Lecture 22: Apoptosis-programmed cell death Flashcards
Programmed Cell Death
• Programmed cell death is a vital process to the overall good of a multicellular organism
- Apoptosis is one form of programmed cell death
- Up to half of the neurons in a developing vertebrate organism die soon after they are formed (target selection)
- Some tissues are formed and most are maintained at a consistent size by selective apoptosis
Morphological changes during apoptosis
- Cells shrink in size
- Chromatin condenses and fragments
- The cytoskeleton disassembles
- The nuclear envelope collapses
- The cell surface distends or “blebs”
- The plasma membrane is chemically altered to promote phagocytosis
____ results from acute injury and the cells typically swell and burst causing an inflammatory response.
Cell necrosis
_____ are essentially degraded from the inside out without damage to neighboring cells
Apoptotic cells
Cell death is induced by the absence or low amounts of “_______”
survival factors
Biochemical changes associated with apoptosis
- DNA fragmentation
- Loss of phospholipid asymmetry in the plasma membrane.
– Phosphatidylserine flips to the outer leaflet
- Cell surface modifications that promote phagocytosis
- Loss of Proton Motive force in mitochondria
- Some of these changes can be detected in dying cells
_____ that are activated as a result of apoptosis cut genomic DNA into a characteristic ladder patterns due to cleavage in the linker regions between nucleosomes
Endonucleases
How does the TUNEL assay detect apoptosis?
The newly generated DNA ends that result from genomic cleavage can be detected in cells by modifying the ends of DNA with a fluorescent nucleotide.
TUNEL=TdT mediated dUTP nick end labeling
TdT=terminal deoxynucleotide transferase
____ is responsible for the apoptosis that is involved in limb formation
BMP
The primary mechanism of destruction is the activation of proteases called ____ (active site cys, cleave at specific asp residues)
caspases
Not all caspases are involved in apoptosis, some are important for the _____
inflammatory response
An activation cascade is thought to be set in motion by _____ that are activated in response to a signal through their _____.
initiator caspases
caspase recruitment domain (CARD)
Once activated, these initiator caspases propagate the cascade of activation through ______
executioner caspases
Caspases involved in inflamation
– Caspase 1 (Interleukin-1 converting enzyme, ICE), 2, 4
Initiator caspases
– Extrinsic pathway
• Caspases 8, 10
– Intrinsic pathway
• Caspases 2, 9
Executioner Caspases
– Caspases 3, 6, and 7
Activation of Caspases
Caspase Cascade
How do caspases trigger DNA fragmentation
-executioner caspases cleave iCAD, cleave iCAD releases CAD which cuts DNA between nucleosomes
Extrinsic pathway
– Activation of Cell surface receptors (death receptors)
Intrinsic pathway
– Triggered in response to injury or stress
– DNA damage
– Lack of oxygen, nutrients, or survival signals
Extrinsic pathway of Apoptosis
- Cell surface receptors (death receptors) activate the extrinsic pathway
- Single span membrane proteins with an extracellular ligand binding domain and an intracellular death domain
- Receptors are homotrimers of the tumor necrosis factor (TNF) family
- Includes the TNF receptors itself and the Fas death receptor
Induction of apoptosis by extracellular stimuli
Extrinsic pathway by activation of Fas death receptors
Intrinsic Pathway of Apoptosis
- Depends on the release of mitochondrial intermembrane space proteins into the cytosol
- The release of cytochrome c, the electron carrier between ETS complex III and cytochrome oxidase, can trigger apoptosis
- Released cytochrome c binds to a procaspase activating adapter called Apaf-1 (apoptotic protease activating factor), driving its oligomerization into a wheel like heptamer called the apoptosome
- The aggregated Apaf-1 binds and activates caspase-9 by induced proximity
- This initiator caspase-9 activates downstream executioner caspases
Induction of apoptosis by intracellular stimuli
_____ proteins control the release of cytochrome c from mitochondria
Bcl-2 (B-cell lymphoma-2)
_____ inhibit apoptosis (anti-apoptotic)
(Bcl-2 and BclXL)
Other family members _____ promote apoptosis (pro-apoptotic)by increasing cytochrome c release from mitochondria.
(Bax and Bac)
anti-apoptotic Bcl2 family proteins have:
all 4 BH domains
pro-apoptotic Bcl2 family proteins have
BH 1-3 domains
BH3 only pro-apoptotic proteins include:
Bad, Bim, Bid, Puma, Noxa
___ is tightly bound to the mitochondrial membrane even in the absence of an apoptotic signal
Bak
___ is mainly located in the cytosol an translocates to the mitochondria in response to an apoptotic signal
Bax
Upon activation, Bax and Bak generate….
pore-forming proteins in the mitochondrial outer membrane
Bcl2 mechanism of cytochrome C release
Bcl-2 and Bcl-XL
• Located on the cytosolic surface of the mitochondrial outer membrane
• Binds to bax to prevent bax oligomerization
BH3 only proteins
- proapoptotic
- Binds to Bcl2 to inhibit their ability to bind and inhibit Bak or Bax
____ is transcriptionally upregulated in the absence of cell survival signals
Bim
___ and ___ are transcriptionally upregulated in response to DNA damage
Puma and Noxa
Bid is cleaved to tBid by activated ___ coordinating the extrinsic and intrinisic pathways in some cells
caspase 8
Inhibitors of Apoptosis (IAPs)
• Caspase inhibitors
- Used by viruses to prevent the host cell form committing suicide prior to viral replication
- IAPs can be countered by anti-IAPs in some cells
- Anti-IAPs bind to IAP and prevent them from inhibiting caspase
- Genetic manipulation of IAP or anti-IAPs have dire consequneces in flies indicating the delicate balance needed for apoptosis during development
Three methods of survival factor inhibition of apoptosis
- increased production of anti-apoptotic Bcl2 family protein
- inactivation of pro-apoptotic BH3-only protein
- inactivation of anti-IAPs
Apoptotic cells are cleared by ____
phagocytosis
