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NECROPTOSISS.Gramatikova 2 PhD, K.Gramatikoff 2PhD, J.Mountzouris

1PhD, T.Gilliam 1 & C.Wu

1PhD

(1) Abgent Inc., 10239 Flanders Ct, San Diego, CA 92121(2) Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037

Cell Death Survey

siRNA transfection of L929cells targeting 16,873 genes

zVAD - induced necroptosis

Viability assay

Selection of 666 genes required for zVAD - induced necroptosis

TNF - induced necroptosis

zVAD - induced necroptosis

TNF+CX - inducedapoptosis

32 genes

7 genes positive in:TNF / zVAD / TNF+CX

prim

ary

scre

ense

cond

ary

scre

en

32 genes 432 genes

Caspase 8

zVAD inhibitor

BID

BCL2

Cytochrome c release

AIF release

Apoptosome formation

PARPRIP1

DNA fragmentation

AIF release

BMF

Cathepsins

TLR signaling

TNFR activation

Type 1 IFN family

Necrostatins

Ca2+ overload

ROS

Energy collapseATP depletion

CYLD

Deubiquitination

Membranepermeabilization

BCL2

CypD

Caspase 3

Calpains

BAX

BAX

NECROPTOSIS

NECROPTOSIS

APOPTOSIS

Numb

er of

prote

in pa

rtner

s >193>43

13 10 7 6 6 5 4 4 2 2 2 1 1 1 1 1 1 1 1 1

GRB2

TNFR

SF1A MAG

RAB2

5

PVR

BMF

PARP

2

CYLD

DEFB

1

SPAT

A2

EIF5B

DPYS

L4

KCNI

P1

TMEM

57

JPH3

COMM

D4

TXNL

4B

HSPB

AP1

HOXA

3

GALN

T5

ATP6

V1G2

TNFA

IP8I1

TNFR

TNFα

Death domain

Kinase domain

RIP1

ROS formation &mitochondrial dysfunction

Autophagy Lipoxygenases

Phospholipase A

Sphingomyelinase

JNK activation

Membrane

Survivin (BIRC5)

Oxidoreductase, 10Extracellular matrix, 10

Protease, 11

Transporter, 13

Cytoskeleton, 14

Hydrolase, 16

Signaling, 21

Transferase, 20

Regulatory, 24Transcription, 43

Receptors, 50

Nucleic acid binding, 65

Apoptosis, 10Lipid, fatty acid, steroid metabolism, 13Carbohydrate metabolism, 13

Proliferation & differentiation, 13Neuronal activities, 13

Cell structure & motility, 18

Intracellular protein traffic, 19

Transport, 26

Sensory perception, 27

Immunity & defense, 28

Signaling, 95

Nucleic acid metabolism, 80

Cell cycle, 18

Development, 38Protein metabolism & modification, 48

CaspaseKinaseRegulatory protein

CASP3

Survivin

CASP9

XIAP

CASP7

CDK4

CDCA8

DIABLO

AURKB

HSP90AA1

INCENPCDC2

XPO1

RASA1

TNF

TRAF2

RIPK1

TNFRSF1A*CASP3*

TRAF1

TRAF3

CASP8*

CASP10*

TRAF6 CYLD*

TRAF7TTRAP

TRAF5

IKBKG

TRAIP

SPATA2*

HSP90AA1

PARP2

GRB2

CASP7*

KCNIP1 BCL2* BMF

Caspase Necroptosis-related TNF-signaling associated Other

Fig. 2 siRNA screening for genes re-quired in necroptosis. siRNA screen of the mouse genome for regulators of necroptosis identified a set of 432 genes that regulate necroptosis, a subset of 32 genes that are associated with RIP1 kinase, 32 genes required for apoptosis, and 7 genes involved in both necroptosis and apoptosis. zVAD, carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone; CX, cycloheximide (7).

Fig. 3 Classification of 432 selected pro-teins from the necroptosis screen into (A) molecular function and (B) biological process catego-ries. Genes for which no annotations could be assigned were excluded from the analysis. Categories with at least ten genes are displayed. The number of genes assigned to each category are shown in gray (7).

Fig. 4 Molecular interactions of TNF-induced necroptotic proteins. A, Crosstalk between necroptosis-related proteins, positively identi-fied in the secondary screen, with apoptotic (*) and other signaling molecules. The network was generated by using data curated from the literature. B, Number of protein binding partners for necroptotic proteins se-lected from the secondary screen.

Protein abbreviations: ATP6V1G2, vacu-olar ATP synthase subunit G2; CASP, caspase; COM-MD4, COMM domain containing 4; EIF5B, eukaryotic translation initiation factor 5B; DEFB1, defensin, beta 1; DPYSL4, dihydropyrimidinase-like 4; JPH3, junctophilin 3; HOXA3, homeobox A3; HSPBAP1, heat shock associated protein 1; GALNT5, N-acetyl-galactosaminyltransferase 5; GRB2, growth factor receptor-bound protein 2; KCNIP1, Kv channel inter-acting protein 1; MAG, myelin associated glycoprotein; RAB25, RAB25, member RAS oncogene family; SPA-TA2, spermatogenesis associated 2; TMEM57, trans-membrane protein 57; TNFAIP8L1, tumor necrosis factor, alpha-induced protein 8-like 1; TNFRSF1A, tumor necrosis factor receptor superfamily, member 1A; TRAIP, TRAF interacting protein; TRAF, TNF receptor-associated factor 1; TTRAP, TRAF and TNF receptor as-sociated protein; TXNL4B, thioredoxin-like 4B.

1. Bredesen DE. (2007) Key note lecture: toward a mechanistic taxonomy for cell death programs. Stroke 38(2 Suppl), pp.652-660.

2. Galluzzi L and Kroemer G. (2008) Necroptosis: a specialized pathway of programmed necrosis. Cell 135(7), pp.1161-1163.

3. Degterev A and Yuan J. (2008) Expansion and evolution of cell death programmes. Nat Rev Mol Cell Biol. 9(5), pp.378-390.

4. Golstein P and Kroemer G. (2007) Cell death by necrosis: towards a molecular definition. Trends Biochem Sci. 32(1), pp.37-43.

5. Zong WX and Thompson CB. (2006) Necrotic death as a cell fate. Genes & Dev. 20, pp.1-15.

6. Beneke S, Bürkle A. (2007) Poly(ADP-ribosyl)ation in mammalian ageing. Nucleic Acids Res. 35(22), pp.7456-7465.

7. Degterev A, Hitomi J, Germscheid M, Ch’en IL, Korkina O, Teng X, Abbott D, Cuny GD, Yuan C, Wagner G, Hedrick SM, Gerber SA, Lugovskoy A and Yuan J. (2008) Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol. 4(5), pp.313-321.

8: Sarkar S. (2008) Till death do us part. Med Econ. 85(11), pp.42-46.

9: Hitomi J, Christofferson DE, Ng A, Yao J, Degterev A, Xavier RJ and Yuan J. (2008) Identification of a molecular signaling network that regulates a cellular necrotic cell death pathway. Cell 135(7):1311-1323.

10. Schreiber V, Dantzer F, Ame JC and de Murcia G. (2006) Poly(ADP-ribose): novel functions for an old molecule. Nat Rev Mol Cell Biol. 7(7), pp.517-528.

11. Chiarugi A and Moskowitz MA. (2002) Cell biology. PARP-1-- a perpetrator of apoptotic cell death? Science 297(5579), pp.200-201.

12. Pommier Y. (2006) Topoisomerase I inhibitors: camptothecins and beyond. Nat Rev Cancer 6(10), pp.789-802.

13. Altieri DC. (2008) Survivin, cancer networks and pathway-directed drug discovery. Nat Rev Cancer 8(1), pp.61-70.

Product Abbreviations

G. H. I. J. K. L. M.

Figure Target Tissue/Cell line Cat #

A. BAD Human lung carcinoma AP1314b

B. PUMA Human breast carcinoma AP1317a

C. CASP1 Human hepatocarcinoma AT1400a

D. CASP3 Human lung carcinoma AP7563c

E. DNMT1 Human carcinoma (HeLa) AT1805a

F. CBX5 Human carcinoma (HeLa) AT1411a

G. CASP6 Mouse liver tissue AP1313b

H. MLL3 Transfected 293T cells AP6184a

I. AIF1 Human leukemia cells K562 AT1077a

J. BIRC5 Transfected 293T cells AT1299a

K. SPP1 Human carcinoma (HeLa) AT4028a

L. JUN Transfected 293T cells AP1984d

M. PARP1 Transfected 293T cells AP6373a

Selected Abgent Products

Protein Associations

Protein Markers

293T293THeLa293TK562293TLiver

55

3625

20

15

150

37

250

15

130

55

25072

541

A. B.

C. D.

E. F.

References

BAD: BCL2-associated agonist of cell death; BCL-X/BCL-2 binding protein; BCL2-binding component 6; BBC2PUMA: BCL2 binding component 3; BBC3CASP1, -3, -6: caspase 1, -3, -6 DNMT1: DNA (cytosine-5-)-methyltransferase 1; CXXC finger protein 9; DNA methyltransferase 1CBX5: chromobox homolog 5 (HP1 alpha homolog, Drosophila); heterochromatin protein 1 homolog alphaMLL3: myeloid/lymphoid or mixed-lineage leukemia 3; histone-lysine N-methyltransferase, H3 lysine-4 specificAIF1: allograft inflammatory factor 1; interferon gamma responsive transcript; ionized calcium-binding adapter BIRC5: baculoviral IAP repeat-containing 5; apoptosis inhibitor 4; survivin variant 3 alphaSPP1: secreted phosphoprotein 1; bone sialoprotein I; osteopontinJUN: jun oncogene; Jun activation domain binding protein; v-jun avian sarcoma virus 17 oncogene homologPARP1: poly (ADP-ribose) polymerase 1; ADP-ribosyltransferase (NAD+; poly (ADP-ribose) polymerase)

CHARACTERISTICS APOPTOSIS NECROPTOSIS AUTOPHAGICCALCIUM- MEDIATED

AIF/PARP-DEPENDENT

ONCOSIS

MorphologyChromatin condensation,nuclear fragmentation,

apoptotic bodies

Mitochondrial dysfunction, membrane rupture, ER

swelling, increase of ROS

Autophagic vacuoles, membrane

rupture

Membrane whorls Mild chromatin condensation

Cellular swelling

TriggersOxidative stress, death

receptors, viral infections, hypoxia, etc.

Trophotoxicity, TNF,damage-induced lesions,

ischemia, antimycin A

Serum, amino acid starvation, protein

aggregates

Calcium entry,CDK5 signaling

deg mutants

DNA damage,glutamate, NO

Ischemia,excitotoxicity

Mediators Caspases, BH family, etc. ERK2, NUR77 Atg orthologs Calpains, cathepsins PARP, AIF JNK

InhibitorsCaspase inhibitors,

TOP1 inhibitors, survivin, VEGF, zVAD, NO, etc.

Necrostatins, Ca2+ chelators, PARP inhibitors, U0126,

DN NUR77, CypD inhibitors

3-Methyladenine, bafilomycin A1, mTOR,

JNK inhibitors?

Calreticulin, calpain inhibitors

PARP inhibitors JNK inhibitors,glycine

Examples Type I and nuclear pcd Type III and cytoplasmic pcd Type II pcd C. elegansdeg mutants

Some excito-toxic pcd Ischemic pcd

Kinase Transcription factor Other

NFκB

DNA damage or drugs

TOP1 cleavage complex

Covalent TOP1 complex

ReplicationTranscription

ATM ATR

CDC25

Cell-cycle arrestDNA repair Apoptosis

CDK

CHK1/2

BER complex

RAD51

PARPXRCC1

DNA ligase DNA polβ

APEX1

XRCC2/3RAD52

PI3K

AKT

XIAP

p53

p21

Comparison of different cell death programs

Fig. 1. Crosstalk between apoptosis and programed necrosis (necropto-sis). Caspase 8-mediated degradation of RIP1 (receptor-interacting protein kinase 1) is a major molecular switch between apoptosis and necroptosis. Necroptosis centers on the activation of RIP1. As opposed to apoptosis, necroptosis does not engage apoptotic regulators such as caspases, BCL2 family members, or cytochrome c (1-7).

55

36

17

95250

10

Cell Death

Table 1. Alternative programed cell death (pcd) processes. Necroptosis is a cellular mechanism of necrotic cell death induced by apoptotic stimuli under conditions where apoptotic and/or autophagic execution are prevented. Abbreviations for Fig. 1 and Table 1: AIF, apoptosis-inducing factor; BAX, BCL2-as-sociated X protein; BCL2, B-cell CLL/lymphoma 2; BID, BH3 interacting domain death agonist; BMF, Bcl2 modifying factor; CDK5, cyclin-dependent kinase 5; CYLD, cylindromatosis (turban tumor syndrome); CypD, cyclophilin D; deg, degenerin; ERK2, mitogen-activated protein kinase 1; IFN, interferon; JNK, mitogen-activated protein kinase 8; mTOR, mechanistic target of rapamycin; NO, nitric oxide; NUR77, nuclear receptor; DN Nur77, dominant negative Nur77; PARP, poly (ADP-ribose) polymerase; ROS, reactive oxygen species; TLR, Toll-like receptor; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; TOP1, DNA topoisomerase 1; U0126, inhibitor of MEK kinase; zVAD, carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone, a caspase inhibitor (1-7).

poly (ADP-ribose) polymerase 1 (PARP1)

Fig. 5 Modulation of chromatin structure by PARP1. A, PARP1 is a chromatin-associated enzyme, which modifies proteins by poly (ADP-ribosyl)ation, indicated in blue. Poly (ADP-ribosyl)ation is a postranslational modifi-cation in response to DNA strand-breaks. B, Application of ABGENT’s PARP1 monoclonal antibody #AT3183a for immu-nofluorescent detection of PARP1 in HeLa cells.

DNA topoisomerase I(TOP1)

Necroptosis

Identification Protocol Statistical Distribution Inferred Network

Molecular interactionsof necroptosis proteins

Fig.6 Signaling events associated with TOP1. DNA damage and drugs induce reversible TOP1 cleavage complexes, subsequently converted to irreversible by replication and tran-scription. Irreversible TOP1 complexes can also be formed during apoptosis. DNA repair engages multiple signaling events, including the BER (base excision repair) complex and the RAD51/RAD52-associated homologous recombination. Cell-cycle arrest facilitates DNA repair and involves ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and RAD3 related) kinases, as well as the checkpoint homologs CHK1 and CHK2. Additional molecules, associated with checkpoint regulation, include CDC25, histone H2AX, MRN complex, and BRCA1. p53 activates apop-tosis both directly and by transactivating pro-apoptotic genes. p21 is a p53-inducible gene, which inhibits CDKs (cyclin-dependent kinases). TOP1 inhibitors suppress apoptosis by activating nuclear factor NFkB, which induces the expression of anti-apoptotic genes (12). Fig.7 Molecular interactions between survivin and its protein part-ners. The network was generated by using protein-protein interaction data curated from the literature.

Protein abbreviations: AKT, v-akt murine thymoma viral oncogene homolog; APEX1, nuclease; AURKB, aurora kinase B; BCL2, B-cell CLL/lymphoma 2; BUB3, budding uninhibited by benzimidazoles 3 homolog; CASP, caspase; CDC, cell division cycle; CDKN1A or p21, cyclin-dependent kinase inhibitor 1A; CENPA, centromere protein A; DIABLO, diablo homolog; INCENP, inner centromere protein antigens; MYBL2, v-myb myeloblastosis viral oncogene ho-molog (avian)-like 2; PCNA, proliferating cell nuclear antigen; PI3K, phosphoinositide-3-kinase; RAD51, RAD51 homolog; XIAP, X-linked inhibitor of apoptosis; XPO1, exportin 1; XRCC, X-ray repair complementing defective repair in Chinese hamster cells.

ABGENT has hundreds of cell death/survival-related antibodies which cover key targets for apoptosis,autophagy, necroptosis, and other programed cell death processes. Visit www.abgent.com for a complete listing.

A

Fig. 2 Fig. 3 Fig. 4

A

B

B

Fig. 7

PARP1 & PARP2protein partners

DNA REPAIR

p53; DNA ligase III; XRCC1, 5 & 6; PCNA;Werner syndrome

CELL CYCLE

CENPA & B; MYBL2;Nucleophosmin 1; BUB3

APOPTOSIS

Caspase 1, 3, 6, 7 & 8;Granzyme B; PARP3; BCL2; NFkB; CDKN1A

TRANSCRIPTION

Transcription factors;Retinoid receptors; DNA topoisomerase I

PARP1

A

B

Packed chromatin Relaxed chromatin

DNA repair ON, Transcription ONDNA repair OFF, Transcription OFF

Fig. 5 Fig. 6

Necroptosis_Poster_v4.indd 1 6/8/09 8:07:55 AM