Cell Death

How can apoptosis contribute to disease?

Either Excessive or Insufficient Apoptosis Can Contribute to Disease

Extrinsic Pathway of Apoptosis

Extracellular signal proteins binding to cell-surface death receptors trigger this pathway; FAS ligand dependent binding of TNF to its receptor and eventual activation of caspases - pathway initiated by engagement of plasma membrane death receptors on a variety of cells signal = cytokine ---> TNF Death receptor pathway (extrinsic); TNF = Tumor Necrosis Factor; FAS = Focal AdhesionS; FADD = Fas-Associated Death Domain; DISC = Death Inducing Signaling Complex (DISC)

The Intrinsic pathway of apoptosis

Intracellular apoptotic stimuli cause mitochondria to release cytochrome c, which interacts with Apaf1. the binding of cytochrome c causes Apaf1 to unfold partly, exposing a domain that interacts with the same domain in other activated Apaf1 molecules. Seven activated Apaf1 proteins form a large ring complex called the apoptosome. Each Apaf1 protein contains a caspase recruitment domain (CArD), and these are clustered above the central hub of the apoptosome. the CArDs bind similar domains in multiple caspase-9 molecules, which are thereby recruited into the apoptosome and activated. (the mechanism of caspase-9 activation is not clear: it probably results from dimerization and cleavage of adjacent caspase-9 proteins, but it might also depend on interactions between caspase-9 and Apaf1. ) Once activated, caspase-9 cleaves and thereby activates downstream executioner caspases. Note that the CArD is related in structure and function to the death effector domain of caspase-8

p53

Molecule that prevents mitosis in cells with damaged DNA. Changes in its activity can result in cancer. this tumor suppressor gene causes cell cycle arrest in G1, providing time for DNA repair. If repair is successful, cells re-enter the cycle. If unsuccessful, apoptosis acts as transcription regulator of cdk inhibitor p21 (blocks Cdk-S)

apoptosome

Multiprotein complex that consists of cytochrome c molecules and Apaf-1 and helps to initiate apoptosis by activating procaspase 9 into caspase 9. a large quaternary protein structure formed in the process of apoptosis. Its formation is triggered by the release of cytochrome c from the mitochondria in response to an internal (intrinsic) or external (extrinsic) cell death stimulus. A complex formed by the aggregation of cytochrome c and molecules of a protein called Apaf-1, that cleaves and activates the initiator caspase, caspase 9, thus triggering the caspase activation cascade in the intrinsic pathway of apoptosis.

hyperproliferative signals

Myc actites ARF (cell cycle inhibitor)

Apaf-1

Normally induces the activation of caspases. If Bcl-2 is overexpressed (e.g., follicular lymphoma), then Apaf-1 is overly inhibited have caspase9 recruitment domain (Apoptotic Protease Activating Factor) ; binds with cytochrome c, then activates caspase 9

CARD domain

caspase recruitment domain

p21

cell cycle regulatory protein that inhibits the cell cycle; its levels are controlled by p53

BH123 activation

forms pores activated by Bax/Bak

How can apoptosis contribute to disease? excessive apoptosis

excessive numbers of cells undergo apoptosis and thereby contribute to tissue damage: -Among the most dramatic examples are heart attacks and strokes. -In these acute conditions, many cells die by necrosis as a result of ischemia (inadequate blood supply), but some of the less affected cells die by apoptosis. It is hoped that, in the future, drugs that block apoptosis—such as specific caspase inhibitors—will prove useful in saving such cells.

Fas

extrinsic apoptotic pathway protein, found on the surface of a target cell by Fas ligand on the surface of a killer (cytotoxic) lymphocyte /NKT cells initiates DISC cascade

Ca2+ levels high intracellularly

induces apoptosis, cell death

IAPs Help Control Caspases

inhibitors of apoptosis (IAPs). have one or more BIR (baculovirus IAP repeat) domains, which enable them to bind to and inhibit activated caspases. Some IAPs also polyubiquitylate caspases, marking the caspases for destruction by proteasomes. In this way, the IAPs set an inhibitory threshold that caspases must overcome to trigger apoptosis. Anti-IAPs are released from the mitochondrial intermembrane space when the intrinsic pathway of apoptosis is activated, blocking IAPs in the cytosol and thereby promoting apoptosis. However, mice appear to develop normally if they are missing either the major mammalian IAP (called XIAP) or the two known mammalian anti-IAPs (called Smac/Diablo and Omi). Worms do not even contain a caspase- inhibiting IAP protein. Apparently, the tight control of caspase activity is achieved by different mechanisms in different animals.

Intrinsic pathway of apoptosis

mitochondrial leakage of cytochrome c into the cytosol with eventual activation of caspases; cytochrome c, a water-soluble component of the mitochondrial electron-transport chain. When released into the cytosol : it binds to Apaf1 (apoptotic protease activating factor-1), causing the Apaf1 to oligomerize into a wheel-like heptamer called an apoptosome. The Apaf1 proteins in the apoptosome then recruit initiator caspase-9 proteins, which are thought to be activated by proximity in the apoptosome, just as caspase-8 is activated in the DISC. The activated caspase-9 molecules then activate downstream executioner caspases to induce apoptosis -Involved in tissue remodeling in embryogenesis. Occurs when a regulating factor is withdrawn from a proliferating cell population (e.g., decreased IL-2 after a completed immunologic reaction --> apoptosis of proliferating effector cells). Also occurs after exposure to injurious stimuli (e.g., radiation, toxins, hypoxia). -Changes in proportions of anti- and pro- apoptotic factors increasing mitochondrial permeability and cytochrome c release. BAX and BAK are proapoptotic proteins; Bcl-2 is antiapoptotic. Bcl2 proteins regulate this path in mammals : regulate the intrinsic pathway of apoptosis mainly by controlling the release of cytochrome c and other intermembrane mitochondrial proteins into the cytosol. -Bcl-2 prevents cytochrome c release by binding to and inhibiting Apaf-1. Apaf-1 normally induces the activation of caspases. If Bcl-2 is overexpressed (e.g., follicular lymphoma), then Apaf-1 is overly inhibited, caspase activation and tumorigenesis.

Apoptosis is regulated by

mitogens, growth factors, and survival factors Bcl2 family proteins; mitochondria p53 produced in response to dna damage (typically G1 phase) or acute cellular stress causes cell cycle to arrest; sensiesence; apoptosis

Fas ligand

molecule expressed on cytotoxic T cells and NK cells that binds to the fas molecule on a target cell and induces it do undergo apoptosis

p53 and apoptosis

p53: transcripton factor effect activating effect on apoptosis Induced apop: 1. increaseproapop genes 2. Activating Bax to bind membrane - DNA damage > p53 > cell cycle inhibitor p21 - p53 > pro-apoptotic Bcl-2 - p53 > Fas receptor (CD95) - p53 > IGFBP-3 (sequesters cell survival proteins away from receptors)

Extracellular Survival Factors Inhibit Apoptosis in Various Ways Survival Factors

part of the normal "social" controls that ensure that individual cells behave for the good of the organism as a whole—in this case, by surviving when they are needed and killing themselves when they are not. Some extracellular signal molecules stimulate apoptosis, whereas others inhibit it. (Fas ligand that activate death receptors and thereby trigger the extrinsic pathway of apoptosis). Most animal cells require continuous signaling from other cells to avoid apoptosis (helps ensure that cells survive only when and where they are needed). e.g. Nerve cells, are produced in excess in the developing nervous system and then compete for limited amounts of survival factors that are secreted by the target cells that they normally connect to (see Figure 21-81). Nerve cells that receive enough survival signals live, while the others die. In this way, the number of surviving neurons is automatically adjusted so that it is appropriate for the number of target cells they connect with (Figure 18-11). A similar competition for limited amounts of survival factors produced by neighboring cells is thought to control cell numbers in other tissues, both during development and in adulthood. usually bind to cell-surface receptors, which activate intracellular signaling pathways that suppress the apoptotic program, often by regulating members of the Bcl2 family of proteins. -Some survival factors, for example, stimulate the synthesis of anti-apoptotic Bcl2 family proteins such as Bcl2 itself or BclXL. -Others act by inhibiting the function of pro-apoptotic BH3-only proteins such as Bad (Figure M18-12B). In Drosophila, some survival factors act by phosphorylating and inactivating anti-IAP proteins such as Hid, thereby enabling IAP proteins to suppress apoptosis (Figure 18-12C). Some develop- ing neurons, like those illustrated in Figure 18-11, use an ingenious alternative approach: survival-factor receptors stimulate apoptosis—by an unknown mecha- nism—when they are not occupied, and then stop promoting death when survival factor binds. The end result in all these cases is the same: cell survival depends on survival factor binding.

How pro-apoptotic BH3- only and anti-apoptotic Bcl2 family proteins regulate the intrinsic pathway of apoptosis.

(A) In the absence of an apoptotic stimulus, anti-apoptoticBcl2 family proteins bind to and inhibit the effector Bcl2 family proteins on the mitochondrial outer membrane (and in the cytosol—not shown). (B) In the presence of an apoptotic stimulus, Bh3-only proteins are activated and bind to the anti-apoptotic Bcl2 family proteins so that they can no longer inhibit the effector Bcl2 family proteins; the latter then become activated, aggregate in the outer mitochondrial membrane, and promote the release of intermembrane mitochondrial proteins into the cytosol. Some activated Bh3-only proteins may stimulate mitochondrial protein release more directly by binding to and activating the effector Bcl2 family proteins. Although not shown, the anti- apoptotic Bcl2 family proteins are bound to the mitochondrial surface.

apoptosis function

-Helps body get rid of cells it doesn't need -Removes genetically damaged cells -Plays an important role in the development of the embryo and the maintenance of adult tissues Eliminates excess cells, carving out structures and weeds out aging or defective cells. ELIMINATES UNWANTED CELLS functions as a quality-control process in development, eliminating cells that are abnormal, misplaced, nonfunctional, or potentially dangerous to the ani- mal. e.g. in the vertebrate adaptive immune system, where apoptosis eliminates developing T and B lymphocytes that either fail to produce potentially useful antigen-specific receptors or produce self-reactive receptors that make the cells potentially dangerous it also eliminates most of the lymphocytes activated by an infection, after they have helped destroy the responsible microbes. embryological development

proteins that are cleaved by caspases during apoptosis:

-nuclear lamins, the cleavage of which causes the irreversible breakdown of the nuclear lamina. -iCAD--Another target is a protein that normally holds a DNA- degrading endonuclease in an inactive form; its cleavage frees the endonuclease to cut up the DNA in the cell nucleus . -Other target proteins include components of the cytoskeleton and cell-cell adhesion proteins that attach cells to their neighbors; the cleavage of these proteins helps the apoptotic cell to round up and detach from its neighbors, making it easier for a neighboring cell to engulf it, or, in the case of an epithelial cell, for the neighbors to extrude the apoptotic cell from the cell sheet.

Three ways that extracellular survival factors can inhibit apoptosis. Apoptosis Inhibition

1. increased production of anti-apoptotic Bcl2 family protein 2. inactivation of pro- apoptotic BH3-only protein 3. inactivation of anti-IAPs (A) Some survival factors suppress apoptosis by stimulating the transcription of genes that encode anti- apoptotic Bcl2 family proteins such as Bcl2 itself or BclXL. (B) Many others activate the serine/threonine protein kinase Akt, which, among many other targets, phosphorylates and inactivates the pro-apoptotic Bh3-only protein Bad (see Figure 15-53). When not phosphorylated, Bad promotes apoptosis by binding to and inhibiting Bcl2; once phosphorylated, Bad dissociates, freeing Bcl2 to suppress apoptosis. Akt also suppresses apoptosis by phosphorylating and inactivating transcription regulatory proteins that stimulate the transcriptionof genes encoding proteins that promote apoptosis (not shown). (C) In Drosophila, some survival factors inhibit apoptosis by stimulating the phosphorylation of the anti- IAp protein hid. When not phosphorylated, hid promotes cell death by inhibitingIAps. Once phosphorylated, hid no longer inhibits IAps, which become active and block apoptosis. MAp kinase, mitogen- activated protein kinase.

how many different capsases are there?

14 kinds 3, 6, 7 = executioner caspases

procaspase activation by cleavage

2 main pathways: 1) death receptor pathway (extrinsic); and 2) mitochondrial pathway (intrinsic). Both pathways branch into many pathways, lead to death, and rely on the caspase family. procaspase = inactive form activated by proteolytic precursors by apoptotic signals which cleaves part of inactive procaspases (removal of domains activates capsase) dimer ----> active tetramer

initiator caspases how is this first activated?

2, 8, 9, 10 begin the apoptotic process. They normally exist as inactive, soluble monomers in the cytosol apoptotic signal triggers the assembly of large protein platforms that bring multiple initiator caspases together into large complexes. Within these complexes, pairs of caspases associate to form dimers, resulting in protease activation Each caspase in the dimer then cleaves its partner at a specific site in the protease domain, which stabilizes the active complex and is required for the proper function of the enzyme in the cell. The major function of the initiator caspases is to activate the executioner caspases. this is first activated in response to an apoptotic signal- The two best-understood activation mechanisms in mammalian cells are called the extrinsic pathway and the intrinsic, or mitochondrial, pathway. Each uses its own initiator caspase and activation system.

executioner caspases

3, 6, 7 These normally exist as inactive dimers. When they are cleaved by an initiator caspase at a site in the protease domain, the active site is rearranged from an inactive to an active conformation. One initiator caspase complex can activate many executioner caspases, resulting in an amplifying proteolytic cascade. Once activated, executioner caspases catalyze the widespread protein cleavage events that kill the cell.

Bcl2 family proteins (B-Cell Lymphoma cells)

A major class of intracellular regulators of the intrinsic pathway is the Bcl2 family of proteins, which, like the caspase family, has been conserved in evolution from worms to humans; a human Bcl2 protein, for example, can suppress apoptosis when expressed in the worm Caenorhabditis elegans. participate in the tight regulation to ensure that cells kill themselves only when it is appropriate. Mammalian Bcl2 family proteins regulate the intrinsic pathway of apoptosis mainly by controlling the release of cytochrome c and other intermembrane mitochondrial proteins into the cytosol. Some Bcl2 family proteins are pro-apoptotic and promote apoptosis by enhancing the release, whereas others are anti-apoptotic and inhibit apoptosis by blocking the release (Bcl2, BclXL). The pro-apoptotic and anti-apoptotic proteins can bind to each other in various combinations to form heterodimers in which the two proteins inhibit each other's function. (BAX & BAK)---->death promoters The balance between the activities of these two functional classes of Bcl2 family proteins largely determines whether a mammalian cell lives or dies by the intrinsic pathway of apoptosis.

Death inducing signalling complex (DISC)

A multiprotein complex that is formed by recruitment of adaptor proteins to death receptors upon ligand binding. The complex recruits initiator procaspase molecules, which are autoactivated. activate executioner caspases 3, 6, 7

FADD

Adapter protein that binds inactive caspases, activating them after crosslinking of Fas and FasL binds FAS death receptor protein tails death effector domain recruits initiator caspases (8,10, or both)

Phenobarbital

Anticonvulsant, Barbiturate causes liver to swell, apoptosis returns it to normal size

Carnegie Stages of Human Development

Based on both internal and external development and not on age or size. By comparing common stages between organisms you can compare the development time between species. apoptosis participates in limb sculpture (combo of regeneration and apoptosis)

Death receptors

Cell surface receptors to which ligands that activate the extrinsic apoptotic pathway bind. The ligands may be secreted factors or molecules expressed on the surface of other cells. are transmembrane proteins containing an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular death domain, which is required for the receptors to activate the apoptotic program. The receptors are homotrimers and belong to the tumor necrosis factor (TNF) receptor family, which includes a receptor for TNF itself and the Fas death receptor. The ligands that activate the death receptors are also homotrimers; they are structurally related to one another and belong to the TNF family of signal proteins.

the Intrinsic pathway of Apoptosis Depends on Mitochondria

Cells that activate their apoptosis program from inside the cell, often in response to stresses, such as DNA damage, or in response to developmental signals. governed by the intrinsic, or mitochondrial, pathway of apoptosis, which depends on the release into the cytosol of mitochondrial proteins that normally reside in the intermembrane space of these organelles (see Figure 12-19). Some of the released proteins activate a caspase proteolytic cascade in the cytoplasm, leading to apoptosis. A key protein in the intrinsic pathway is cytochrome c, a water-soluble component of the mitochondrial electron-transport chain. When released into the cytosol (Figure 18-6), it takes on a new function: it binds to an adaptor protein called Apaf1 (apoptotic protease activating factor-1), causing the Apaf1 to oligo- merize into a wheel-like heptamer called an apoptosome. The Apaf1 proteins in the apoptosome then recruit initiator caspase-9 proteins, which are thought to be activated by proximity in the apoptosome, just as caspase-8 is activated in the DISC. The activated caspase-9 molecules then activate downstream executioner caspases to induce apoptosis Bcl2 proteins facilitates/mediates (tightly regulate) this path to ensure that cells kill themselves only when it is appropriate. A major class of intracellular regulators of the intrinsic pathway is the Bcl2 family of proteins, which, like the caspase family, has been conserved in evolution from worms to humans; a human Bcl2 protein, for example, can suppress apoptosis when expressed in the worm Caenorhabditis elegans.

death-inducing signaling complex (DISC).

Complex in which initiator caspases are brought into close proximity and activated in the extrinsic pathway of apoptosis extrinsic path Fas protein dependent

Reaper, Grim, and hid

IAP

Multiple robust mechanisms exist to ensure that proteases are activated only when appropriate since activation of a caspase cascade leads to certain death. what are they?

IAPs Survival Factors Inhibit Apoptosis in Various Ways mitogens growth factors

How do the Bcl2 proteins regulate the (extrinsic/intrinsic?) pathway of Apoptosis?

INTRINSIC regulate the intrinsic pathway of apoptosis mainly by controlling the release of cytochrome c and other intermembrane mitochondrial proteins into the cytosol. Some Bcl2 family proteins are pro-apoptotic and promote apoptosis by enhancing the release, whereas others are anti-apoptotic and inhibit apoptosis by blocking the release. The pro-apoptotic and anti-apoptotic proteins can bind to each other in various combinations to form heterodimers in which the two proteins inhibit each other's function. The balance between the activities of these two functional classes of Bcl2 family proteins largely determines whether a mammalian cell lives or dies by the intrinsic path- way of apoptosis.

Bax and Bak

Proapoptotic proteins; change in concentration affects mitochondrial permeability and cytochrome c release (increased BAX and BAK = increased mitochondrial permeability and cytochrome c release)

BAX & BAK

Proapoptotic proteins; change in concentration affects mitochondrial permeability, and insert inside of omm and causes the cytochrome c release into cytoplasm (increased BAX and BAK = increased mitochondrial permeability and cytochrome c release) adaptor protein (e.g. Apaf1) will bind cytochrome c (ACTIVATES Apaf1) E required ATP hydrolysis as ATP is hydrolyzed, CytochromeC-APAF1 complex conjoin with several other CytochromeC-APAF1 complexes to form apoptosome apoptosome recruits procaspase9 molecules and activate them to caspase9 BAX/BAK mediates the release of cytochrome c and other mm matrix proteins

necrosis pathway

Reversible injury ( with possibility of recovery) --> Myelin appears, swelling of mitochondria and ER, membrane blebs --> (Progressive injury) --> Breakdown of plasma membrane, organelles, nucleus and leakage of cellular contents, amorphous densities in mitochondria ( Necrosis) --> Inflammation

TNF

Secreted by macrophages. Mediates septic shock. Causes leukocyte recruitment, vascular leak. facilitates apoptosis; a cytokine TNFR Superfamily; TRADD = TNFR-Associated Death Domain; FADD = Fas Associated Death Domain

involution

Shrinking of the uterus (womb) to its normal size after childbirth. starts within 12-24-48 hrs of weaning infant off of milk cells of milk duct prod inhibitory proteins which leads to activation of STAT3 (TF); activation of apoptotic genes once activated, cells of milk duct start to die (apoptotic bodies are shoved into alveolar lumen of ducts)

caspase cascade

Signal that becomes progressively more intense; insures that once trigger is pulled the process pushes forward with full force An Enzymatic Cascade Culminates in *DNA Degradation and Apoptosis* - one initiator --> activates executioners --> activates more executioners - as caspases are activated, they BREAK DOWN cell components and ACTIVATE other caspases

BH3-only proteins provide the crucial link between apoptotic stimuli and the intrinsic pathway of apoptosis

Some extracellular survival signals, for example, block apoptosis by inhibiting the synthesis or activity of certain BH3-only proteins Similarly, in response to DNA damage that cannot be repaired, the tumor suppressor protein p53 accumulates (discussed in Chapters 17 and 20) and activates the transcription of genes that encode the BH3-only proteins Puma and Noxa. These BH3-only proteins then trigger the intrinsic pathway, thereby eliminating a potentially dangerous cell that could otherwise become cancerous. in some cells the extrinsic apoptotic pathway recruits the intrinsic pathway to amplify the caspase cascade to kill the cell. The BH3-only protein Bid is the link between the two pathways. Bid is normally inactive. How- ever, when death receptors activate the extrinsic pathway in some cells, the initia- tor caspase, caspase-8, cleaves Bid, producing an active form of Bid that trans- locates to the outer mitochondrial membrane and inhibits anti-apoptotic Bcl2 family proteins, thereby amplifying the death signal.

effector Bcl2 family proteins for anti-apoptotic path

The anti-apoptotic Bcl2 family proteins such as Bcl2 itself and BclXL are also located on the cytosolic surface of the outer mitochondrial membrane, where they help prevent inappropriate release of intermembrane proteins. inhibit apoptosis mainly by binding to and inhibiting pro-apoptotic Bcl2 family proteins—either on the mitochondrial membrane or in the cytosol. On the outer mitochondrial membrane, for example, they bind to Bak and prevent it from oligomerizing, thereby inhibiting the release of cytochrome c and other intermembrane proteins. There are at least five mammalian anti-apoptotic Bcl2 family proteins, and every mammalian cell requires at least one to survive. Moreover, a number of these proteins must be inhibited for the intrinsic pathway to induce apoptosis; the BH3-only proteins mediate the inhibition.

cytochrome c

The enzyme to which electrons are transferred in complex III of the electron transport chain. Involved in energy transfer, it is a protein released from mitochondria when cell is stressed. When released into the cytosol: it binds to an adaptor protein called Apaf1 (apoptotic protease activating factor-1), causing the Apaf1 to oligomerize into a wheel-like heptamer called an apoptosome. The Apaf1 proteins in the apoptosome then recruit initiator caspase-9 proteins, which are thought to be activated by proximity in the apoptosome, just as caspase-8 is activated in the DISC. The activated caspase-9 molecules then activate downstream executioner caspases to induce apoptosis

capsases

The enzyme which is responsible for disassembly of intracellular proteins during apoptosis. They have a cysteine in the active site which cleaves target proteins at aspartic acid sites. apoptosis is reliant upon these to mediate proteolytic cascade; -this cascade is not only destructive and self- amplifying but also irreversible, so that once a cell starts out along the path to destruction, it cannot turn back. correlate to proteolytic activity of specific components of a cell -- lamins, actin (cytoskeleton; results in blebbing) involved in intrinsic and extrinsic pathways

Extrinsic apoptosis pathway

The extrinsic pathway of apoptosis activated through Fas death receptors. trimeric Fas ligands on the surface of a killer lymphocyte interact with trimeric Fas receptors on the surface of the target cell, leading to clustering of several ligand-bound receptor trimers receptor clustering activates death domains on the receptor tails, which interact with similar domains on the adaptor protein FADD (FADD stands for Fas-associated death domain). Each FADD protein then recruits an initiator caspase (caspase-8) via adeath effector domain on both FADD and the caspase, forming a death-inducing signaling complex (DISC). Within the DISC, two adjacent initiator caspases interact and cleave one another to form an activated protease dimer, which then cleaves itself in the region linking the protease to the death effector domain. this stabilizes and releases the active caspase dimer into the cytosol, where it activates executioner caspases by cleaving them.

BH3-only proteins

The largest subclass of Bcl2 family proteins. Produced or activated in response to an apoptotic stimulus and promote apoptosis mainly by inhibiting anti-apoptotic Bcl2 family proteins. A) INACTIVE PATH-- BAX/BAK are inhibited by anti apoptotic proteins (also Bcl2 fam) B)ACTIVATION PATH (to prevent mitochondrial leakage)--BAX/BAK bind to PRO apoptotic Bcl2 proteins Pro-apoptotic Bcl2 proteins which bind to and inactivating anti-apoptotic Bcl2, thus allowing the oligomerization of effector Bcl2 proteins within the outer mitochondrial membrane BH3 domain binds to a long hydrophobic groove on anti-apoptotic Bcl2 family proteins, neutralizing their activity. This binding and inhibition enables the aggregation of Bax and Bak on the surface of mitochondria, which triggers the release of the intermembrane mitochondrial proteins that induce apoptosis these proteins provide the crucial link between apoptotic stimuli and the intrinsic pathway of apoptosis, with different stimuli activating different BH3- only proteins. A

The role of pro-apoptotic effector Bcl2 family proteins (mainly Bax and Bak) in the release of mitochondrial intermembrane proteins in the intrinsic pathway of apoptosis:

When activated by an apoptotic stimulus, the effector Bcl2 family proteins aggregate on the outer mitochondrial membrane and release cytochrome c and other proteins from the intermembrane space into the cytosol by an unknown mechanism.

mitochondrial pathway

activated by acute stress (including necrotic activation factors) -- low O2/cytotoxins/etc) involves an imbalance of the Bcl family, which induce leakage of mitochondrial proteins (Bax, Bak). Some other Bcl family members (Bcl-2, Bcl-x) are anti-apoptotic proteins ("regulators") that inhibit mitochondrial leakiness and cytochrome c-dependent caspase activation.c

Apoptosis is dependent upon

an Intracellular proteolytic Cascade that Is Mediated by Caspases

apoptotic signal activation mechanisms

apoptotic signal activates an extrinsic pathway and the intrinsic, or mitochondrial, pathway. Each uses its own initiator caspase and activation system.

The BH123 proteins Bax & Bak promote apoptosis by

apoptotic stimulus activates BH3-only protein and binds to antiapoptotic Bcl2 protein (causing it to dissociate from BH123 proteins (Bcl2)) ----> BH123 proteins aggregate to from pores / channels in the omm membrane aggregate to form channels in the mitochondrial outer membrane Bcl2 protein

apoptosis fxn In adult tissues that are neither growing nor shrinking,

cell death and cell division must be tightly regulated to ensure that they are exactly in balance. Thus, the liver is kept at a constant size through the regulation of both the cell death rate and the cell birth rate. Animal cells can recognize damage in their various organelles and, if the dam- age is great enough, they can kill themselves by undergoing apoptosis. An important example is DNA damage, which can produce cancer-promoting mutations if not repaired. Cells have various ways of detecting DNA damage, and undergo apoptosis if they cannot repair it.

cell necrosis

cellular death due to stressors of insults that overwhelm the cell's ability to survive, it is irreversible. You get an enlarged cell size, disrupted plasma membrane, cellular contents leak out of cell and frequent adjacent inflammation cells that die in response to an acute insult, such as trauma or a lack of blood supply cells swell and burst, spilling their contents over their neighbors and eliciting an inflammatory response; e.g. nucleases, proteases this process in most cases is likely to be caused by energy depletion, which leads to metabolic defects and loss of the ionic gradients that normally exist across the cell membrane. affect cell membrane integrity

Pro-apoptotic proteins

consist of two subfamilies—the effector Bcl2 family proteins and the BH3-only pro- teins. The main effector proteins are Bax and Bak, which are structurally similar to Bcl2 but lack the BH4 domain. The BH3-only proteins share sequence homology with Bcl2 in only the BH3 domain. :BAX and BAK stim in response to loss of signals, ER stress, DNA damage, Bim, Bid, Bad- have Bcl-2 homology domain activate Bax and Bak- form oligomers-insert in mito mem-> channels that leak into cyto BH3 only proteins block Bcl-2/-x

Apoptotic signals / triggers

cytokines; radiation; activating signals (physiological & pathological); withdraw from hormones and GF signals; chemotherapy

why do macrophages not phagocytose healthy cells?

despite the fact that healthy cells normally expose some phosphatidylserine on their surfaces. Healthy cells express signal proteins on their surface that interact with inhibitory receptors on macrophages that block phagocytosis. Thus, in addition to express- ing cell-surface signals such as phosphatidylserine that stimulate phagocytosis, apoptotic cells must lose or inactivate these "don't eat me" signals that block phagocytosis.

FLIP protein

inhibitory proteins that inhibit extrinsic apoptosis pathway -- resembles an initiator caspase but has no protease activity because it lacks the key cysteine in its active site. dimerizes with caspase-8 in the DISC; although caspase-8 appears to be active in these heterodimers, it is not cleaved at the site required for its stable activation, and the apoptotic signal is blocked. Such inhibitory mechanisms help prevent the inappropriate activation of the extrinsic pathway of apoptosis.

Perforin/Granzyme Pathway

involves synthesis of special killing proteins -in response to virally infected cells 1. Cytotoxic T-cells secretes perforin & granzymes 2. Perforin forms pores in target cell 3. Granzyme B enters cell and activates caspase 10 by cleavage = inactivation of apoptosis inhibitors 4. Also activates caspase 3 = execution of apoptosis binding of a virus-infected cell by an active cytotoxic T cell. After perforin and granzymes have been released from Tc cell vesicles, granzymes enter infected cell through the perforin complex for and activate the enzyme of apoptosis

Necropoptosis

is a form of programmed cell death that is triggered by a specific regulatory signal from other cells, although we are only just beginning to understand the underlying mechanisms.

what removes an apoptotic cell?

phagocytes / macrophages the apoptotic cell and its fragments do not break open and release their contents, but instead remain intact as they are efficiently eaten—or phagocytosed—by neighboring cells, leaving no trace and therefore triggering no inflammatory response engulfment process depends on chemical changes on the surface of the apoptotic cell, which displays signals that recruit phagocytic cells. An especially important change occurs in the distribution of the negatively charged phospholipid phosphatidylserine on the cell surface. -This phospholipid is normally located exclusively in the inner leaflet of the lipid bilayer of the plasma membrane (see Figure 10-15), but it flips to the outer leaflet in apoptotic cells. -The external exposure of phosphatidylserine is likely to depend on caspase cleavage of some protein involved in phospholipid distribution in the membrane. A variety of soluble "bridging" proteins interact with the exposed phosphatidylserine on the apoptotic cell. These bridging proteins also interact with specific receptors on the surface of a neigh- boring cell or macrophage, triggering cytoskeletal and other changes that initiate the engulfment process.

membrane blebbing

process that keeps an apoptotic cell from losing its membrane integrity

Apoptosis

programmed cell death cell systematically destroys itself from within and is then eaten by other cells, leaving no trace; irreversible nucleus and cytoplasm condense; membrane blebbing; nuclear envelope disappears; chromatin and dna fragment, cytoskeleton collapses; cells fragment (apoptotic bodies) shrink and condense, the cytoskeleton collapses, the nuclear envelope disassembles, and the nuclear chromatin condenses and breaks up into fragments apoptotic bodies. The surface of the cell or apoptotic bodies becomes chemically altered, so that a neighboring cell or a macrophage rapidly engulfs them, before they can spill their contents

death receptors stimulate the extrinsic pathway of apoptosis:

requires: the activation of Fas on the surface of a target cell by Fas ligand on the surface of a killer (cytotoxic) lymphocyte. When activated by the binding of Fas ligand, the death domains on the cytosolic tails of the Fas death receptors bind intracellular adaptor proteins, which in turn bind initiator caspases (primarily caspase-8), forming a death-inducing signaling complex (DISC). Once dimerized and activated in the DISC, the initiator caspases cleave their partners and then activate downstream executioner caspases to induce apoptosis (Figure 18-5). In some cells, the extrinsic pathway recruits the intrinsic apoptotic pathway to amplify the caspase cascade and kill the cell. many cells produce inhibitory proteins that act to inhibit the extrinsic pathway. e.g. some cells prod/release the protein FLIP, which resembles an initiator caspase but has no protease activity because it lacks the key cysteine in its active site. FLIP dimerizes with caspase-8 in the DISC; although caspase-8 appears

apoptotic bodies

small membrane-bound vesicles produced during the programmed death of a cell by apoptosis have glycoprotein markers w/ affinity for phagocytic cells (macrophages)

effector Bcl2 family proteins for Pro-apoptotic path

stimulus triggers apoptotic path pro-apoptotic effector Bcl2 family proteins become activated and aggregate to form oligomers in the mitochondrial outer membrane, inducing the release of cytochrome c and other intermembrane proteins by an unknown mechanism In mammalian cells, Bax and Bak are the main effector Bcl2 family proteins, and at least one of them is required for the intrinsic pathway of apoptosis to operate: mutant mouse cells that lack both proteins are resistant to all pro-apoptotic sig- nals that normally activate this pathway. Whereas Bak is bound to the mitochondrial outer membrane even in the absence of an apoptotic signal, Bax is mainly located in the cytosol and translocates to the mitochondria only after an apoptotic signal activates it.

How can apoptosis contribute to disease? insufficient apoptosis

too few cells die by apoptosis. -Mutations in mice and humans, for example, that inactivate the genes that encode the Fas death receptor or the Fas ligand prevent the normal death of some lymphocytes, causing these cells to accumulate in excessive numbers in the spleen and lymph glands. In many cases, this leads to autoimmune disease, in which the lymphocytes react against the individual's own tissues. Decreased apoptosis also makes an important contribution to many tumors, as cancer cells often regulate their apoptotic program abnormally. -The Bcl2 gene, for example, was first identified in a common form of lymphocyte cancer in humans, where a chromosome translocation causes excessive production of the Bcl2 protein; indeed, Bcl2 gets its name from this B cell lymphoma. -The high level of Bcl2 protein in the lymphocytes that carry the translocation promotes the development of cancer by inhibiting apoptosis, thereby prolonging lymphocyte survival and increasing their number; it also decreases the cells' sensitivity to anticancer drugs, which commonly work by causing cancer cells to undergo apoptosis. Similarly, the gene encoding the tumor suppressor protein p53 is mutated in about 50% of human cancers so that it no longer promotes apoptosis or cell-cycle arrest in response to DNA damage. The lack of p53 function therefore enables the cancer cells to survive and proliferate even when their DNA is damaged; in this way, the cells accumulate more mutations, some of which make the cancer more malignant (discussed in Chapter 20). As many anticancer drugs induce apoptosis (and cell-cycle arrest) by a p53-dependent mechanism (discussed in Chapters 17 and 20), the loss of p53 function also makes cancer cells less sensitive to these drugs. If decreased apoptosis contributes to many cancers, then we might be able to treat those cancers with drugs that stimulate apoptosis. This line of thinking has recently led to the development of small chemicals that interfere with the function of anti-apoptotic Bcl2 family proteins such as Bcl2 and BclXL. These chemicals bind with high affinity to the hydrophobic groove on anti-apoptotic Bcl2 family proteins, blocking their function in essentially the same way that BH3-only pro- teins do (Figure 18-13). The intrinsic pathway of apoptosis is thereby stimulated, which in certain tumors increases the amount of cell death. Most human cancers arise in epithelial tissues such as those in the lung, intestinal tract, breast, and prostate. Such cancer cells display many abnormalities in their behavior, including a decreased ability to adhere to the extracellular matrix and to one another at specialized cell-cell junctions. In the next chapter, we dis- cuss the remarkable structures and functions of the extracellular matrix and cell junctions.