Cell Death

Question 1

Learning Outcomes

Answer 1

• Understand the concept of apoptosis
• Understand the role of caspase in apoptosis
• describe the intrinsic and extrinsic pathways

Question 2

Reading:

Answer 2

• Alberts et al. Molecular Biology of the Cell, Ch10
• Alberts et al. Essential Cell Biology, Ch11

Answer 3

Necroptosis – recent studies identified a regulated cell death with necrotic phenotypes
(caspase independent)

Question 3

Apoptosis vs. Necrosis

Answer 3

• Swell and burst
• Spill contents to the neighbours and cause inflammation
• Energy depletion leads to metabolic defects and loss of the ionic
  gradients that normally exist across the cell membrane

Answer 3

Necrosis – cell death derived by acute insults (unregulated) e.g., a trauma and a lack of
blood supply – normally necrotic cells swell and burst

Question 4

Cell death

Answer 4

The growth (proliferation), development and maintenance (homeostasis) of multicellular
organisms depend not only on the production of cells (by cell cycle/division) but also on
mechanisms to destroy them. E.g., the maintenance of tissue size requires that cells die at
the same rate as they are produced.
Programmed cell death occurs by a process called apoptosis (highly regulated by caspase
signaling)

Question 5

Apoptosis vs. Necrosis

Answer 5

• Nuclear chromatin condenses and breaks up into fragments
• Cytoskeleton collapses
• Nuclear envelope disassembles
• If large, breaks up into membrane-enclosed fragments,
  apoptotic bodies
• Phagocytic cells (e.g., macrophages) engulfs them quickly before
  spilling contents (lead inflammation)
• Adult human loses ~ 50-70 billion cells (out of ~ 37 trillion) each
  day due to apoptosis

Question 6

Apoptosis eliminates unwanted cells

Answer 6

Apoptosis can shape during development
* Self destructing cells with abnormalities
* Self destructing cells not required anymore for normal development

Question 7

Apoptosis eliminates unwanted cells

Answer 7

Sculpting the digits in the developing mouse paw by apoptosis
* Special bright green labelled for apoptotic cells (A)
* Interdigital cell death eliminated the tissue after one day (B)
forming individual digits

Question 7

Caspase mediate apoptosis

Answer 7

caspases = proteases with a cysteine at their active site and cleave their target proteins at
specific aspartic acids

Question 8

Apoptosis eliminates unwanted cells

Answer 8

Metamorphosis of a tadpole to a frog
* Massive cells death in tadpole tail
* Eliminating unwanted tissues (cells)

Question 8

Caspase mediate apoptosis

Answer 8

Apoptosis is triggered by members of a family of specialized intracellular proteases,
caspases, which cleave specific sequences in numerous proteins inside the cells, thereby
bringing about the dramatic changes that lead to cell death and engulfment.

Question 9

Caspase mediate apoptosis

Answer 9

Caspases are synthesized in the cell as inactive precursors and are activated only during
apoptosis

Question 10

Caspase mediate apoptosis

Answer 10

2 major classes of apoptotic caspases: initiator caspases and executioner caspases

Question 10

Initiator caspase and executioner caspase

Answer 10

Executioner caspase
* Normally inactive dimer
* Cleaved by initiator caspases at a site in the
protease domain = activation
* One initiator caspase can activate many
executioner caspases = amplifying proteolytic
cascade
* Once activated, executioner caspases catalyse
the widespread protein cleavage events that kill
the cell

Question 10

Initiator caspase and executioner caspase

Answer 10

Question 11

Initiator caspase and executioner caspase

Answer 11

Initiator caspase
* Begin the apoptotic process
* Assemble active caspase by forming dimer of
initiator caspases and adaptor proteins
* Dimer then cleaves its partner at a specific site in
the protease domain to stabilise the active complex
* Major function = activate executioner caspase

Question 12

Caspases cause irreversible breakdown of proteins

Answer 12

Nuclear lamina rupture during cell division
(prometaphase – telophase) and during apoptosis

Question 12

Caspases cause irreversible breakdown of proteins

Answer 12

Nuclear envelope rupture during apoptosis
* Caspase 3 dependent
* Caspase can also inhibit cytoskeleton and cell-cell
adhesion molecules to make engulfing easier

Question 13

Caspase also mediate DNA fragmentation

Answer 13

• Endonuclease CAD associated with its inhibitor, iCAD in
  healthy cells
• Activated executioner caspase cleaves iCAD leading CAD
  activation
• Active CAD produce fragmented DNA

Question 14

Caspases cause irreversible breakdown of proteins

Answer 14

Nuclear envelope rupture during cell division
* Disassembly of the nuclear lamina and M phasespecific phosphorylation of lamins by Cdc2 kinase
(Cdk1)

Question 15

Extrinsic vs. intrinsic pathways

Answer 15

Cells use at least two distinct pathways to activate initiator caspase and trigger a caspase
cascade leading to apoptosis:
* Extrinsic pathway – activated by extracellular ligands binding to cell-surface death
receptors
* Intrinsic pathway – activated by intracellular signals generated when cells are stressed

Question 16

Caspase also mediate DNA fragmentation

Answer 16

Question 17

Extrinsic pathway via death receptors

Answer 17

• Extracellular signal proteins (Fas ligand
  in killer lymphocyte) bind to cellsurface death receptors (Fas death
  receptor)
• Death receptors are homotrimers and
  belong to the tumor necrosis factor
  (TNF) receptor family e.g., TNF and Fas
  * Fas ligand-death receptor binding
  activates the cytosolic tails of Fas death
  receptors to form DISC with
  intracellular adaptor proteins leading
  to the binding of initiator caspases
  (primarily caspase-8)
• Activated initiator caspases cleave their
  partners and activate executioner
  caspases to induce apoptosis

Question 17

Extrinsic vs. intrinsic pathways

Answer 17

Each pathway uses its own initiator caspases, which are activated in distinct activation complexes: * Extrinsic pathway – death receptors recruit caspase-8 via adaptor proteins to form the DISC (death-inducing signaling complex) * Intrinsic pathway – cytochrome c released from the intermembrane space of mitochondria activates Apaf1, which assembles into an apoptosome and recruits and activates caspase-9

Question 18

Extrinsic pathway via death receptors

Answer 18

* Some cells use the extrinsic pathway to recruit the intrinsic apoptotic pathway to amplify the caspase cascade * To restrain the extrinsic pathway, some cells produce FLIP proteins, which resembles an initiator caspase but has no protease activity (lacking the key cysteine) – preventing inappropriate activation of the extrinsic pathway of apoptosis

Question 18

Intrinsic pathway depends on mitochondria

Answer 18

* Cells can also activate apoptosis from inside, often in response to stresses, such as DNA damage, or in response to developmental signals * Depends on the release of mitochondrial proteins from intermembrane of mitochondria to cytosol

Question 19

Extrinsic pathway via death receptors

Answer 19

Question 20

Intrinsic pathway depends on mitochondria

Answer 20

Question 21

The intrinsic pathway of apoptosis

Answer 21

Question 22

Bcl2 family controls the release of cytochrome c

Answer 22

* Mammalian Bcl2 family proteins regulate the intrinsic pathway of apoptosis mainly by controlling the release of cytochrome c and other intermembrane mitochondrial proteins into the cytosol * Some Bcl2 family proteins are pro-apoptotic and promote apoptosis by enhancing the release * Others are anti-apoptotic and inhibit apoptosis by blocking the release * Pro- and anti-apoptotic proteins can bind to each other in various combinations to form heterodimers in which the two proteins inhibit each other’s function – will eventually decide whether cell lives or dies by the intrinsic pathway of apoptosis

Question 22

Bcl2 family controls the release of cytochrome c

Answer 22

Question 23

BH3-only and anti-apoptotic Bcl2 in the intrinsic pathway

Answer 23

Inactive intrinsic pathway * In the absence of an apoptotic stimulus, anti-apoptotic Bcl2 family proteins bind to and inhibit the effector Bcl2 family proteins on the mitochondrial outer membrane (1) and in the cytosol (2) – inhibiting the oligomerisation of pro-apoptotic effector Bcl2 family proteins

Question 23

Pro-apoptotic effector in the intrinsic pathway

Answer 23

Question 24

BH3-only and anti-apoptotic Bcl2 in the intrinsic pathway

Answer 24

Activated intrinsic pathway * In the presence of an apoptotic stimulus, BH3-only proteins are activated and bind to the anti-apoptotic Bcl2 family proteins – no longer inhibiting the formation of effector Bcl2 family proteins oligomers

Question 25

BH3-only and anti-apoptotic Bcl2 in the intrinsic pathway

Answer 25

Question 26

Survival factor can control cell number

Answer 26

* Extracellular signal molecules can stimulate apoptosis - stimulation can be important during vertebrate development: a surge of thyroid hormone in the bloodstream, for example, signals cells in the tadpole tail to undergo apoptosis at metamorphosis

Question 27

Survival factor can control cell number

Answer 27

* However, extracellular signal molecules can also inhibit apoptosis – survival factor * Nerve cells are produced in excess in the developing nervous system then compete for limited amounts of survival factors that are secreted by the target cells that they normally connect to * Nerve cells that receive enough survival signals live, while the others die – the number of surviving neurons is automatically adjusted by the number of target cells they connect with

Question 27

Survival factor can control cell number

Answer 27

Question 28

Survival factors inhibit apoptosis

Answer 28

Increase anti-apoptotic Bcl2 family protein * Activated receptor upon binding of survival factor activates and translocates transcription regulator to nucleus * Increased encoding of anti-apoptotic Bcl2 proteins inhibit apoptosis

Question 29

Survival factors inhibit apoptosis

Answer 29

Inactivate pro-apoptotic BH3-only protein * Activated receptor upon binding of survival factor activates Akt kinase * Active Akt kinase dissociate the binding of Bcl2 (anti-apoptotic) and BH3-only protein (e.g., Bad) * Phosphorylated Bad become inactive while dissociated Bcl2 (antiapoptotic) become active * Active anti-apoptotic Bcl2 proteins inhibit apoptosis

Question 30

Survival factors inhibit apoptosis

Answer 30

Question 31

Phagocytes remove the apoptotic cell

Answer 31

Apoptotic cell death is a remarkably tidy process (esp. compared to necrosis) * cell and its fragments do not break open and release their contents * remain intact as they are efficiently eaten – or phagocyted– by neighboring cells * leave no trace – no inflammatory response

Question 32

Phagocytes remove the apoptotic cell

Answer 32

Engulfment process depend on chemical changes on the surface of the apoptotic cells * Distribution of the negatively charged phospholipid, phosphatidylserine on the cell surface * Apoptotic cells show flipping of phosphatidylserine from inner leaflet of the lipid bilayer to the outer leaflet

Question 33

Phagocytes remove the apoptotic cell

Answer 33

Question 34

Chemotherapy by stimulating apoptosis

Answer 34

Stimulating apoptosis = treating cancers * decreased apoptosis contributes to many cancers * Small molecule inhibiting anti-apoptotic Bcl2 family proteins such as Bcl2 and BclXL * Chemicals with high affinity to the hydrophobic groove on anti-apoptotic Bcl2 family proteins – blocking their function in essentially the same way that BH3-only proteins do

Question 35

Chemotherapy by stimulating apoptosis

Answer 35