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Apoptosis

(Programmed Cell Death)


Lecture 20
Nov 17, 2015
Anath Das
dasxx002@umn.edu

Learning Objectives
Describe apoptosis including its features and
importance
Understand TUNEL assays to assess apoptosis

Describe caspases and their roles in apoptosis


Describe the extrinsic apoptotic pathway
Describe the intrinsic apoptotic pathway
Appreciate how aberrations in apoptosis can
lead to disease
2

Lecture Outline
Define apoptosis, describe its features, and
discuss its importance
Discuss caspases, their activation, and their
roles in apoptosis
The extrinsic apoptotic pathway
The intrinsic apoptotic pathway
Apoptosis and disease

Types of Cell Death


Necrosis - cell death due to some type of acute
injury such as trauma or lack of blood supply
Cells swell, burst, and spill their guts
Causes an inflammatory response at the site of injury
and potentially a dangerous systemic response

Apoptosis - programmed cell death: activation of a


specific biochemical pathway leading to cell death
and removal by a macrophage or neighboring cell
Apoptosis is inherent to all metazoan cells
300 million cells die in our bodies every minute (blood,
intestinal, skin cells) (37.2 trillion cells in human adult*)
Also occurs with genetically damaged cells
These are exactly replaced by cell replication
4
*Bianconi et al. (2013) Ann. Hum. Biol. 40:463

When Apoptosis Is a Good Thing


Development/Regeneration

body part sculpting during development

death to neurons that dont connect (about 50% of neurons)

death to immune cells that are duds or recognize self

turnover of intestinal, skin, and bone marrow cells

Cancer prevention

death to cells that have incurred heavy DNA damage, are


not well fed, or have suffocated

Body Sculpting:

A developing mouse paw


5

1 day later

When Apoptosis Is Not So Good


Neurodegenerative diseases (Alzheimer's
disease; Parkinsons disease; ataxias; etc.)
AIDs
Ischemic injury (myocardial infarction; stroke)
Alcohol-induced liver disease

Comparison of Cells Undergoing


Apoptosis and Necrosis
Apoptotic
Cell

Phagocytosis
of Apoptotic
Cell

Necrosing
Cell

Symptoms of Apoptosis
Cytoskeleton collapses; cells becomes more compact
Nuclear DNA breaks up into nucleosomal-sized fragments
Nuclear envelope disassembles and chromatin condenses
Phosphatidylserine flips from the inside to the outside of
the plasma membrane bilayer
Mitochondrial function is lost
Plasma membrane is altered (blebbing), allowing dying cell
to be engulfed by macrophages via phagocytosis

Phosphatidylserine Moves From the


Inner Leaflet to the Outer Leaflet
During Apoptosis

Having phosphatidylserine
on the outside of the cell
serves as an eat me
signal, which is why
phagocytes engulf it.

Apoptotic Nuclei Can Be Detected Using


TUNEL Assay

TUNEL assay of
apoptosis in a
developing chick
bud when the
digits are being
formed.

TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assays add
a fluorescent nucleotide to DNA ends. The DNA ends are extended by adding
fluorescently labeled dUTPs using the template-independent DNA polymerase
terminal deoxynucleotidyl transferase (TdT). Large numbers of labeled
10 DNA
fragments leads to bright fluorescent dots in apoptotic cells.

Lecture Outline
Define apoptosis, describe its features, and
discuss its importance
Discuss caspases, their activation, and their
roles in apoptosis
The extrinsic apoptotic pathway
The intrinsic apoptotic pathway
Apoptosis and disease

11

Two Pathways Regulate Apoptosis:


Extrinsic & Intrinsic
RTK

GPCR

Cytokine

Src

PI3K
Grb2/SOS

G-protein
Adenylyl
Cyclase

PLC

PKA

PKC

AKT

Ras

mdm2

Raf

p53

PTEN

Jak

mTOR NF- BAD STAT


B

R-Smad
Smad4

MAPK

Caspase 8

Caspase 3

Guanylyl
Cyclase
PKG
NO

Dsh
Mito

Caspase 9

ANP

Guanylyl
Cyclase

MEK

CREB

FADD

TGF

APC + GSK3
Gene Regulation

Cytochrome C

Apoptosis

Death Receptors (FasR)

-catenin

Cell Proliferation
NR

NUCLEUS
12

Wnt

Caspases - the Mediators of Apoptosis


(and Inflammation)
Apoptosis is mediated by a cascade of highly specific
proteolytic cleavages by a family of proteases known as
Caspases.
Caspases utilize an active site cysteine residue as the
mechanism of hydrolysis.
Caspases cleave the peptide bond at a specific four amino
acid sequence on the C-terminal side of an aspartate
residue.
(Caspase)
Some caspases (caspases 1, 4, and 5) are involved in
inflammation rather than apoptosis.
Caspases are synthesized as inactive proenzymes that
need to be activated by cleavage to remove an inhibitory
pro-peptide segment.
13

Some Human Caspases


Caspases Involved in Inflammation
Caspases 1 (ICE), 4, and 5
Caspases Involved in Apoptosis
Initiator Caspases (start the caspase cascade)
Caspases 2, 8, 9, and 10
Exist as inactive, soluble monomers in the cytosol
before the apoptotic signal
Function is to activate the executioner caspases
Executioner (Effector) Caspases (cut target proteins)
Caspases 3, 6, and 7
Exist as inactive dimers in the cytosol before the
apoptotic signal
14

A Few Examples of Target Proteins


Degraded by Executioner Caspases
CAD (caspase-activated DNase): Enzyme responsible for
DNA cleavage during apoptosis
CAD is normally inhibited by an inhibitor (iCAD)
Executioner caspases cleave iCAD inactivating it
This releases an active CAD, which fragments DNA between
nucleosomes

Nuclear Lamins: Structural proteins responsible for the


nuclear envelope and nuclear integrity
Mdm2: E3 ubiquitin ligase that targets p53 for
ubiquitination and thus proteasomal degradation. Without
Mdm2, p53 level remains high and can signal cells to stop
proliferating and to die.
15

Activation of CAD by an Executioner


Caspase

Cells in culture
were treated
with Fas ligand
(FasL) to initiate
apoptosis at time
0.

After various
times, DNA was
isolated and
analyzed by
agarose gel
electrophoresis.

DNA cut by CAD forms a ladder of fragments based on the


16
frequency of cutting between nucleosomes

Overview of Caspase Activation


Caspases are originally
made in an inactive,
procaspase form.
FADD or Apaf1

procaspase

Extrinsic or intrinsic signals


trigger the assembly of
adaptor proteins with the
procaspases.
Upon binding of an adaptor
protein (FADD or Apaf), the
procaspases dimerize and
are activated, cleaving the
procaspase into a large and
small subunit. (The
prodomain is degraded.)
The initiator caspases then
clip the executioner caspase
dimer, and the dimer
rearranges and is active.
17

Caspases Function in Cascades that


Can Greatly Amplify the Initial Signal
Initiator caspases
activate the executioner
(effector) caspases by
clipping off their
prodomains.
The catalytic activation
creates an amplification
step as one initiator
caspase can activate
many executioner
caspases.
18

The Procaspases Are Clipped at Different


Sequences and Active Caspases Recognize
Different Target Sequences

(Val-Glu-Thr-Asp)
(Leu-Glu-Thr-Asp)

19

Lecture Outline
Define apoptosis, describe its features, and
discuss its importance
Discuss caspases, their activation, and their
roles in apoptosis
The extrinsic apoptotic pathway
The intrinsic apoptotic pathway
Apoptosis and disease

20

There Are Two Pathways to Apoptosis


All metazoan cells express procaspases and thus express
the proteins necessary for their own destruction
Initiation of apoptosis can occur in response to either
external signals (extrinsic pathway) or internal signals
(intrinsic pathway)
In either case, protein:protein interactions mediated by
specific modular interaction domains are particularly
important to these signaling pathways
DD = death domain
DED = death effector domain
CARD = caspase-associated recruitment domains
21

The Extrinsic Apoptotic Pathway


RTK

GPCR

Cytokine

Src

PI3K
Grb2/SOS

G-protein
Adenylyl
Cyclase

PLC

PKA

PKC

AKT

Ras

mdm2

Raf

p53

PTEN

Jak

mTOR NF- BAD STAT


B

R-Smad
Smad4

MAPK

Caspase 8

Caspase 3

Guanylyl
Cyclase
PKG
NO

Dsh
Mito

Caspase 9

ANP

Guanylyl
Cyclase

MEK

CREB

FADD

TGF

APC + GSK3
Gene Regulation

Cytochrome C

Apoptosis

Death Receptors (FasR)

-catenin

Cell Proliferation
NR

NUCLEUS
22

Wnt

The Extrinsic Apoptotic Pathway


By definition, it is activated by something outside
of the cell
Death signals
TNF (tumor necrosis factor ) is secreted by macrophages and
triggers cell death in chronic inflammatory diseases
Fas ligand (FasL) is a cell surface protein produced by activated
natural killer cells and cytotoxic T lymphocytes to kill off viruses,
foreign graft cells, some tumor cells, etc.
Both TNF and Fas ligand are cell surface proteins and they bind
to specific receptors on adjacent cells (contact-dependent
signaling) that are members of the TNF receptor family (TNF, FAS,
TRAIL), also known as the death receptors.

23

Death Receptor Structure


Receptor: Transmembrane
Protein; Homo-trimer.
Ligand BD(outside)TM DD(inside)
membrane

DD: Death Domain


DED: Death Effector Domain
Receptors bound to ligands
form clusters of trimers and
interact with adaptor proteins
FADD (Fas-associated death
domain). FADD has DD and
DED domains.
FADD recruits an initiator
caspase (caspase 8) via a
DED in both proteins.
24

Death by FasL Is the Best Characterized


Extrinsic Pathway
The extrinsic pathway starts with a
ligand trimer binding to a death
receptor trimer such as Fas. This
leads to clustering of ligand bound
trimers.
The receptor has a DD that
attracts an adaptor protein such as
FAAD that has both DD and DED.
The adaptor protein attracts an
initiator caspase (typically
caspases 8 and/or 10) with a DED.

25
Figure 18-5 Molecular Biology of the Cell ( Garland Science 2015)

Assembly of The Death Inducing Signaling


Complex (DISC)
The aggregate of receptor,
adaptor and procaspase is
called DISC (death-inducing
signaling complex).
The aggregation of the
procaspases causes them to
cross-activate each other.
Procaspase
8/10

26
Figure 18-5 Molecular Biology of the Cell ( Garland Science 2015)

The Initiator Caspases Activate the


Executioner Caspases, Notably Caspase-3
Within the DISC two adjacent
initiator caspapses interact and
cleave one another to form an
active dimer.
The dimer cleaves itself in the
region that links it to the DD
freeing up the initiator caspase
dimer.
The activated caspase move
around in the cytosol and
activate executioner caspases.
27
Figure 18-5 Molecular Biology of the Cell ( Garland Science 2015)

The Extrinsic Pathway: Activation of


Caspases by Clustering of the Initiator
Procaspases so They Activate Each Other

28

More Notes on the Extrinsic Pathway


Six different types of receptors that can trigger apoptosis are
known; all have a related death domain in their cytosolic
region
Procaspases-8 and -10 can activate themselves because the
procaspase has a small amount of basal proteolytic activity;
when many molecules are brought together, one will activate
its neighbor, initiating a chain reaction
The extrinsic pathway is called into play in chronic
inflammatory diseases, tissue grafts/transplants, immune
response
Many viruses express proteins that bind to the death domains
of receptors to inhibit interaction with FADD. Why?
29

Lecture Outline
Define apoptosis, describe its features, and
discuss its importance
Discuss caspases, their activation, and their
roles in apoptosis
The extrinsic apoptotic pathway
The intrinsic apoptotic pathway
Apoptosis and disease

30

The Intrinsic Apoptotic Pathway


RTK

GPCR

Cytokine

Src

PI3K
Grb2/SOS

G-protein
Adenylyl
Cyclase

PLC

PKA

PKC

AKT

Ras

mdm2

Raf

p53

PTEN

Jak

mTOR NF- BAD STAT


B

R-Smad
Smad4

MAPK

Caspase 8

Caspase 3

Guanylyl
Cyclase
PKG
NO

Dsh
Mito

Caspase 9

ANP

Guanylyl
Cyclase

MEK

CREB

FADD

TGF

APC + GSK3
Gene Regulation

Cytochrome C

Apoptosis

Death Receptors (FasR)

-catenin

Cell Proliferation
NR

NUCLEUS
31

Wnt

The Intrinsic Pathway of Apoptosis


Is Activated by the Cell Itself
Intrinsic (internal) pathway:
Signals coming from inside the cell because of
irreparable damages
Signals include:
Extensive DNA damage
Lack of O2
Lack of nutrients
Absence of growth factor signaling
The intrinsic pathway is activated by release of
cytochrome c from inside the mitochondria.
32

The Intrinsic Pathway: Cytochrome C


Activates Apaf 1

Released cytochrome c
binds to Apaf-1(apoptotic
protease activating factor
1), an adaptor protein
with a CARD domain
(caspase-associated
recruitment domain).

33

CytC Bound Apaf1 Aggregates to Form


Apoptosome

CytC

34
Structure 18: 571 (2010)

The CARD Domains in Apoptosome Recruit and


Activate the Initiator Caspase, Caspase 9

Mechanism of
activation is not
known. Activation is
probably through
cleavage by adjacent
Caspase 9 proteins
or intearction of
Caspase 9 and Apaf1.

35

The Intrinsic Pathway: Activation of the


Initiator Procaspase 9 by Clustering

Apaf1 is an adaptor protein. CARD = caspase-associated recruitment


domain
36

What Triggers The Release Of


Cytochrome C From The Mitochondria?
Bcl2 family members control cytochrome c release
Bcl2 family: A family of 15 protein members
Bcl-2 family members come in two functional classes:
Anti-apoptotic (survival): e.g., Bcl2
Pro-apoptotic (death): e.g., Bad, Bax
Pro- and anti-apoptotic Bcl2 proteins bind to each other to
form hetero-oligomers.
The balance between the activities of the pro- and antiapoptotic proteins determines whether a cell lives or dies
by the intrinsic pathway.
37

Bcl2 Family Members Share Bcl2


Homology (BH) Domain(s)

Inhibit pore formation by the BH123 proteins

Effectors: Form the pores that let out cytochrome C

Inhibit the anti-apoptotic proteins

BH = Bcl2 homology domain

The BH3 domain mediates


interactions between pro- and
anti-apoptotic family members

38

Stimulus-Induced Aggregation of Pro-Apoptotic


Effectors On Mitochondrial Outer Membrane
Leads To Cytochrome C Release
Bak

Bax - cytosolic

39

Anti-Apoptotic Bcl2 Family Members


Bcl2 (and other anti-apoptotic family members)
reside on the cytosolic face of the outer
mitochondrial membrane
Act mainly by binding to and inhibiting the action of
pro-apoptotic family members, i.e. Bcl2 is going to
inhibit Bax
Act to prevent inappropriate apoptosis
Five members in mammalian cells; at least one
must be expressed for cell survival!
40

Bcl2 Inhibits Bax (BH123 Proteins) in


the Absence of an Apoptotic Signal
or When RTKs are Activated

Bax Bak

Anti-apoptotic Bcl2
proteins bind to and
inhibit the BH123 (Bax)
proteins, preventing
clustering, cytochrome
c release, and thus
apoptosis.
The Bcl2 proteins are
actually bound to the
mitochondrial surface,
although not shown
here.
41

Pro-Apoptotic BH3 Only Family Members


BH3 only family members (Bad, Bim, Puma, etc.)
are the transducers of the apoptotic signals.
In response to an apoptotic signal the cell produce
or activate them.
BH3 only proteins are the largest subclass of Bcl2
family proteins.
Promotes apoptosis by inhibiting anti-apoptotic
Bcl2 proteins through protein-protein interaction
mediated by the SH3 domain.

42

Inhibition of Anti-Apoptotic Bcl2 Family


Members by BH3 Only Proteins Triggers
Cytochome C Release

Bcl2

Bax

Bad

43

Survival Factors Are Anti-Apoptotic

IAP = Inhibitor of Apoptosis


44

Lecture Outline
Define apoptosis, describe its features, and
discuss its importance
Discuss caspases, their activation, and their
roles in apoptosis
The extrinsic apoptotic pathway
The intrinsic apoptotic pathway
Apoptosis and disease

45

Examples of Aberrant Apoptosis


Contributing to Disease-1
Too many cells die by apoptosis
Neurodegenerative disordersthe progressive loss of
neurons
Parkinsons
Alzheimers
Huntington Chorea
Stroke
Schizophrenia (?)

Ischemic damage (loss of oxygen due to heart attacks


or strokes; often includes both necrosis and apoptosis)
AIDS
46

Examples of Aberrant Apoptosis


Contributing to Disease-2
Too few cells die by apoptosis
Developmental disorders
Disruption of brain development by blocking apoptosis

47
Gilberts, Developmental Biology 9 edition, Figure 3.32
th

Examples of Aberrant Apoptosis


Contributing to Disease-3
Too few cells die by apoptosis
Mutations in FasL or FasR lead to excessive numbers of
lymphocytes , which can cause autoimmune diseases
(when lymphocytes react against the persons own cells:
Rheumatoid Arthritis, Lupus, Multiple Sclerosis,
Myasthenia Gravis, Type I Diabetes, etc.)
Cancer
One of the hallmarks of cancer is that damaged cells
evade apoptosis.

48

Apoptosis and Cancer


The normal defense to loss of growth control in a
cell is the induction of apoptosis.
However, blocking the normal apoptotic pathway
is essential for cancer development and
maintenance.
Therefore, many components of apoptotic
signaling pathways have been identified as
oncogenes or tumor-suppressor genes in human
cancers.

49

Bcl2 Is One Example of an Aberrant Antiapoptotic Protein that Causes Cancer


Bcl2 = B-cell lymphoma2 (follicular lymphoma)
This gene was first identified in a common type of lymphoma, where a
chromosomal translocation between chromosomes 14 and 18 puts the Bcl2
gene under the regulation of the immunoglobulin gene enhancer. The result is
that too much Bcl2 is made, and these cells essentially become immortal.
A number of drugs are being developed to interfere with anti-apoptotic Bcl2
family proteins. These drugs bind with high affinity to the hydrophobic groove
on these proteins and act as a BH3 (Bax) protein mimetic.

The drug ABT-737 inhibits


Bcl2 family members. This
drug elicits apoptosis in any
cell that requires Bcl2 for
survival (damaged cells). It
has been used successfully
in Phase II clinical trials.
50

Mutations in p53 Also Contribute to


Inappropriate Cell Survival
p53 is a transcriptional activator that accumulates in
response to many types of DNA damage
p53 causes cells to arrest the cell cycle in response to
DNA damage until the damage is repaired
If DNA damage is too extensive, p53 induces the
expression of pro-apoptotic family members (e.g., Bax)
to trigger apoptosis
Hence, p53 is pro-apoptotic, and it is the most
commonly mutated tumor suppressor gene in humans
51

Active p53 Promotes the Expression of


Proteins that will Either Stop DNA
Replication or Tell Cells to Die
Mdm2 is an E3
ubiquitin ligase.
p53 is a
transcription factor.

BAX is a proapoptotic
BH123 family
member

BAX
BAX
Figure 17-62 Molecular Biology of the Cell ( Garland Science 2015)

52

p53 Is A Transcription Factor Of BH123


And Pro-apoptotic BH3-only Genes

Bax

BH123

Puma

BH3-only

Noxa

BH3-only

53

p53 Increases in Response to DNA


Damage and Increases Transcription of
Pro-Apoptotic Genes
DNA
damage
p53
Increased txn of pro-apoptotic
genes
Release of mitochondrial
cytochrome c
Cell
death

54

Take Home Messages - 1


Apoptosis or programmed cell death is a specific
biochemical pathway invoked to remove cells that are no
longer needed or are extensively damaged
Apoptosis is mediated by caspases - a family of proteases
that are produced as inactive proenzymes and are activated
by specific proteolytic cleavage
Caspases activate each other in proteolytic cascades that
begin with initiator caspases and end with executioner
(effector) caspases
The extrinsic pathway is initiated by a variety of signal
molecules (e.g., FAS) that result in receptor clustering and
auto-cleavage of the initiator caspases, caspase- 8 and/or
-10
55

Take Home Messages - 2


The intrinsic pathway is initiated by release of cytochrome
c from the mitochondria, its binding to the adaptor protein
Apaf-1, clustering and auto-cleavage of initiator caspase 9.
Release of cytochrome c from the mitochondria is
controlled by members of the Bcl2 family
Pro-apoptotic BH123 proteins (Bax) oligomerize in the
mitochondrial membrane in response to apoptotic signals to
release cytochrome c
Anti-apoptotic Bcl2 proteins bind to and inhibit the BH123 proteins
Pro-apoptotic BH3-only proteins (Bad) are produced or activated in
response to apoptotic signals and inhibit actions of anti-apoptotic
Bcl2

p53, one of the most important tumor suppressor genes in


human cancer, is activated by DNA damage and in turn
activates expression of pro-apoptotic BH3-only factors
56
(Bad)