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

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

 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.
 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.
 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

Nature 407, 770-776 (12 October 2000)

 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
 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
 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)

 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

Nature 407, 770-776 (12 October 2000)

 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 cells 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
 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 ))
Nature 407, 770-776 (12 October 2000)
Intrinsic Pathway
 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

Nature 407, 770-776 (12 October 2000)

 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
Nature 407, 770-776 (12 October 2000)
 Extracellular ligands inducing apoptosis via
diverse signal transduction pathways

Stimulus Sensor Adaptor Initiator Effector

FasL Fas FADD Caspase 8 Caspase 3

TNF- TNFR1 TRADD- Caspase 8 Caspase 3

FADD
Apo3L DR3 TRADD- Caspase 8 Caspase 3
(Apo3) FADD
UV ? Apaf1 Caspase 9 Caspase 3
 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-1)
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
Savill & Fadok Nature (2000) 407:784-788
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.
 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
 PI 3-kinase/Akt signaling cascade

2000 by Geoffrey M. Cooper

 Inducers of apoptosis

Physiological Damage related Toxins

TNF family (TNF, FasL) Heat Shock Some chemotherapeutic
TGF-beta Viral infection drugs
Neurotransmitters Bacterial toxins Ethanol
Growth factor Oncogenes Beta-amyloid peptide
withdrawal Tumor supressors (p53)
Loss of matrix Cytolytic T cells
attachment Oxidants
Sustained rise in Free radicals
Calcium
Nutrient deprivation
Glucocorticoids
UV radiation
Gamma radiation
 Inhibitors of apoptosis

Physiological Viral gene products Pharmacological

Growth factors Adenovirus E1B agents
ECM Baculovirus p35 Calpain inhibitors
CD40L Cowpox virus crmA Cysteine protease
inhibitors
Neutral a.a. Epstein-Barr virus BHFR1,
LMP1 Tumor promoters e.g.
Zinc
phenobarbital
Estrogen African swinve fever virus
Androgens LMW5-HL
Herpesvirus gamma 1
34.5
 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
Apoptosis and Pathogens: Theileria parva

Tick transmitted intracellular

apicomplexans
Worldwide infection in
mammals
Major constraint in livestock
development, especially
in tropical regions
 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
 Apoptosis and Pathogens: Enterovirus 71
Enterovirus 71 Infection Induces Fas Ligand Expression
and Apoptosis of Jurkat Cells Journal of Medical Virology 78:780786 (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
 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)
 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
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
 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
 Infection routes of T. gondii

Undercooked meat

Oocysts

Reproductive Host

Asexual Host
predation
 Tachyzoite

Intracellular

Bradyzoite

Extracellular