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, TNF,
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 (TNF)
(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 X-linked Inhibitor of Apoptosis (XIAP), act by inhibiting procaspase activity
Weak activityProcaspase
Very weak activityProcaspase 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
IAPProcaspase-9
Inhibitors of apoptosis protein (IAPs)
IAP
Procaspase-9
Smac
IAPProcaspase-9 Smac
+
IAP
Inactive
Procaspase-9+ +
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)
IAPSmac
IAPProcaspase-9 Smac
Procaspase-9+ +
Active
IAPSmac
IAPProcaspase-9 Smac
Procaspase-9+ +
Inactive
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?
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