Cell death

1
Cell death

• Part II Regulation

Eric R. Gauthier, Ph.D. Dept. Chemistry-Biochemist
ry
2
Outline

• Basic regulatory mechanisms
• Turnover
• Compartmentalization
• Alternative splicing
• Changes in protein conformation
• Post-translational modifications
• Protein-protein interactions

3
Core apoptotic machinery

• Major control points
• Death receptor signalling
• Bax activation/translocation
• Initiator caspase activation
• Executioner caspase activation.
• Major regulatory mechanisms
• Cellular compartmentalization
• Modulation of protein turnover ( transcription,
  translation, stability)
• Changes in protein conformation
• Post-translational modifications
  (phosphorylation, nitrosylation, deamidation,
  ubiquitylation)
• Alternative splicing
• Protein-protein interaction.

caspase-8
cFLIP
Adapted from Nature Struct. Biol. 2001. 8 5
4
Death receptor regulation

• Death receptors multiple outcomes
• TNF can lead to cell survival or death
  (apoptosis, necrosis)
• Fas induction of cell death is dependent on the
  cell context (e.g. TCR stimulation)
• TRAIL preferentially induces death in tumor
  cells
• Major regulatory mechanisms
• Inhibition of DISC (Death Inducing Signalling
  Complex) formation
• Expression (transcription) of death receptors
  (p53 and Fas)
• Decoy receptors
• Compartmentalization.

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Inhibition of DISC formation

• cFlip Inactive homologs of Casp-8 and Casp-10
• cFlipL Mutation of the active site Cys
• cFlipS Contains only two DED
• Binds FADD and dimerizes with pro-Casp-8
  initially thought to act as a dominant-negative
  inhibitor, leading to cell survival
• However recent data indicates that cFlip can
  also trigger cell death by promoting
  Casp-8/Casp-10 dimerization

c-FlipS
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Inhibition of DISC formation

• Low c-FlipL levels
• Dimerizes with pro-Casp-8 at the DISC
• Contributes to Casp-8 activation c-Flip\Casp-8
  dimers form more efficiently than Casp
  homodimers
• Cell death.
• High c-FlipL
• Incomplete processing of proCasp-8
• Casp-8 activated but remains at the DISC
• Cleave different substrates, leading to
  pro-survival function.

Biochem. J. (2004) 382, e1e3
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Decoy receptors - TRAIL receptors
Cell Research (2004) 14(5)359-372
8
Death receptor regulationCompartmentalization

• Complex 1
• Formed early after receptor activation
• Comprises TNF-R, TRADD, TRAF2, RIP1
• Leads to NF-kB activation by recruitment of the
  I-kB kinase signalsome (IKK1-IKK2-NEMO)
• Complex 2
• Found at later time points (gt2 hrs), possibly
  after receptor internalization
• Dissociation from TNFR, and recruitment of FADD
  and proCasp-8
• In situations where complex-1 formation trigger
  sufficient NF-kB signalling, c-FLIP and other
  anti-apoptotic proteins (e.g. IAPs) are
  synthesized, leading to inhibition Casp-8
  activation in complex II
• Also localization in lipid rafts seems to
  promote the survival signalling function of TNFR,
  as cholesterol depletion favors complex II
  formation.

Immunity, Vol. 21, 461465, October, 2004
9
Core apoptotic machinery

• Major control points
• Death receptor signalling
• Bax activation/translocation
• Initiator caspase activation
• Executioner caspase activation.
• Major regulatory mechanisms
• Cellular compartmentalization
• Modulation of protein turnover ( transcription,
  translation, stability)
• Changes in protein conformation
• Post-translational modifications
  (phosphorylation, nitrosylation, deamidation,
  ubiquitylation)
• Alternative splicing
• Protein-protein interaction.

caspase-8
cFLIP
Adapted from Nature Struct. Biol. 2001. 8 5
10
Bax modulation

• Because of its central role in the intrinsic
  pathway, several mechanisms have evolved to
  control Bax activation
• Transcription (p53)
• Alternative splicing
• Subcellular localization
• Protein-protein interaction
• Inactivation of anti-apoptotic proteins

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Bax activation

• Bax exists as an inactive, cytosolic protein
• C-terminal TMD in the BH1/BH2/BH3 hydrophobic
  groove
• Prevents Bax from anchoring into the
  mitochondrial outer membrane (MOM)
• Prevents interaction of regulatory proteins with
  hydrophobic groove.
• Prevents inappropriate aggregation in the
  cytosol
• N-terminal a1 helix not accessible.
• Mitochondrial targeting sequence?
• Bax activation
• Requires the release of the TMD from the BH
  groove
• Results in the exposure of the NH2 terminus
  (detectable by IP with 4G2 antibody)

Biochimica et Biophysica Acta 1644 (2004) 83 94
Helices a2 (BH3) a45 (BH1) a78 (BH2)
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Bax activation
Biochimica et Biophysica Acta 1644 (2004) 83 94
J Cell Biol. 2004. 164 ( 7) 10211032
13
Bax Activation

• Bax activation can be induced by several
  triggers
• pH alkalinization
• Direct tBid/Bim binding
• Ser 184 dephosphorylation
• Ser 184 PO4 by AKT
• Inactivation of Bcl-2 / Bcl-xL
• PO4 (Bcl-2 Ser 87, Thr 69 - JNK)
• Ubiquitylation (Bcl-2)
• Casp-mediated cleavage
• BH3 protein binding (e.g. Bad, Noxa, Puma)
• Bcl-xL deamidation (Asn 52/66)
• Release of binding by KU70
• Removal of the N-terminal 20 first amino acids
  (calpain-mediated)

SCIENCE. 2002. 298 1346-1347
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Bax modulationInactivation of Bcl-xL
Cell, Vol. 87, 619628, November 15, 1996
Mol. Cell. 2004. 13 627638
Cell, Vol. 91, 231241, October 17, 1997
15
Core apoptotic machinery

• Major control points
• Death receptor signalling
• Bax activation/translocation
• Initiator caspase activation
• Executioner caspase activation.
• Major regulatory mechanisms
• Cellular compartmentalization
• Modulation of protein turnover ( transcription,
  translation, stability)
• Changes in protein conformation
• Post-translational modifications
  (phosphorylation, nitrosylation, deamidation,
  ubiquitylation)
• Alternative splicing
• Protein-protein interaction.

caspase-8
cFLIP
Adapted from Nature Struct. Biol. 2001. 8 5
16
IAPs

• BIR Baculovirus IAP repeat
• RING ubiquitin ligase (E3) domain
• UBC ubiquitin E2 domain
• CARD caspase recruitment domain
• NACHT putative ATP-binding domain

Nature Reviews Molecular Cell Biology 5, 897-907
(2004)
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IAPs

• Mammals
• BIR-3 inhibits active Casp-9
• Linker segment between BIR-1/2 inhibits active
  Casp-3/7
• Drosophila
• BIR1 inhibits Drice (Casp-3 homolog)
• BIR2 inhibits Dronc (Casp-9 homolog)

Nature Reviews Molecular Cell Biology 5, 897-907
(2004)
18
XIAP and caspase inhibition
Biochem. J. (2004) 384, 201232
19
XIAP and effector caspase inhibition
TRENDS in Biochemical Sciences Vol.27 No.2
February 2002 94-101
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XIAP and caspase-9 inhibition

• The BIR-3 domain of XIAP traps Casp-9 in a
  monomeric, inactive conformation
• BIR-3 binds Casp-9 through an interface which is
  required for Casp-9 homodimerization and its
  interaction wiht the apoptosome
• An N-terminal segment of the small subunit of
  Casp-9 (A298-T-P-F301) anchors this interaction
  by binding a conserved groove on BIR-3 (next
  slide).

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XIAP and caspase-9 inhibition
Biochem. J. (2004) 384, 201232
22
DIAP and Dronc inhibitionRecruitment of E2/E3
enzymes
Cell, Vol. 109, 793796, June 28, 2002 793-796
23
Inhibiting the inhibitorsSMAC/DIABLO
Nature Reviews Molecular Cell Biology 5, 897-907
(2004)
24
DIAP and Dronc inhibitionRecruitment of E2/E3
enzymes
Cell, Vol. 109, 793796, June 28, 2002 793-796
25
Core apoptotic machinery

• Major control points
• Death receptor signalling
• Bax activation/translocation
• Initiator caspase activation
• Executioner caspase activation.
• Major regulatory mechanisms
• Cellular compartmentalization
• Modulation of protein turnover ( transcription,
  translation, stability)
• Changes in protein conformation
• Post-translational modifications
  (phosphorylation, nitrosylation, deamidation,
  ubiquitylation)
• Alternative splicing
• Protein-protein interaction.

caspase-8
cFLIP
Adapted from Nature Struct. Biol. 2001. 8 5
26
DIAP and Dronc inhibitionInvolvement of the
N-end rule pathway
NATURE CELL BIOLOGY VOL 5 MAY 2003 373-376
27
Inhibiting the inhibitorsThe case of Drosophila
NATURE STRUCTURAL BIOLOGY. 2003. 10 (9) 892-898