Nov 24 - Lecture 36 ~ Apoptosis

apoptosis is a gentle form of cell death (regulated dissolution)

• compare to necrosis — a cellular explosion

• DNA gets fragmented; nuclear lamins break down & actin rearrangement to form blebbing

cell death is as important to development as cell proliferation

• embryonic development is, of course, dependent upon proliferation of the initial zygote into the multitudes of cells that make up body tissues

• Not all cells in a developing tissue, however, contribute to the final structure

• developing mouse paw cells stained in a yellow-green color are undergoing apoptosis to produce distinct digits

  • Failure of this process in mice (and humans) leaves a persistent webbing between the digits

  • As a tadpole metamorphoses into an adult frog, the cells making up its tail likewise die in a regulated fashion

apoptosis is regulated by caspases (“cleave after aspartate”)

• Apoptosis has a complex integrative regulatory regime (which you will see), but major events are controlled by caspase proteins

• Caspases are cysteine proteases that are activated by proteolytic cleavage

• Each caspase has multiple targets that are cleaved at conserved sequences that end with aspartate

• Initiator caspases are activated early in the process

• Executioner caspases are activated by initiator caspases and drive apoptosis by cleaving their own targets

  • E.g. inhibitor of Caspase-Activated DNase (iCAD)

capase-activated DNase (CAD)

different caspases deliver death signals from different parts of the cell

• extrinsic and intrinsic pathway

pro-apoptotic signals can come from other cells

• The extrinsic apoptotic pathway relies on activation of plasma membrane proteins collectively termed death receptors

• The group of proteins recruited to death receptors is collectively referred to as the Death-Inducing Signaling Complex (DISC)

  • The best characterized death receptor is Fas. Cytotoxic T cells kill virally infected cells, in part, by signaling through FasL — contact-dependent pathway

immune cell stimulation of apoptosis

• natural killer cells and cytotoxic (CD8) T cells can kill abnormal or sick cells with Fas ligand

• activation of Fas creates a Death-Inducing Signaling Complex (DISC) consisting of procaspase-8 and Fas-Associated Death Domain (FADD)

the intrinsic pathway requires release of cytochrome c from mitochondria

• Cytochrome c (part of the electron transport chain) assembles with other proteins to form a complex called the apoptosome which activates initiator caspase-9

• Cytochrome c and dATP (deoxy-ATP) bind and activate Apaf1, which assembles via its CARD domain into a wheel-like structure

  • Caspase-9 is recruited via its own CARD domain and activated

    

interaction domains of anti- and pro- apoptotic proteins

Bak oligomers promote Mitochondrial Outer Membrane Permeabilization (MOMP)

Bcl2 family proteins inhibit Bak oligomerization

• BH3-only proteins competitively inhibit Bcl2 proteins

  • BH3 -| Bcl2

pro-survival signaling acts through inhibitors of apoptosis

• Normal animal cells require regular pro-survival signaling

• these pathways suppress apoptosis by stimulating the production and/or activation of apoptosis inhibitors

• Most promoters of apoptosis are always present – only continuous suppression keeps the cell alive

  • this makes apoptosis a nimble process – a cell that is damaged or in danger of becoming a cancer can readily be induced to self-destruct

limited survival factors prune developing nervous systems

• Many infant animals (including humans) are born with more neurons in their brains than adults

• The target cells produce a finite amount of pro-survival factors

• As the nervous system develops postnatally, neurons die and, ultimately, the number of neurons and target cells are balanced

Inactivation of flippase and activation of scramblase relocalizes membrane lipids

• In healthy cells, most of phosphatidylserine and phosphatidylethanolamine is on the cytosolic leaflet of the plasma membrane

• Executioner caspases cleave both flippases and scramblases on the plasma membrane

• This inactivates flippases, but activates scramblases

• Altered flippase and scramblase activity allows cytosolic phospholipids to appear in greater numbers on the extracellular leaflet

• Extracellular phosphatidylserine is an “eat me” signal that stimulates phagocytosis of apoptotic cells

Summary / Key Concepts

• Apoptosis is a regulated form of cell death

  • in contrast to necrosis, which is simply disruption of the plasma membrane, apoptosis gradually breaks down cell contents and packages them into vesicles for consumption by phagocytes

• Caspases are major drivers of apoptosis

  • initiator caspases are activated by different cellular events

  • executioner caspases activate mediators (like Caspase-Activated DNase) through proteolytic cleavage

• The extrinsic pathway is activated by death-inducing ligands (like FasL)

  • death receptors signal through caspase-8

• Pro-apoptotic factors include members of the Bak and BH3-only family

  • Bcl2 family members are anti-apoptotic

• The intrinsic pathway of apoptosis relies on release of cytochrome c from the mitochondria via Bax and Bak;

  • these proteins are inhibited by Bcl2

  • Cytochrome c forms a complex with other proteins that activates initiator caspase-9

• pro-survival signaling suppresses apoptosis

  • limitations in survival factors during development prune cells from forming tissues

• Alterations in flippase and scramblase activity via Caspase cleavage allows formerly cytosolic phospholipids to appear on the outside of cells