Don't think of death as an ending. Think of it as a really effective way of cutting down your expenses.

Woody Allen

6/12/12

Apoptosis

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Introducti on Function Induction Caspases Mechanis ms Regulatio n Recogniti 6/12/12 on

For every cell, there is a time to live and a time to die. There are two ways in which cells die: 1.Cell death by injury (NECROSIS) - Mechanical damage - Exposure to toxic chemicals - Lack of Oxygen - Extremes of temperature 2. Cell death by suicide

APOPTOSIS
Provoking stimuli Programmed tissues remodelling Genomic damage

NECROSIS
Metabolic stresses Changes in pH, temperature Hypoxia, anoxia Injuries

Morphological changes Affected cells Cell volume Chromatin Lysosomes Mitochondria Inflammatory response Cell fate

Individual cells Decreased Condensed Unaffected Normal initially None Apoptotic bodies are phagocytated

Group of cells Increased Fragmented Abnormal Aberrant morphologically Marked Lysis

Molecular changes Gene activity Chromosomal DNA Ca2+ intracellular Ion pumps

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Required Cleaved at specific sites Increased Functioning

Not needed Random cleavage Unaffected Lost

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Why should a cell commit 1. Apoptosissuicide? proper is needed for

development -The resorption of the tadpole tail at the time of its metamorphosis into a frog occurs by apoptosis. -The formation of the fingers and toes of the fetus requires the Introducti on Function Induction Caspases Mechanis ms Regulatio n Recogniti 6/12/12 on removal, by apoptosis, of the tissue between them. -The formation of the proper connections (synapses) between neurons in the brain requires that surplus cells be eliminated by apoptosis. -Formation of reproductive organs (Mllerian duct--uterus, deleted in males;

Resorption of tail during metamorphosis Development of toes

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Incomplete apoptosis

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2. Apoptosis is needed to destroy cells Cells infected with viruses One of the methods by which cytotoxic T lymphocytes (CTLs) kill virusinfected cells is by inducing apoptosis . (And some viruses mount countermeasures to thwart it ) Cells of the immune

What makes a cell decide to commit suicide?

1. Withdrawal of positive signals

The continued survival of most cells requires that they receive continuous stimulation from other cells and, for many, continued adhesion to the surface on which they are growing. Some examples of positive signals: growth factors for neurons Interleukin-2 (IL-2), an essential factor for the mitosis of lymphocytes.

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Introducti on Function Induction Caspases Mechanis ms Regulatio n Recogniti 6/12/12 on

2.Receipt of negative signals increased levels of oxidants within the cell damage to DNA by these oxidants or other agents like ultraviolet light x-rays chemotherapeutic drugs accumulation of proteins that failed to fold properly into their proper tertiary structure molecules that bind to specific receptors on the cell surface and signal the cell to begin the apoptosis program. These death activators include: Tumor necrosis factor-alpha (TNF-) that binds to the TNF receptor; Lymphotoxin (also known as TNF-) that also binds to the TNF receptor; Fas ligand (FasL), a molecule that binds to a cell-surface receptor named Fas (also called CD95).

Apoptosis is induced by signaling cascades

The pathway for apoptosis, which was first elucidated in Caenorhabditis elegans, involves three so called ced gene products. CED-4 protein, activates the protease CED-3 which then initiates the destruction of the cell; CED-9 function is to inactivate CED-4. EGL-1 functions to inhibit CED-9 and hence over expression induces apoptosis.
Major players of Apoptosis

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C. caenorhabditis CED-3 CED-4 CED-9 EGL-1

Mammals Caspases Apaf-1 Anti-apoptotic Bcl-2 Pro-apoptotic Bcl-2

Caspases(for cysteine aspartate-specific proteases)

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22 heterotetramers that consist of two large subunits(~300 residues) and two small subunits(~100 residues) Expressed as zymogens (procaspases) that have three domains: an N-terminal prodomain that is proteolytically excised on activation, followed by

Caspase activation

Prodomai n

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Types of caspases

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11 caspases have been discovered out which 6 are involved in apoptosis and 5 in cytokine activation and hence control of inflammation. There are two classes of apoptotic caspases: 1. Initiator caspases 2. Effector caspases

1.Initiator Caspases(caspase-8,9,10)

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These have long prodomains Caspase-8 and-10 each contains Death Effector Domains, through which they bind to DEDs on their target adaptor proteins Caspase-9 contains caspase recruitment domain(CARD) that promotes the

2.Effector Caspases(caspase3,6 and-7)

These have short prodomains and are activated by initiator caspases They cleave a wide variety of cellular proteins, thereby bringing about apoptosis.

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Over 60 cellular proteins have been identified as caspase substrates that includes:

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Cytoskeletol proteins - actin Protein components of intermediate filaments forming nuclear envelope - lamins Those involved in cell cycle regulation-cyclin A, pRB Those involved in DNA replicationTopoisomerase I Those involved in signal transduction -RasGAP and protein kinase C Transcription Factors- NF-B

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During apoptosis, caspaseactivated Dnase (CAD) cleaves the chromosomal DNA at the sites often followed by nucleosomes to yield a series of DNA fragments that differ in their lengths by increments of ~200bp. CAD is expressed in all tissues in complex with its inhibitor ICAD, which on induction of

Caspase activate the degradation of chromosomal DNA

The degradation of chromosomal DNA

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Apoptosis: Intrinsic and extrinsic pathways

INTRINSIC (Mitochondrial pathway) Involves release of (Death receptor pathway) cytochrome c (and other proteins) from mitochondria Introducti Activated by the engagement of death receptors on cell on surface Function EXTRINSIC

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Activation of caspases

Mechanisms of Apoptosis

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1.Extrinsic

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Pathway -Triggered by binding of extracellular signal proteins to cellsurface death receptors. -Death receptors are transmembrane proteins containing: an extracellular ligandbinding domain

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For example binding of FasL (homotrimeric protein on the surface of a killer lymphocyte) to Fas( death receptor on target cell) causing the Fas cytoplasmic domains to trimerize. -Trimerized Fas recruits 3 molecules of adapter protein known as FADD that in turn, recruits procaspases-8 and -10 via the DED in their prodomains to form the
v

Extrinsic Pathway

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Many cells produce inhibitory proteins that Act either extracellularly or intracellularly to restain the extrinsic pathway.
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-Some produce decoy receptors which have a ligand binding domain but not the death domain, so they competitively inhibit death

2.Intrinsic

Pathway
-Neighboring cell secrete lot of cytokines that regulate cells growth, differentiation, activity and survival; withdrawal of this chemical support or loss of direct cell-cell interactions induce cell to undergo apoptosis. The intrinsic pathway of

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Introducti on Function Induction Caspases Mechanis ms Regulatio n Recogniti 6/12/12 on

For example: cytochrome C (a water soluble component of ETC). It binds to a procaspase activating adaptor protein called Apaf1(Apoptotic Protease Activating Factor-1), causing Apaf1 to oligomerize into a wheel-like heptamer called apoptosome. -The Apaf1 proteins in the apoptosome then recruits initiator procaspase
v

Intrinsic Pathway

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Apoptosome (wheel of death)

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Intrinsic pathway is regulated by Bcl-2 family receptors

Classified into 3 groups 1. Group I -Bcl-2 and Bcl-XL -have four short regions of homology BH1 to BH4 -C-terminal hydrophobic segments

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Bcl-2 family proteins

ANTI-APOPTOTIC I Group
BCL-2 BCl-XL BCL-w A1 MCL1

PRO-APOPTOTIC

Group II
BAX BAK BOK BID BIM BIK BAD BMF NOXA PUMA

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Group III

Regulation of Intrinsic Pathway

-The anti-apoptotic Bcl-2

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proteins such as BclXL are located on the cytosolic surface of the outer mitochondrial membrane. -These inhibit apoptosis by binding to and inhibiting proapoptotic

Regulation of Intrinsic Pathway

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The link between two pathways

The BH-3 only protein Bid is the link between two pathways.When death receptor activate the extrinsic pathway initiatorcaspase (-8) cleaves Bid, producing t-Bid which translocates to mitochondria and inhibit anti-

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IAPs (Inhibitors of Apoptosis)

These proteins act inhibiting caspase activity in 2 different ways: Direct binding inhibiting the proteolytic activity of caspases Marking caspases for ubiquitination and so degradation Smac (second mitochondria-derived activator of caspases) and DIABLO (Direct IAP-binding protein with low pI) binds to BIR domains of IAPs so as to prevent them from binding to caspases.

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Techniques to recognize apoptosis

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Morphological assessment Measurement of DNA fragmentation Flow cytometry of apoptotic cells Specific probes for apoptotic cells Annexin V Detection of electrical potential across the inner membrane of mitochondria

1.Morphological Assesment

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2.DNA fragmentation - biochemical hallmark of apoptosis

DNA cleaved into non-random fragments 180-200 bp fragments & multiples of this unit
1.DNA from apoptotic cell 2.DNA from necrotic cell 3. No sample 4. DNA from normal

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3.Flow cytometry detection of changes in cell size and granularity

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Side scatter shows a decrease in granularity Forward scatter shows a decrease in cell size

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4.Annexin V - specific apoptosis probe Based on observation that phosphatidylserine is translocated to outside leaflet of the plasma membrane during apoptosis Annexin V preferentially binds PS As PS translocates to outer leaflet of the plasma membrane, time dependent increase in annexin binding

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5.Detection of electrical potential across the inner membrane of mitochondria Cells undergoing apoptosis often lose the electrical potential that normally exists across the inner membrane of mitochondria. This membrane potential can be measured by the use of positively charged fluorescent dyes that accumulate in mitochondria. A decrease in labeling of

1.Apoptosis and
Cancer
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Resistance mechanisms: Expression of antiapoptotic proteins (Bcl2 overexpression infollicular B-cell lymphoma; over-

p53 and apoptosis

BC L-2 BC LxL

p53transcriptional targets

Direct binding of p53

BAX NOX A PUM A APAF -1 BC L-2

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p53
Transcriptional repression

I Survivin A

progression

2.HIV

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The progression of the human immunodeficiency virus infection to AIDS is primarily due to the depletion of CD4+ Thelper lymphocytes, which leads to a compromised immune system. One of the mechanisms by which T-helper cells are depleted is apoptosis, which results from a series of biochemical pathways:

3.Apoptosis : a way of life for plants Xylogenesis

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The parenchymatous cells differentiates in phloem and xylem cells, where apoptosis plays a vital role in preferential elimination of cells Reproduction In maize, sex determination involves the selective killing of the female reproductive primordia in order that the male floral structures (the stamens) can develop in the tassel. Senescence An important process for defense mechanism, where apoptosis plays a vital role Also involved in fruit ripening

Apoptosis : a way of life for plants

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Pathogenesis Plants can recognize certain pathogens and activate defense (called the resistance response) that result in the limitation of pathogen growth at the site of infection. A Ubiquitous Process in Plants In addition to the

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