Chapter 12 & 14 only summarized from the lectures!
Apoptosis:
- The regulated and orderly destruction of a cell through a genetically encoded process
also known as programmed cell death
- Apoptosis is a type of ‘cell suicide’ that is intrinsic to the cell
➔ Function:
• Developmental morphogenesis
• Controls cell numbers
• Removal of damaged cells
• Negative and positive selection of lymphocytes
• Cytotoxic effect of radio- and chemotherapy
7.1 Molecular mechanisms of apoptosis
Apoptosis exists of two pathways; the extrinsic and intrinsic way. Important proteins in both
pathways are caspases, that act like molecular scissors to cleave intracellular proteins at
separate residues
The extrinsic pathway: mediated by membrane
death receptors
This pathway for triggering cell death shares some
common features with pathways involved in triggering
cell growth.
It begins with a death factor, such as
- Fas ligand
- Tumour necrosis factor (TNF)
that is received by a transmembrane death receptor
such as Fas receptor or TNF receptor. The complex the
ligand and receptor make exposes so-called death
domains that enable intracellular adaptor proteins (such
as FADD and TRADD).
➔ The function of adaptor proteins is to transduce
the death signal from the receptor to caspases.
The adaptors recruit several molecules of pro-caspase-
8 via death effector domains. Caspase-8 is known as
an initiator caspase, as it is the first link between the
receptor and the apoptotic proteases, and it is key to
the extrinsic pathway.
DISC complex: death-inducing signalling complex. This complex consists of the death
ligands, receptors, adaptors, and initiator caspase together.
1
,Caspase-8 initiates a cascade of caspase activation (caspase- Caspases
3, -6 and -7). The cascade ultimately causes the cleavage of - Cysteine-proteases
specific protein targets and results in apoptosis. synthesized as zymogens
But, this process can be inhibited by c-Flip. It can bind to - Requirement for an
adaptor FADD or caspase-8 and inhibit caspase-8 recruitment aspartiatic acid at the P1
and activation. position
- 14 family members cloned
The breakdown of the cell results from the proteolysis of the - Involved in apoptosis: 2, 3, 6,
target proteins. These include 7, 8, 9, 10
- Nuclear lamins allowing for nuclear shrinkage
- Cytoskeletal proteins (actin and intermediate filaments)
- Specific kinases for cell signalling
- Other enzymes (such as DNase)
The intrinsic pathway: mediated by the mitochondria
This pathway does not depend on external stimuli. Stimuli from
inside the cell, such as DNA damage and oxidative stress,
induce the intrinsic pathway of apoptosis through the Bcl-2
family of proteins that act at the outer mitochondrial
membrane.
Bcl-2 family
Two groups within this family have opposing functions: one
group inhibits apoptosis, and another group promotes
apoptosis. It is the permeabilization of the outer mitochondrial
membrane and the release of stored pro-apoptotic factors that
are critical in triggering the intrinsic pathway of apoptosis. The
balance of the activities of the pro- and anti-apoptotic proteins
is what regulates the release of important molecular apoptotic
mediators from the mitochondria.
The activity of the Bcl-2 proteins can also be regulated by
phosphorylation.
• The Bcl-2 family forms hetero- or homodimers through
a shared domain (Bcl-2 homology domain ((BH)3))
Anti-apoptotic Pro-apoptotic Pro-apoptotic
members members members (BH3 only)
Bcl-2 Bax Bad The BH3 only
Bcl-XL Bok/Mtd Bik/Nbk/Blk group is really
Bcl-w Bcl-x Bid important for
A1 Bak Hrk/DP5 cancer treatment
and development
Mcl-1 Bcl-G Bim/Bod
Boo Bmf
Noxa
Puma/Bbc3, BNIP3(L)
BH3 only proteins means that these members only can bind to one region on the protein.
The others have more regions to bind to. (Apoptosis Regulation by Genes | Bcl-2 Family -
YouTube)
2
,Remember that BAD and Noxa can only bind specifically to a couple
factors, but BIM, BID and PUMA to all!
Upon activation
Upon activation by an apoptotic signal, BH3-only proteins, Bid and
Bim, bind to, and activate, Bax. This induces a conformational
change in Bax, as it translocates from the cytoplasm to the
mitochondria. Further, the mitochondrial membrane increases the permeability of the outer
mitochondrial membrane and therefore allows the release of apoptotic mediators.
- Cytochrome C and procaspase-9 are released into the cytoplasm and assemble
into a complex called an apoptosome, along with dATP bound to Apaf-1. The binding
of cytochrome c to Apaf-1 triggers the formation of a complex that
facilitates the recruitment of procaspase-9 via protein domains. Caspase-8 is key to
- Apaf-1 is a protein co-factor that is required for activation of pro- the extrinsic pathway
caspase-9. Caspase-9 is an initiator caspase activated by procaspase Caspase-9 is key to
aggregation that begins another caspase cascade activating the intrinsic pathway
downstream caspase-3, -6, and -7.
C-Flip regulation
In the picture depicted right, shown is that caspase 8 (as
a homodimer) can activate apoptosis.
- When caspase-8 forms a heterodimer with
cFLIPlong, limited caspase-8 activity is induced.
- When caspase-8 forms a heterodimer with
cFLIPshort, caspase 8 activation is being inhibited
and apoptosis as well.
Overview regulators of the apoptosis pathways
Bcl-2 family:
Inhibitors: Bcl-2, Bcl-XL
Activators: Bax, Bid
C-Flip:
c-Flip short: inhibitor of activation of caspase 8
c-Flip long: controversial, inhibitor/inducer of apoptosis
IAPs (inhibitors of apoptosis proteins):
cIAP1, cIAP2, XIAP
Cross-talk between extrinsic and intrinsic pathways
The two pathways converge at the activation of downstream caspases.
F.e. caspase-8 can cleave and activate Bid, that then stimulates the
intrinsic pathway of apoptosis by directly activating Bax and Bak,
facilitating the release of cyt c from the mitochondria and inducing the
subsequent activation of downstream caspases.
P53 and apoptosis
As a transcription factor, p53 induces the expression of genes that
code for death receptors and pro-apoptotic members of the Bcl-2
family. Essential targets of p53 is a member of the Bcl-2 family called
‘PUMA’. In PUMA knock-out mice apoptosis and cell stress were
blocked when induced by DNA-damaging drugs, irradiation and
oncogenic activation.
3
, 7.2 Apoptosis and cancer
Apoptotic signals stimulate procaspase processing in normal cells, while apoptotic signals
stimulate the cessation of inhibition of processed caspases in cancer cells.
TRAIL receptors
TRAIL receptors are a subfamily of TNF receptors, and have been found to elicit a differential
sensitivity to apoptosis between normal cells and cancer cells. Its ligand (TRAIL) induces
apoptosis in many cancer cells, but not in most normal cells. As TRAIL and its receptors are
expressed in most organs, it is hypothesized that the addition or loss of regulatory molecules
determines whether apoptosis will be induced in particular cell types.
Mutations that affect the extrinsic pathway
• UV exposure causes the clustering of Fas death receptors and activation of the
caspase cascade. Mutation in the Fas pathway may reduce the apoptotic response
and increase the risk of skin cancer.
• Suppression of caspase gene expression in the development of particular cancers,
such as small-cell lung carcinoma and neuroblastomas, has also been demonstrated.
• Also mutations and epigenetic loss in/off caspase 8 and 10 are seen.
Mutations that affect regulators of the intrinsic pathway
Alterations of the intrinsic pathway of apoptosis are much more common than in
the extrinsic pathway. Mutations are
- A major contribution to intrinsic pathway alterations occurs through found a lot in
mutations that affect the p53 pathway. The p53 mutations provide the ✓ P53
cancer cells with a survival advantage by disrupting apoptosis. ✓ Apaf-1
o Also, mutations in genes involved in upstream & downstream ✓ Bcl-2
regulation of p53 have been identified in human tumours. ✓ Bax
- Overexpression of the anti-apoptotic protein Bcl-2 leads to insufficient ✓ XIAP
apoptotic turnover and accumulation of B-cells. ✓ survivin
Alternative death pathways
• Autophagy
o Acts as a recycling system for the cell whereby proteins and components of
damaged organelles that require degradation are targeted to the lysosomes.
Cells can recycle the resulting products of degradation (important under
starvation).
• Mitotic catastrophe
o Caused by aberrant mitosis
7.3 Apoptosis and chemotherapy
1. Anti-cancer drugs kill tumour cells by apoptosis activation
2. Apoptosis is deregulated in cancer, and correlates with aggressive phenotype
3. Inhibition of apoptotic pathways make cells resistant to anti-cancer treatments.
In order for chemotherapy to be successful, cells must be capable of undergoing apoptosis.
Many tumours have defective apoptotic pathways and are inherently resistant to
chemotherapies. This type of resistance contrasts the classical acquired mechanisms that
are associated with drug accumulation and drug stability.
➔ Upregulation of the anti-apoptotic members of the Bcl-2 family and the
downregulation of the pro-apoptotic members of the Bcl-2 family in tumours are
associated with an increased resistance to chemotherapies.
4
