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Cell Death Pabio 552 5/11/06. Classical View of Cell Death: Apoptosis vs Necrosis . Murder?. Suicide?. www.imm.ki.se/ sft/bilder/Image1.jpg. Types of cell death.

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Cell Death Pabio 552 5/11/06

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Cell death pabio 552 5 11 06 l.jpg

Cell DeathPabio 5525/11/06


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Classical View of Cell Death: Apoptosis vs Necrosis

Murder?

Suicide?

www.imm.ki.se/ sft/bilder/Image1.jpg


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Types of cell death

Necrosis – non-apoptotic accidental cell death (common definition) <or> morphology seen after a cell has already died (pathology; has nothing to do with biochemistry of how the cell died)

Autophagy – degradation of cellular components within dying cells in an autophagic vacuole; begins with sequestration of cytoplasmic material within phagosomes, under control of GTPases and phosphatidylinositol kinases

Oncosis – prelethal pathway leading to cell death; accompanied by cellular swelling, organelle swelling, blebbing, and increased membrane permeability; oncotic cells proceed to necrosis with lysis and spillage of contents before being recognized by phagocytosis; inflammation results

Pyroptosis – induced by infection with Salmonella and Shigella; inherently proinflammatory; dependent on caspase 1

Apoptosis…..


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Fink et al Inf. Imm. Apr.2005: 1907-16


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Apoptosis

An active programmed process of autonomous cellular dismantling that avoids eliciting inflammation

Characterized by:

- exposure of phosphotidyl serine on cell surface

- cytoplasm shrinkage

- membrane blebbing

- chromatin condensation

- cleavage of DNA at internucleosome site

- caspase mediated

- corpse clearance via phagocytosis

Fink et al Inf. Imm. Apr.2005: 1907-16


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Biological Roles for Apoptosis

Development

Metamorphosis

Regulation of cell number in tissues (homeostasis and tumorigenesis)

Immune defense (cytotoxic T cell activity)

Develoment of B and T cells via negative selection

Disease: Cancer, autoimmunity, infectious disease etc.


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Caspases

Cysteine-dependent aspartate specific proteases

Have a cysteine at the active site

Cleave target just after aspartic acid residues

Substrate specificity is determined by the 4 residues upstream of cleavage site

Exist in cytosol as single chain proenzymes (procaspases) which are activated when cleaved by other caspases

Once activated, cleave other caspases – results in proteolytic cascade

Also cleave key proteins in the cell, causing the characteristic morphology and biochemistry of apoptosis.


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Caspases…

Procaspases - Contain N terminal pro-domain followed by region that forms a 2 subunit catalytic effector domain

Prodomain: for prot-prot interactions; allows it to bind upstream regulators and effector proteins; examples include:

DED – death effector domain (e.g. caspase 8)

CARD – caspase activation and recruitment domain (e.g. caspase 9)

Active caspases – heterotetramers composed of two large and two small subunits with two active sites per molecule

Nature407, 770-776 (12 October 2000)


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Caspases…

Two types:

- those related to caspase 1 (Caspases 1, 4, 5, 13, and 14); role in cytokine processing during inflammation

- those involved in apoptosis (Caspases 2, 3, 6, 7, 8, 9 and 10)

Initiators – activate downstream effector caspases to initate activation cascades

Effectors - cleave target proteins resulting in morphological and biochemical markers of apoptosis


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Effector Caspases

Activated effector caspases cleave target proteins:

Nuclear Lamins – scaffold proteins of nuclear envelope; leads to nuclear shrinkage and fragmentation

Cytoskeleton proteins – e.g.:

Fodrin; leads to loss of cell shape and membrane blebbing;

Gelsolin (an actin depolymerizing enzyme); cleaved by caspase 3; role in cell morphology during apoptosis (blebbing etc.)

ICAD (inhibitor of Caspase Activated Dnase) – DNA now cut up by CAD

Components of focal adhesion complex; leads to detachment of apoptotic cells from other cells

Other caspase dependent features:

Cleavage of PAK2 (member of p21-activated kinase family); results in formation of apoptotic bodies and other signaling cascades

Exposure of phosphatidylserine on outer membrane; probably due to down-regulation of phospholipid translocase activity and/or activation of lipid scramblase


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Caspase Pathways

Intrinsic pathway – mitochondria mediated; caspase 9

Extrinsic pathway – involves death receptors (TNFreceptor, Fas); caspase 8

Converge to active executioner caspases 3 and 7

Hail et al. Apoptosis (2006)


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Intrinsic Pathway

Usually initiated by cellular stress (UV, cytotoxic drugs etc.) usually causing alterations in mitochondria membrane potential (MMP).

Mitochondria-dependent:

mitochondria sequester pro-apoptotic proteins, e.g. cytochrome C.

Regulated by Bcl-2 family member of proteins – regulate the release of pro-apoptotic factors from mitochondria.

Changes in MMP free cytochrome c from intermembrane space out into the cytosol

Cytochrome c then combines with dATP, APAF-1 (apoptotic protease activating factor-1), and caspase 9 to form a catalytic complex called the apoptosome

(Caspase 9 co-factor is APAF-1 which must be bound by cytochrome c to drive Caspase 9 into its active conformation)

Apoptosome activates caspases 3 and 7 (effector caspases)

Effector

caspases

Nature407, 770-776 (12 October 2000)


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Regulation of intrinsic pathway

Bcl-2 family of proteins (gene orig. isolated from a B-cell lymphoma):

Pro apoptotic effects: e.g. Bax (indirectly regulated by tumor repressor P53), Bad, and Bak

Anti apoptotic effects: e.g. Bcl-2, Bcl-XL

These proteins may be localized to mitochondria intermembrane or targeted to the organelle in response to stimulus

Regulate release of pro-apoptotic factors from mitochondria, especially cytochrome c.

Also release Smac (second mitochondria-derived activator of caspases) and DIABLO (direct IAP-binding protein with low pI) which bind to IAPs (Inhibioros of Apoptosis proteins)

Battle between pro and anti levels to determine cell’s response to apoptotic stimuli such as ROS (reactive oxygen species), Ca++, radiation etc.

Also released from mitochondria is AIF – apoptosis inducing factor; once released, it translocates to nucleus to induce chromatin condensation and DNA fragmentation


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How do proteins get released from mitochondria?

Several hypotheses for how Bcl-2 proteins regulate release of cytochrome C:

By forming channels in mitochondria membrane

By interacting with other proteins to form channels

By inducing rupture of the mitochondrial membrane

By oligomerizing to form a weakly selective ion channel.

BcL-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC (voltage dependent anion channel) Nature 399, 483-487 (1999))


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Nature407, 770-776 (12 October 2000)


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Intrinsic Pathway


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Extrinsic Pathway

Initiated by death receptors:

Start with ligand binding, clustering or aggregation of death receptors

Cytoplasmic tails of death receptor bind adaptor proteins

Adaptor proteins recruit and activate procaspase 8 to yield caspase 8 (active)

Caspase 8 triggers downstream effector caspases, such as caspase 3

Activation of death receptors may also activate intrinsic pathway

Death Receptors:

TNFR family

Fas (CD95)

Death Ligands:

TNF

CD95L(FasL) –

only expressed by

activated T cells

Nature407, 770-776 (12 October 2000)


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Fas – Fas ligand system

Fas

Prototypical cell death receptor of the TNF receptor superfamily

No intrinsic enzymatic activity

Signals via adaptor proteins

Fas ligand (FasL)

Only expressed by activated T cells

Transmembrane TNF-like protein

When the TCR (T cell receptor) of an antigen specific CTL (cytotoxic T lymphocyte) binds antigen on MHC I, the expression of FasL is induced on the T cell

FasL binds Fas (present on most cells of body) that is on the presenting cell to induce death of that cell

Cytoplasmic tail of Fas binds its adaptor protein FADD (Fas Associated Death Domain protein)

Fas-FADD complex binds to and activates caspase 8

Initiates lethal proteolytic cascade


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Nature407, 770-776 (12 October 2000)


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Extracellular ligands inducing apoptosis via diverse signal transduction pathways


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Corpse clearing

A defining point of apoptosis is to clear the dying cell before it can release inflammatory molecules

Macrophages ingesting apoptotic cells release anti-inflammatory and immunosuppressive cytokine transforming growth factor-beta1 (TGF-β1)

Macrophages ingesting necrotic cells will release pro-inflammatory mediators

Cells undergoing apoptosis show changes in surface of plasma membrane.

These “eat me” signals are recognized by phagocytes

Exposure of phosphatidyl serine (PS) – PS receptor is expressed on phagocytes

Change in cell surface sugars – detected by lectins on phagocyte cell

Sites that bind “bridging molecules” e.g. C1q – C1q deficiency leads to impaired phagocytosis of apoptotic cells

ICAM-3 – binds alternate receptor on macrophages; possibly CD14

Macrophages thought to “tether” dying cells by using CD14 or beta integrin before engaging receptors that drive apoptosis


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Savill & Fadok Nature (2000) 407:784-788


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Jurkat T-cell targets were labelled with 5-(and 6)-carboxytetramethylrhodamine succinimidyl ester and irradiated to induce apoptosis. The macrophages were stained with fluorescein isothyocyanate-conjugated phalloidin to identify actin filaments. Nature (407) pp 784-788.


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Cell survival

Many signaling pathways exist to promote cell survival

Often dependent on growth factors or cell-cell interactions (survival factors)

Example: PI 3-Kinase initiated signaling cascase

PI3K is activated by tyrosine kinases or G coupled protein receptors

PI3K phosphorylates PIP2 to form PIP3 which activates the serine threonine kinase Akt

Akt phosphorylates many proteins involved in regulation of apoptosis:

Bad (induces release of cytochrome c from mitochondria); phosphorylation of Bad creates binding sites for proteins to sequester Bad in the cytosol to keep it from going to the mitochondrial membrane

Caspase -9

Transcription factors e.g. NFkB

GSK-3; affects metoblism and protein synthesis

Other signaling pathways include: Ras/Raf/MAP kinase


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PI 3-kinase/Akt signaling cascade

© 2000 by Geoffrey M. Cooper


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Physiological

TNF family (TNF, FasL)

TGF-beta

Neurotransmitters

Growth factor withdrawal

Loss of matrix attachment

Sustained rise in Calcium

Glucocorticoids

Damage related

Heat Shock

Viral infection

Bacterial toxins

Oncogenes

Tumor supressors (p53)

Cytolytic T cells

Oxidants

Free radicals

Nutrient deprivation

UV radiation

Gamma radiation

Inducers of apoptosis

Toxins

Some chemotherapeutic drugs

Ethanol

Beta-amyloid peptide


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Physiological

Growth factors

ECM

CD40L

Neutral a.a.

Zinc

Estrogen

Androgens

Viral gene products

Adenovirus E1B

Baculovirus p35

Cowpox virus crmA

Epstein-Barr virus BHFR1, LMP1

African swinve fever virus LMW5-HL

Herpesvirus gamma 1 34.5

Inhibitors of apoptosis

Pharmacological agents

Calpain inhibitors

Cysteine protease inhibitors

Tumor promoters e.g. phenobarbital


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Assays for detecting apoptosis

Histological stains to look at condensed chromatin

DNA fragmentation assessed by gel electrophoresis - CAD (caspase activated DNase) is present in cells bound to its inhibitor (ICAD - inhibtor CAD); activation of CAD occurs when ICAD is cleaved; CAD cleaves genomic DNA to generate strands ~ 180 bp; forms “DNA ladder” used as a marker for an apoptosing cell

Annexin V staining - stains phosphatidyl serine on outer membrane; when fluorescently conjugated, can be sorted by FACS

TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) – uses activity of the terminal deoxynucleotidyl transferase enzyme to label the 3’ ends of DNA strand breaks which may then be identified by microscopy

Caspase activation – can be demonstrated by Western using Abs against caspase substrates; also measured by using colorimetric and fluorometirc assays based on proteolysis of conjugated tetrapeptide substrates mimicking caspase cleavage sites

Cytochrome C release – assays measure cytochrome c in mitochondria vs cytosol

Alterations in mitochondrial membrane potential – various assays to look at this; involve labeling specific molecules that move across mitochondria membrane; aid in calculating membrane potential


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Apoptosis and Pathogens: Theileria parva

Tick transmitted intracellular apicomplexans

Worldwide infection in mammals

Major constraint in livestock development, especially in tropical regions


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T. parva vs. B. taurus leukocyte

T. parva prevents apoptosis in host cells

Upon infection, get induced expression of NFkB

How? Parasite recruits IKK complex to its cell surface, get oligomerization causing “proximity induced activation”

IKK complex stimulation results in phosphorylation of inhibitory IkB

NFkB is now free to translocate to the nucleus

Get upregulation of anti-apoptotic proteins e.g. cFLIP

Alberts ch-17


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Apoptosis and Pathogens: Enterovirus 71

Enterovirus 71 Infection Induces Fas Ligand Expression and Apoptosis of Jurkat CellsJournal of Medical Virology 78:780–786 (2006)

EV71 is a +RNA virus

Transmission – fecal-oral

Clincial features: meningitis, encephalitis, pulmonary edema, death

Px: increased cytokine levels, significant decrease in T cells

EV71 induced FasL expression in Jurkat cells and increases Jurkat cell apoptosis


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TUNEL assay

A and B: mock infection of

Jurkat cells

C and D: EV71 infected

Jurkat cells

TUNEL labeling in green

Cell nuclei in red (propidium

idodide stain)


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DNA fragmentation assay

Jurkat cells incubated with or without

EV71 for 48 hours. DNA samples run

on 2% agarose gel and stained with

ethidium bromide


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Let down the bars, O death!

The tired flocks come in

Whose bleating ceases to repeat,

Whose wandering is done.

Thine is the stillest night,

Thine the securest fold;

Too near thou art for seeking thee,

Too tender to be told.

Emily Dickinson


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Inhibition of caspase activation and a requirement for NF-kB function in the Toxoplasma gondii-mediated blockade of host apoptosis

T. Matthew Payne, Robert E. Molestina and Anthony P. Sinai


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Infection routes of T. gondii

Undercooked meat

Oocysts

Reproductive Host

Asexual Host

predation


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Tachyzoite

Intracellular

Bradyzoite

Extracellular


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