BMOL 6106 - W2005 1

Cell death

Part II: Regulation

Eric R. Gauthier, Ph.D.Dept. Chemistry-Biochemistry

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Outline

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

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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.

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caspase-8

cFLIP

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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…

Oncogene (2003) 22, 8634–8644

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, e1–e3

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Decoy receptors - TRAIL receptors

Cel

l Res

earc

h (

2004

); 1

4(5)

:359

-372

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Death receptor regulationCompartmentalization

• Complex 1: Formed early after receptor activation; Comprises: TNF-R, TRADD, TRAF2, RIP1 Leads to NF-B activation by recruitment of the

I-B kinase signalsome (IKK1-IKK2-NEMO)

• Complex 2: Found at later time points (>2 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.

Imm

un

ity,

Vo

l. 2

1, 4

61–4

65,

Oct

ob

er,

2004

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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.

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caspase-8

cFLIP

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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 1 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);

Biochim

ica et Biophysica

Acta 1644

(2004) 83– 94

Helices: 2 (BH3) ; 4+5 (BH1); 7+8 (BH2)

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Bax activation

Biochim

ica et Biophysica

Acta 1644

(2004) 83– 94

J C

ell B

iol.

2004

. 16

4 (

7):

102

1–10

32

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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, 619–628, November 15, 1996

Cell, Vol. 91, 231–241, October 17, 1997

Mo

l. Cell. 2

00

4. 13: 62

7–638

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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.

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caspase-8

cFLIP

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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)

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XIAP and caspase inhibitionB

ioch

em

. J.

(20

04)

384

, 2

01–2

32

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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).

Nat. Rev. Mol. Cell Biol. 5, 897-907 (2004)

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XIAP and caspase-9 inhibitionB

ioch

em

. J.

(20

04)

384

, 2

01–2

32

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DIAP and Dronc inhibitionRecruitment of E2/E3 enzymes

Cel

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ol.

109

, 7

93–7

96,

Jun

e 28

, 20

02:

793-

796

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Inhibiting the inhibitors:SMAC/DIABLO

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

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DIAP and Dronc inhibitionRecruitment of E2/E3 enzymes

Cel

l, V

ol.

109

, 7

93–7

96,

Jun

e 28

, 20

02:

793-

796

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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.

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caspase-8

cFLIP

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DIAP and Dronc inhibitionInvolvement of the N-end rule pathway

NATURE CELL BIOLOGY VOL 5 MAY 2003: 373-376

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Inhibiting the inhibitors:The case of Drosophila

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