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Forms of cell death

Necrosis is characterized by loss of plasma membrane integrity, organelle

and cell swelling, and ultimately, cell lysis
Apoptosis, named after the Greek word for falling off, e.g., of dead tree leaves. The
process of apoptosis is characterized morphologically by cell shrinkage, plasma membrane
blebbing, nuclear condensation, and internucleosomal DNA fragmentation. The dead cell
is packaged into membrane-bound apoptotic bodies, which are engulfed and removed by
neighboring cells or tissue phagocytes. Apoptosis is usually associated with minimal
Necroptosis - It is thus named because it shares some critical inductive-phase
features with apoptosis but possesses many of the morphological characteristics of
accidental necrosis.

Like necrosis, necroptosis is characterized by swelling of the cell and its organelles, particularl
the mitochondria, and by consequent cell implosion.
The upstream molecular signaling pathways that trigger necroptosis are ordered and
strictly controlled.

Chapter X

In the embryo, apoptosis contributes to the sculpting of various tissues and organs; in
the adult animal, it supports the operation and maintenance of key physiological
functions, including the immune, digestive, endocrine, and nervous systems.

Genetic evidence suggests that necroptosis also may be operative during

development, although it may have a more prominent postdevelopmental role in
combating infection by intracellular pathogens.
Unscheduled apoptosis of certain cell types contributes to diseases such as diabetes,
immune deficiency, and neurodegeneration.
Undue necroptosis may worsen tissue injury caused by ischemia and/or reperfusion in
the heart, brain, liver, or kidney and may exacerbate certain autoimmune inflammatory
conditions, including Crohns disease.
Insufficient apoptosis can promote autoimmunity and
cancer, whereas too little necroptosis may exacerbate infection by certain viruses.

Apoptosis is a process of programmed cell suicide, mediated by a group of

specialized proteincleaving enzymes dubbed caspases.

Kerr, Wyllie, and Currie first identified the unique morphology of this form of cell
death in the 1970s.
A few decades later, studies by Horvitz and coworkers in Caenorhabditis
elegans nematodes demonstrated that apoptosis is a gene-controlled cell
death program.
The seminal discovery of specific cell death (CED) genes in nematodes enabled the
identification of CED homologs in mammals.

CED-3, represents the caspase family of proteases, of which several members

play crucial roles in apoptosis initiation or execution.
CED-9, represents the mammalian BCL-2 gene family, of which multiple
members play critical roles in apoptotic control.
CED-4, is homologous to mammalian Apaf-1a component of the
apoptosome complex, which drives caspase activation in many contexts.
Caspasesa subset of the cysteine-dependent aspartate-specific protease family
(Thornberry & Lazebnik 1998).
Apoptotic caspases fall into two major categories: initiator caspases (e.g.,
caspase-8 and -9) and executioner, or effector, caspases (e.g., caspase-3 and -7)
(Salvesen & Ashkenazi 2011). Caspase proteins are constituent in
the cytoplasm in zymogen form.
Apoptotic stimuli induce activation of one ormore of the initiator
caspases through specific oligomerization platforms.

The initiators then trigger a cascade-like proteolytic stimulation of effector caspase

In turn, the latter products drive the execution phase of the apoptotic death program
by cleaving hundreds or even thousands of structurally and functionally critical proteins
within the cell.

Apoptosis Pathways
(a) the intrinsic pathway, gated through proteins encoded by the BCL-2
gene family
which control th release of specific caspase-activating factors from
(b) the extrinsic pathway, governed by specialized death receptors (, which
transmit signals from extracellular death ligands across the plasma membrane
to engage the intracellular caspase machinery.

Mechanisms of Apoptosis
The CED-9/BCL-2 gene family encodes two major subclasses of apoptosis-regulating
factors, which contain different numbers of BCL-2 homology (BH) motifs:
(a) single BH motif (BH3-only) proteins (e.g., BAD, BIK, BMF, BID, PUMA, and NOXA),
which typically act as death agonists,
(b) multi-BH motif proteins, which possess three or four BH regions and act
respectively as agonists (e.g., BAX, BAK, BOK) or antagonists (e.g., BCL-2, BCL-XL,
BCL-w, A1, MCL-1) of apoptotic stimulation.
DNA damage activates tumor suppressor p53 protein, which upregulates
mRNA transcription of PUMA and NOXA. These BH3-only proteins then activate BAX
and BAK, either through direct interaction or indirectly by binding to and sequestering
BCL-2 or BCL-XL, tipping the balance toward apoptotic activation.
BAX and BAK reside in the mitochondrial outer membrane; their homo-
oligomerization upon activation creates membrane pores, releasing mitochondrial
cytochrome C into the cytoplasm.

Chapter X

Mechanisms of Apoptosis
Cytochrome C triggers a conformational change in Apaf-1, resulting in (d)ATP
exchange and oligomerization to form the caspase-9 activating apoptosome.
In turn, caspase-9 activates the effectors caspase-3 and -7 to
execute the cells apoptotic demise.
Caspase-3, -7, and -9 are kept in check by X-linked inhibitor of apoptosis protein
(XIAP); however, BAX and BAK also release from mitochondria a protein
called SMAC/DIABLO, which sequesters XIAP, thereby facilitating effector caspase
More often, however, caspase-8 drives apoptotic execution less directly, by cleaving
and thereby stimulating the BH3- only protein BID. Truncated BID then engages the
cell-intrinsic pathway via BAX and BAK.
Caspase-10 is a close structural relative of caspase-8, present in zebrafish and primates but
not in rodents (Eimon & Ashkenazi 2010). Yet another close relative of caspase-8 and -10 is
cellular FLICE inhibitory protein (cFLIP). cFLIP lacks protease function; it is an important
regulator of apoptotic as well as nonapoptotic functions of caspase-8

Mechanisms of Apoptosis
Ligation of death receptors on susceptible target cells leads to activation of
the apoptosis-initiating protease caspase-8.
In a minority of cell types, caspase-8 can then directly activate caspase-3 and
-7 to drive apoptosis execution.
More often, however, caspase-8 drives apoptotic execution less directly, by
cleaving and thereby stimulating the BH3- only protein BID. Truncated BID
then engages the cell-intrinsic pathway via BAX and BAK
Caspase-10 is a close structural relative of caspase-8, present in zebrafish and
primates but not in rodents. Another close relative of caspase-8 and -10 is
cellular FLICE inhibitory protein (cFLIP). cFLIP lacks protease function; it is an
important regulator of apoptotic as well as nonapoptotic functions of caspase-8.

Mechanisms of Apoptosis (Extrinsic Pathway)

TNFR1 can trigger apoptosis under specific alternative circumstances, namely

(a) in the presence of transcriptional or translational inhibitors
(b) when NF-B activation is blocked
(c) upon depletion of cellular inhibitor of apoptosis proteins (cIAPs), or
(d) in the face of unmitigated DNA damage.
If caspase activation is blocked in conjunction with one of the above conditions,
TNFR1 ligation by TNF typically induces necroptotic cell death.
DR3 mainly regulates noncanonical NF-B signaling but can activate apoptosis
indirectly via TNF.

Upon ligation, the death receptors Fas, DR4, or DR5 assemble a primary death inducing
signaling complex (DISC).
In epithelial cells, K63-linked ubiquitination of caspase-8 mediated by CUL3 augments
activation, whereas K48-linked ubiquitination mediated by TRAF2 promotes proteasomal
inactivation of caspase-8. BID activates BAX and BAK to induce release of cytochrome C and
Smac/DIABLO from mitochondria.
Cytochrome C associates with Apaf1 to form an apoptosome that drives activation of
caspase-9, which then stimulates caspase-3 and -7.
Smac further augments the apoptotic signal by preventing X-linked inhibitor of apoptosis
protein (XIAP) from blocking caspase -3, -7, and -9. The same receptors also may assemble a
secondary cytoplasmic complex (Complex II), which mediates activation of prosurvival and
proinflammatory pathways or other cell functions via NF-B, JNK, and ERK.
By contrast, upon ligation by TNF, TNFR1 assembles a distinct primary complex (Complex I)
at the plasma membrane. Complex I mediates NF-B, JNK, and;ERK signaling, supporting cell
survival or other non-death functions.

Tumor necrosis factor (TNF) is capable of eliciting necrotic cell death.
In 2000, the laboratories of Tschopp and Nagata described a similar, necrotic type of cell death,
triggered in response to the death ligand FasL.
Electron microscopic examination revealed a necrosis-like morphology of the dying cells.
Surprisingly, this form of cell death was found to be independent of caspase activity.
It requires receptor-interacting protein 1 (RIP1, also called RIPK1)a serine/
threonine kinase previously known to be involved in mediating nuclear factor-B (NF-B)
activation by TNF receptor 1 (TNFR1).
These findings therefore suggested the existence of a specific molecular mechanism for inducing
regulated necrosis.

A small molecule compound, called Necrostatin-1 (Nec-1) inhibits necroptosis.
RIPK1 is the molecular target of Nec-1.
The process of regulated necrosis is now referred to as necroptosis or programmed necrosis.
Necroptosis occurs when caspase activity is suppressed.
Necroptosis provides an important innate mechanism of defense against infectious intracellular
pathogens, including certain viruses that can block apoptosis activation.
The biochemical events that mediate necroptotic death execution are less well understood.
Production of reactive oxygen species by mitochondria, as well as lysosomal leakage and lipid
peroxidation, plays a role in necroptotic cell demise.
Several proapoptotic death ligands including TNF, FasL can induce necroptosis, particularly
when caspase activation is prevented.
Damage-associated molecular patterns such as bacterial lipopolysaccharide or viral nucleic
acid also are capable of inducing necroptotic signaling as are interferons
Several intracellular proteins involved in apoptotic signalingespecially components of the cell-
extrinsic pathwayparticipate also in controlling necroptotic stimulation, suggesting molecular
coordination between the two types of regulated cell ablation.


The TNFR superfamily consists of more than 20 proteins that typically possess a type-I
transmembrane topology with two to four cysteine-rich extracellular domains. Death receptors
form a subgroup within this superfamily, distinguished by the presence of an approximately 80-
aminoacid- long death domain in the cytoplasmic portion.
Six death receptors can regulate apoptosis either directly or indirectly: TNFR1 (TNFRSF1A;
cognate ligands: TNF/TNFSF2 and lymphotoxin /TNFSF1), Fas (APO1/CD95/TNFRSF6;
cognate ligand: FasL/CD95L/TNFSF6), DR3 (APO3/TNFRSF25; cognate ligand: TL1A/
TNFSF15), DR4 (TRAILR1/TNFRSF10A; cognate ligand: Apo2L/TRAIL/TNFSF10), DR5
(TRAILR2/TNFRSF10B; cognate ligand: Apo2L/TRAIL/TNFSF10), and DR6 (TNFRSF21)
(Gonzalvez & Ashkenazi 2010, Wilson et al. 2009).
The ectodysplasin A receptor EDAR (TNFRSF19) and the p75 neurotrophin receptor (an atypical
TNFR superfamily member) also possess discernible death domain motifs. Ligand binding to
some of the death receptors is counter-regulated by decoy receptors, e.g., DcR1 (TNFRSF10C)
and DcR2 (TNFRSF10D), which bind to Apo2L/TRAIL, and DcR3 (TNFRSF6B), which binds to FasL

Caspase-8, likely in its heterodimeric association with cFLIP, suppresses necroptosis by

cleaving RIPK1, among other substrates; Inhibition of caspase-8 permits RIPK1 to
recruit RIPK3 and form a third complex called the necrosome. RIPK1 phosphorylates
RIPK3, driving recruitment and phosphorylation of MLKL and thereby triggering

Phosphorylation of MLKL by RIPK3 causes its oligomerization and translocation to the plasma
membrane, where it perturbs membrane integrity, thereby promoting necroptotic cell death.
Phosphorylation of RIPK1 is dispensable for Complex I activity but essential for necrosome
activity; in some circumstances, e.g., in response to autocrine TNF signaling driven by excessive
DNA damage, RIPK1 phosphorylation is also important for assembly and proapoptotic activity of
Complex II.
The intrinsic pathwaycontrolled by the Bcl-2 protein familyis activated by various types of
cell distress, including DNA or microtubule damage or metabolic stress. Damage sensors, such
as the p53 protein or the lipid kinase AKT, activate specific BH3 proteins by inducing their mRNA
transcription or post-translational modification.
Activated BH3 proteins stimulate proapoptotic BAX and BAK, either directly or by counteracting
their inhibition by antiapoptotic Bcl-2 family members, such as Bcl-2 and Bcl-XL. BAX and BAK
permeabilize the mitochondrial outer membrane, facilitating release of cytochrome C and
Smac/DIABLO to augment stimulation of caspase-3 and -7 and apoptotic cell demise.

Upon ligation, the death receptors Fas, DR4, and DR5 typically signal apoptosis in
responsive cells. Alternatively, depending on the status of key intracellular signaling
components, these receptors may induce necroptosis or even engage pathways that
promote cell survival or other cellular functions. These latter pathways, including the
IKK/NF-B, JNK/c-Jun, and p38/MAPK/AP-1 cascades, are engaged more readily when
apoptosis stimulation is intercepted. On the other hand, TNFR1 mainly signals activation
of the IKK/NF-B, JNK/c-Jun, and p38/MAPK pathways.

Cells of the innate and adaptive immune systems are the main producers of death
ligands such as FasL, Apo2L/TRAIL, and TNF.
These ligands are often expressed as type-II transmembrane proteins at the surface
of activated immune cells; alternatively, they can be released as soluble ligands into
the extracellular space through the action of specific cell-surface proteases.
In turn, these ligands can directly kill target cells that express cognate death receptors
and are primed for death. Target cells may be primed for apoptotic or necrotic
stimulation as part of their normal differentiation program, or owing to damage,
infection, or oncogenic transformation. Indeed, the most salient biological function of
death receptors and their ligands is their involvement in regulation and activity of the
innate and adaptive immune system.
Although death-receptor activation typically requires ligand binding, recent evidence
uncovers ligand-independent intracellular activation of DR5 in response to
unmitigated endoplasmic reticulum stress.

Fas, DR4, andDR5 directly trigger the apoptotic cascade by assembling a death-inducing
signaling complex (DISC), which minimally contains the adaptor protein Fas-associated death
domain (FADD) and the apoptosis-initiating protease caspase-8.
The corresponding death ligands, FasL and Apo2L/TRAIL are homotrimeric proteins; the basic
ligand-receptor complex consists of one trimeric ligand and three receptor molecules.
Upon ligation, death receptors multimerize in the cell membrane, leading to a
conformational change in the receptors intracellular domain.

This, in turn, enables recruitment of the adaptor protein FADD, which contains both a death
domain that interacts with the receptor and a death effector domain (DED) that recruits caspase
8 or -10 and/or cFLIP.
Caspase-8 dimers form the basic catalytic unit of the protease.
Apoptosis commitment often requires further clustering of the initial ligand-receptor complex.

Certain posttranslational modifications of death receptors (palmitoylation of Fas near its

transmembrane domain and O-linked glycosylation of DR4 and DR5 in their ectodomains can
facilitate ligand-induced clustering and apoptosis activation.
Clathrin-mediated internalization of DR5 upon ligation limits signaling and dampens caspase
Caspase-8-mediated cleavage of the clathrin adaptor AP2 counteracts internalization,
reinforcing apoptosis stimulation.
In lymphoma cells, an excess of caspase-8 molecules over FADD is recruited to the DISC
via sequential DED interactions of caspase-8.
In epithelial cells, the homotypic adhesion protein E-cadherin interacts with ligated DR4 and
DR5; E-cadherin couples these death receptors to the actin cytoskeleton via its interacting
partner -catenin, which augments DISC assembly.

When epithelial cancer cells undergo epithelial-to-mesenchymal transition, they

downregulate E-cadherin expression, which makes them less susceptible to apoptosis
activation via DR4 and DR5.
Furthermore, in epithelial cells an E3 ligase complex based on cullin 3 (CUL3) promotes
caspase-8 activation at the DISC by mediating ubiquitination on the small catalytic
domain of caspase-8 ( Jin et al. 2009). Indeed, CUL3 recruitment and caspase-8
ubiquitination are augmented by the interaction between E-cadherin and DR4/DR5

Activation modes of caspase-8.

Caspase-8 and cFLIPL share the same molecular architecture, but FLIPL lacks catalytic
residues. Both proteins possess caspase cleavage sites (Asp) between the large and small
subunits of the catalytic domain, but FLIPL lacks the caspase cleavage site between the
prodomain (DEDs) and the catalytic domain.
Hetero- or homodimerization can lead to some of the species indicated above, but the
relative abundance of these species remains unclear.
Mutation of the caspase-8 interchain linker in mice abrogates apoptotic signaling, but
survival signaling is retained, which suggests that cleavage at this site is necessary to
promote apoptosis.
Subsequent cleavage of the homodimer (c) and release separates the catalytic domains from
the DISC, presumably allowing their decay by monomerization. The heterodimer would not
be expected to dissociate from the DISC (d ) because the FLIPL unit lacks a caspase cleavage
site in its prodomain linker.