© 2009 ABGENT. All Rights Reserved 10239 Flanders Court San Diego, CA 92121, USA Tel: 858.622.0099 www.abgent.com [email protected] NECROPTOSIS S.Gramatikova 2 PhD, K.Gramatikoff 2 PhD, J.Mountzouris 1 PhD, T.Gilliam 1 & C.Wu 1 PhD (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 L929 cells 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 - induced apoptosis 32 genes 7 genes positive in: TNF / zVAD / TNF+CX primary screen secondary screen 32 genes 432 genes Caspase 8 zVAD inhibitor BID BCL2 Cytochrome c release AIF release Apoptosome formation PARP RIP1 DNA fragmentation AIF release BMF Cathepsins TLR signaling TNFR activation Type 1 IFN family Necrostatins Ca 2+ overload ROS Energy collapse ATP depletion CYLD Deubiquitination Membrane permeabilization BCL2 CypD Caspase 3 Calpains BAX BAX NECROPTOSIS NECROPTOSIS APOPTOSIS Number of protein partners >193 >43 13 10 7 6 6 5 4 4 2 2 2 1 1 1 1 1 1 1 1 1 GRB2 TNFRSF1A MAG RAB25 PVR BMF PARP2 CYLD DEFB1 SPATA2 EIF5B DPYSL4 KCNIP1 TMEM57 JPH3 COMMD4 TXNL4B HSPBAP1 HOXA3 GALNT5 ATP6V1G2 TNFAIP8I1 TNFR TNFα Death domain Kinase domain RIP1 ROS formation & mitochondrial dysfunction Autophagy Lipoxygenases Phospholipase A Sphingomyelinase JNK activation Membrane Survivin (BIRC5) Oxidoreductase, 10 Extracellular matrix, 10 Protease, 11 Transporter , 13 Cytoskeleton, 14 Hydrolase, 16 Signaling, 21 Transferase, 20 Regulatory, 24 Transcription, 43 Receptors, 50 Nucleic acid binding, 65 Apoptosis, 10 Lipid, fatty acid, steroid metabolism, 13 Carbohydrate metabolism, 13 Proliferation & differentiation, 13 Neuronal 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, 38 Protein metabolism & modification, 48 Caspase Kinase Regulatory protein CASP3 Survivin CASP9 XIAP CASP7 CDK4 CDCA8 DIABLO AURKB HSP90AA1 INCENP CDC2 XPO1 RASA1 TNF TRAF2 RIPK1 TNFRSF1A* CASP3* TRAF1 TRAF3 CASP8* CASP10* TRAF6 CYLD* TRAF7 TTRAP 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 293T 293T HeLa 293T K562 293T Liver 55 36 25 20 15 150 37 250 15 130 55 250 72 541 A. B. C. D. E. F. References BAD: BCL2-associated agonist of cell death; BCL-X/BCL-2 binding protein; BCL2-binding component 6; BBC2 PUMA: BCL2 binding component 3; BBC3 CASP1, -3, -6: caspase 1, -3, -6 DNMT1: DNA (cytosine-5-)-methyltransferase 1; CXXC finger protein 9; DNA methyltransferase 1 CBX5: chromobox homolog 5 (HP1 alpha homolog, Drosophila); heterochromatin protein 1 homolog alpha MLL3: myeloid/lymphoid or mixed-lineage leukemia 3; histone-lysine N-methyltransferase, H3 lysine-4 specific AIF1: allograft inflammatory factor 1; interferon gamma responsive transcript; ionized calcium-binding adapter BIRC5: baculoviral IAP repeat-containing 5; apoptosis inhibitor 4; survivin variant 3 alpha SPP1: secreted phosphoprotein 1; bone sialoprotein I; osteopontin JUN: jun oncogene; Jun activation domain binding protein; v-jun avian sarcoma virus 17 oncogene homolog PARP1: poly (ADP-ribose) polymerase 1; ADP-ribosyltransferase (NAD+; poly (ADP-ribose) polymerase) CHARACTERISTICS APOPTOSIS NECROPTOSIS AUTOPHAGIC CALCIUM- MEDIATED AIF/PARP- DEPENDENT ONCOSIS Morphology Chromatin 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 Triggers Oxidative 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 Inhibitors Caspase inhibitors, TOP1 inhibitors, survivin, VEGF, zVAD, NO, etc. Necrostatins, Ca 2+ 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. elegans deg mutants Some excito- toxic pcd Ischemic pcd Kinase Transcription factor Other NFκB DNA damage or drugs TOP1 cleavage complex Covalent TOP1 complex Replication Transcription ATM ATR CDC25 Cell-cycle arrest DNA repair Apoptosis CDK CHK1/2 BER complex RAD51 PARP XRCC1 DNA ligase DNA polβ APEX1 XRCC2/3 RAD52 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 95 250 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 interactions of 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 & PARP2 protein 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 ON DNA repair OFF , Transcription OFF Fig. 5 Fig. 6
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© 2009 ABGENT. All Rights Reserved 10239 Flanders Court San Diego, CA 92121, USA Tel: 858.622.0099www.abgent.com [email protected]
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.
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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
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