protein called Fas (CD95), but several others have been described. The mechanism of apoptosis induced by these death receptors is well illustrated by Fas, a death receptor expressed on many cell types (Fig. 4). The ligand for Fas is called Fas ligand (FasL). FasL is expressed on T cells that recognize self antigens (and functions to eliminate self-reactive lymphocytes), and on some cytotoxic T lymphocytes (which kill virus-infected and tumor cells). When FasL binds to Fas, three or more molecules of Fas are brought together, and their cytoplasmic death domains form a binding site for an adaptor protein that also contains a death domain and is called FADD (Fas-associated death domain). FADD that is attached to the death receptors in turn binds an inactive form of caspase-8 (and, in humans, caspase-10), again via a death domain. Multiple pro-caspase-8 molecules are thus brought into proximity, and they cleave one another to generate active caspase-8. The subsequent events are the same as in the mitochondrial pathway, and culminate in the activation of multiple executioner caspases. This pathway of apoptosis can be inhibited by a protein called FLIP, which binds to pro-caspase-8 but cannot cleave and activate the caspase because it lacks a protease domain. Some viruses and normal cells produce FLIP and use this inhibitor to protect themselves from Fas-mediated apoptosis.

Fig4. The extrinsic (death receptor initiated) pathway of apoptosis, illustrated by the events following Fas engagement. FAAD, Fas-associated death domain; FasL, Fas ligand.

The extrinsic and intrinsic pathways of apoptosis involve fundamentally different molecules for their initiation, but there may be interconnections between them. For instance, in hepatocytes and pancreatic β cells, caspase-8 produced by Fas signaling cleaves and activates the BH3- only protein BID, which then feeds into the mitochondrial pathway. The combined activation of both pathways delivers a fatal blow to the cells.

The Execution Phase of Apoptosis

The two initiating pathways converge to a cascade of caspase activation, which mediates the final phase of apoptosis. The mitochondrial pathway leads to activation of the initiator caspase-9, and the death receptor pathway to the initiator caspases-8 and -10. After an initiator caspase is cleaved to generate its active form, the enzymatic death program is set in motion by rapid and sequential activation of the executioner caspases. Executioner caspases, such as caspase-3 and -6, act on many cellular components. For instance, these caspases, once activated, cleave an inhibitor of a cytoplasmic DNase and thus make the DNase enzymatically active; this enzyme induces cleavage of DNA. Caspases also degrade structural components of the nuclear matrix and thus promote fragmentation of nuclei. Some of the steps in apoptosis are not fully defined. For instance, we do not know how the structure of the plasma membrane is changed in apoptotic cells, or how membrane blebs and apoptotic bodies are formed.

Removal of Dead Cells

The formation of apoptotic bodies breaks cells up into “bite-sized” fragments that are edible for phagocytes. Apoptotic cells and their fragments also undergo several changes in their membranes that actively promote their phagocytosis so they are most often cleared before they undergo secondary necrosis and release their cellular contents (which can result in injurious inflammation). In healthy cells, phosphatidylserine is present on the inner leaflet of the plasma membrane, but in apoptotic cells this phospholipid “flips” out and is expressed on the outer layer of the membrane, where it is recognized by several macrophage receptors. Cells that are dying by apoptosis secrete soluble factors that recruit phagocytes. Some apoptotic bodies are coated by thrombospondin, an adhesive glycoprotein that is recognized by phagocytes, and macrophages themselves may produce proteins that bind to apoptotic cells (but not to live cells) and thus target the dead cells for engulfment. Apoptotic bodies may also become coated with natural antibodies and proteins of the complement system, notably C1q, which are recognized by phagocytes. Thus, numerous receptors on phagocytes and ligands induced on apoptotic cells serve as “eat me” signals and are involved in the binding and engulfment of these cells. This process of phagocytosis of apoptotic cells is so efficient that dead cells disappear, often within minutes, without leaving a trace, and inflammation is absent even in the face of extensive apoptosis.

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مواضيع ذات صلة

Damage to DNA and Proteins

Causes of Apoptosis

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Catheter-related Bloodstream Infection (CRBSI)

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Mitochondrial Damage