PRESENTED BY DOCTOR DEATH Apoptosis
Learning Objectives To Die For • To understand the proteins involved in apoptosis. • Know the regulation of apoptotic signaling pathways. • Importance of apoptosis in the immune system and diseases.
Apoptosis (Programmed cell death) • Active process of cell collapse • Requires ATP • Morphological Features • Membrane blebbing • Chromatin condensation • Protein fragmentation • Apoptotic bodies (engulfed by phagocytosis)
Apoptosis • Apoptosis is conserved from worms to mammals • In humans 10 billion cells undergo apoptosis to maintain number of new cells generated each day • In the immune system development, over-production of cells arise followed by death of these cells that fail to have productive antigen specifications • Apoptosis is necessary to purge the body of pathogens invaded cells and eliminate activated or autoimmune cells.
Apoptosis • Apoptosis needs to be tightly regulated since too little or too much cell death may lead to developmental defects, autoimmune disease, or cancer such as leukemia. • There are now over 50,000 paper on apoptosis • Many companies are developing therapeutic drugs targeted against apoptosis for treatment of diseases.
Morphological changes during apoptosis Department of Biochemistry and Immunology at St.George's Hospital Medical School, London.
C. Elegans • First organism in which regulation of apoptosis was discovered. • C. elegans is special since every lineage of cells is known and ultimate fate of each cell is known. • Genetic analysis by elimination of genes revealed four genes essential for apoptoiss • CED 3 • CED 4 • CED 9 • Egl 1
CED 3 CED 4 CED 9 Egl 1 APOPTOSIS
CED 3 • CED 3 is a cystiene protease homologous to proteases in mammals called caspases. • Caspases cleave specific proteins • Proteolysis is irreversible • Regulation involves - substrate availability • - catalytic activity • - protein turnover
Activation involves proteolytic cleavage of the subunits into a tetramer complex Active caspase Caspases Expressed as a proenzyme Prodomain (30-50kDa) Large subunit (20kDa) Small subunit (10kDa)
Caspases • Activated caspases cleave at four amino acids with aspartic acid required at the amino terminal end. • Consensus sites for caspases • Caspase 8 (L/V/D)EXD • Caspase 9 (I/V/L)EHD • Caspase 3 DEXD • Caspase 1 (W/Y/F)EHD
Classifications of Caspases Caspase 8 Caspase 10 Initiation Caspases Caspase 9 Caspase 2 Caspase 1 Intermediate Caspases Caspase 4 Capsase 5 Caspase 3 Terminal (Effector) Caspases Caspase 6 Caspase 7
Caspase • Substrates: • Caspases cleavage many proteins that can be categorized into four general groups • Nuclear proteins • Structural proteins • Signaling proteins • Caspases
Department of Biochemistry and Immunology at St.George's Hospital Medical School, London.
Caspases • Regulation of Effector Caspases • Cascade model • Initiator caspases activate effector caspases through cleavage. • Regulation of Initiator Caspases • Activation involves co-factors • Binding of co-factors triggered by a pro-apoptotic signal • Mediated through two distant structural motifs • Prodomain of caspases and its corresponding co-factor • Caspase 8 pro-domian binds to the adaptor protein FADD through a death effector domain (DED) • Caspase 9 binds to a co-factor through the caspase recruitment domain (CARD) found in Apaf1
Caspases • How do co-factors activated caspases? • Induced proximity or oligomerization model • Based on • Low but detectable levels of activity • Dimerization required for activation • Over-expression and cross-linking leading to activation • Caspases are latant and co-factors bring two or more caspases together allowing for intermolecular auto-proteolytic activation • Faciliated autocataylsis model • Caspase precursors are present in complexes • Co-factors facilitate activation by conformational changes either directly or by removing an inhibitory protein. • Compartmentaliztion • A co-factor is sequester in a different location in the cells than the caspase • Inhibitors binding to caspases prevent binding of co-factors.
CED 4 CED 3 APOPTOSIS
Co-Factor CED 4 • CED 4 contains a CARD domain and is homologous to Apaf1. • Afap1 is localized to the cytoplasmic side of the mitochondria. • Binds to caspase 9 through its CARD domain • Requires cytochrome c and ATP to bind to caspase 9 and activate its activity. • Caspase 9 cleavage might not be necessary for its activation.
Caspase 2 has co-factors! Rastogi et al EXCLI Journal 2009
Co-Factors CED4 • Besides APAF-1, another co-factor exists called FAS associated death domain (FADD). • Binds caspase 8 and caspase 10 • FADD inducibly associates with activated death receptors causes caspase trans-cleavage and activation
Types of Apoptotic Signaling Pathways • Extrinsic Apoptotic pathway • Death receptor activation • Intrinsic Apoptotic pathway • Mitochondria regulated
Death Receptors • Tumor necrosis factor receptor (TNFR) family is involved in the induction of apoptosis. • Members contain a five cysteine rich repeats in the extracellular domain and a death domain in the cytosolic tail. • Ligation of these receptors causes rapid induction of apoptosis.
? TRADD FADD FADD TRADD FADD FADD FADD FADD Caspase8 Caspase 8 Caspase 8 Caspase8 Caspase 8 Caspase 8 Active caspase 8 APOPTOSIS Death Receptor Family FASL TRAIL TNF FAS DR4/5 TNFR
Department of Biochemistry and Immunology at St.George's Hospital Medical School, London.
TRAIL DR4/ DR5 TRAIL DR4/ DR5 DR4/ DR5 DD DD FADD DD pro-caspase 8 Caspase 8 tBid • R.O.S. • DYM • Release of Smac/Diablo Signal Transduction Upstream of Mitochondria TRAIL Cell Membrane Cytosol
FAS Receptor • Expression is increased following cytokine, or lymphocyte activation. • Ligand for FAS receptor is FAS ligand that is a type II transmembrane molecule and its expression is tightly controlled. • FAS is trimerized following FAS ligand binding and is required for its activation. • A complex of proteins associate with the receptor called the death inducing signaling complex (DISC).
FAS Receptor • In Type I • Caspase 8 cleaves and activates caspase 3 • In Type II • Caspase 8 cleaves BID and releases cytochrome c from the mitochondria. • DISC formation is limited and BID cleavage serves to amplify the apoptotic signal. • FASL membrane bound vs soluble might determine type of FAS activation?
Mitochondria Bid tBid caspase-3 APOPTOSIS Beyond Caspase 8 Activation FASL FAS TYPE II FADD Procaspase-8 Initiator Caspase Active caspase-8 TYPE I Effector Caspase
TRAIL (a death receptor ligand) • Binds to death receptor 4 and 5. • Activates caspase 8 through binding of FADD to receptors. • Synergy between TRAIL and chemotherapeutic drugs in cancer cells.
Control EGF TRAIL Pro-Caspase 8 BID MKP-1
Decoy Receptors: STOP THE DESTRUCTION • Expressed on the cell surface and bind to death receptor ligands such as TRAIL • Fail to recruit FADD to the receptor and thereby fails to activate caspase 8. • Blocks death receptor induced apoptosis by sequestering its ligand.
FASL TRAIL DcR1/2 DcR3 FLIP FLIP Active caspase 8 FASL TRAIL FAS DR4/5 ? FADD FADD FADD FADD Caspase8 Caspase 8 Caspase 8 Caspase8 APOPTOSIS
Intrinsic Apoptotic Pathway • Requires mitochondria • Three main events happen in the mitochondria to induce apoptosis • Loss of Dym • Increase reactive oxygen species (ROS) • Release of mitochondrial proteins
Release of Mitochondrial Protein • During apoptosis, cytochrome c is released from the mitochondria. • It binds to caspase 9/Apaf1 complex thereby activating caspase 9 activity • This complex is called the apoptosome (same concept as the DISC). • Leads to effector caspase activation and apoptosis.
Release of Mitochondrial Protein • Smac/Diablo is another protein release from the mitochondria during apoptosis. • Binds to inhibitor of apoptosis proteins (IAP) • IAP constitutively bind to caspases (both initiator and effector). • Smac/Diablo sequestors IAPs away from caspases allowing for caspase activation • Takes the brakes off the apoptotic pathway.
CELL Apoptotic Signal IAPs Healthy Death
Smac/Diablo Omi/HtrA2 FADD APAF1 Caspases Caspases Caspases Caspases Caspases Caspases Caspases GAS CELL CELL CELL CELL CELL CELL CELL Caspases CELL Apoptotic Signal Healthy Death
Other Proteins Released • Apoptosis inducing Factor (AIF) is released from the mitochondria. • Translocates to the nucleus and participates in DNA fragmentation. • Nuclease G is a Dnase that also translocates to the nucleus and degrades DNA.
The Mitochondria and Apoptosis • Different pro and anti-apoptotic signals converge at the mitochondria. • Apoptotic signals activate pro-apoptotic Bcl-2 family members such as Bid, Bax, and Bak. Survival signals work through the action of anti-apoptotic Bcl-2 family members such as Bcl-2, and Bcl-XL. • Key apoptotic events include a loss in the mitochondrial membrane potential DyM, the production of reactive oxygen species R.O.S., and the release of mitochodrial proteins such as Smac/Diablo and cytochrome c.
Role of Mitochondria • Two well defined compartments in the mitochondria • Inner membrane where the electron transport chain is found • Outer membrane that controls solutes and ions into the mitochondria. • Permeable transition pore (PT pore) controls the movement of ions and solutes in and out of the mitochondria. • Changes in this pore during apoptosis reduces the electrochemial gradient and increases reactive oxygen species (ROS). • PT pore might also control release of proteins?
PT Pore Cyclosporin A Hexokinase PBR VDAC CK ANT CycD ? DYm Cytochrome c Smac/Diablo AIF Omi/Hrti Nuclease G - - - - - +++++ Inner Membrane Outer Membrane Mitochondria
CED 9 Egl 1 CED 3 CED 4 APOPTOSIS