Transcript Chapter 13: Vesicular Traffic

Chapter 17: Cell Death
Know the terminology:
Apoptosis, necrosis, Bcl-2, caspase, procaspase,
caspase-activated DNAse (or CAD), death domain,
cytochrome c, mitochondrial permeability
transition pore, Apaf, FAS, TNFa,
The many forms of cell death
Necrosis:
Cell death resulting from physical or chemical
damage. It progresses in an uncontrolled way and
causes local tissue damage or inflammation (in
some species)
Apoptosis:
Controlled form of cell death, where the cell
controls its own demise. First, it degrades its
internal structure, then dies in a way that is
easily handled by local phagocytotic cells.
The many forms of cell death
Why is apoptosis needed?
Apoptosis is needed to:
1. Control the death of irreversibly damaged cells,
preventing local tissue disruption. For example,
-radiation damage
-cell cycle defects
2. Remove cells that are unwanted. For example,
-morphogenesis (tissue formation)
-tissue remodelling
Examples of apoptosis in development
Target cells secrete enough “survival factors” to ensure
the survival of the appropriate numbers of neurons
Examples of apoptosis in development
Embryonic hands/feet start as broad pads, with apoptosis
sculpting the final shape by killing the cells between digits
Examples of apoptosis in development
Metamorphosis hormones trigger apoptosis of trunk
muscles in tail, and induce differentiation of limbs and
appendicular muscle
Intracellular events
Controlled Cell Death:
-cell shrinkage (necrotic cells explode)
-cytoskeletal collapse
-nuclear envelope disassembles
-proteolytic degradation
-membrane phospholipid inversion (PS)
-membrane display of phagocytotic signals
-DNA fragmentation
Caspase activated DNase
CAD cuts DNA between histones, resulting in DNA
fragments of multiples of 280 bp (a DNA ladder).
DNA Ladder
TUNEL
Terminal deoxynucleotidyl
transferase–mediated dUTP
Nick End-Labeling
Meet the executioner: Caspases
Caspases are Cysteine-ASPartate proteases:
-cysteine in in the enzyme active site
-the attack aspartate residues on target
-produced by the cell as inactive proenzymes
(procaspase)
-upon the appropriate signal, the procaspase is
cleaved to form the active caspase
-who cuts up the procaspase? Another caspase.
Caspase cascade
Caspase cascade
Caspase cascade
Initiator caspase
(e.g. caspase 9 or caspase 10)
Executioner caspase
(e.g. caspase 3)
Targets
•Self amplifying
•Irreversible
2 routes of apoptotic induction
1. Intracellular route:
Mitochondrial permeability transition
pore (MPTP)
Recall that the mitochondrial inner membrane has
low permeability (it maintains a proton motive
force).
The outer membrane has porin, which allows small
molecular weight molecules to move freely (less
than about 7,000 daltons)
Mitochondrial compartments
Mitochondrial permeability transition
pore (MPTP)
Cytochrome c (a mobile electron carrier) moves
with the intermembrane space.
Its too big (~12000 daltons) to cross through porin.
When mitochondria completely depolarize, another
pore forms from multiple proteins, allowing
cytochrome c to escape to cytoplasm.
Thus, toxic agents that depolarize mitochondria can
trigger apoptosis.
Bcl2 family
The mitochondria possess a pore that can allow
cytochrome c to escape from intermembrane space
The pore opens with massive membrane
depolarization
Bcl2 (and Bcl XL) are proteins that associate with
the pore and keep it closed.
Bad and Bid are similar in structure to Bcl2 and
bind to anti-apoptotic proteins, blocking their
effects.
Bcl2 family form heterodimers to
cancel out each others effects
Bcl2 family
Bcl2 binds to
mitochondria to prevent
cytochrome c release
Bad binds Bcl2 to
prevent it from
preventing cell death
When Bad is
phosphorylated (PKB) it
can’t bind Bcl2
Bcl2 family
2 routes of apoptotic induction
2. Extracellular route:
Extracellular routes of apoptosis
Extracellular proteins bind to cell membrane
receptors to initiate apoptosis.
Include: membrane proteins such as:
-FAS ligand (killer T cells)
-tumor necrosis factor alpha (TNFa) (macrophages)
Activation of death receptor (FAS protein) recruits
adaptor proteins with “death domain”
Extracellular routes of apoptosis
Adaptor proteins bind (and colocalize) initiator
procaspases (e.g. caspase 8).
Procaspase 8 has weak proteolytic activity but
because they are colocalized, they can attack
each other to form an active caspase.
Inhibitors of apoptosis protein (IAPs)
Procaspases have some low proteolytic activity that
must be held in check in healthy cells
Inhibitor of Apoptosis Proteins (IAPs), such as Xlinked Inhibitor of Apoptosis (XIAP), act by
inhibiting procaspase activity
Weak activity
Procaspase
Very weak activity
Procaspase IAP
Inhibitors of apoptosis protein (IAPs)
Mitochondria can stimulate apoptosis a second way,
by releasing a protein that impairs the effects of
IAPs
Smac (Second Mitochondrial Activator of apoptosis)
Drosophila homologues of Smac include Hid, Grim,
Reaper.
Smac (and its homologues) stimulate apoptosis by
blocking XIAP effects
Inhibitors of apoptosis protein (IAPs)
Procaspase-9 + IAP
Procaspase-9 IAP
Inactive
Procaspase-9 + Smac + IAP
Procaspase-9 Smac IAP
Active
Phosphorylation of Hid
Hid can only bind IAP in
its dephospho form
Phosphorylation of Hid
prevents its ability to
block IAP’s protective
effects
Inhibitors of apoptosis protein (IAPs)
Procaspase-9+Smac + IAP Procaspase-9+Smac + IAP
Procaspase-9 Smac IAP Procaspase-9 IAP
Active
Inactive
Smac
Cancers and apoptosis
Cancer is uncontrolled cell growth.
Many cancer cells are able to proliferate because
they have mutated in a way that prevents the cell
from dying.
Discussion question: What kind of mutations might
disrupt apoptosis?