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6 DNA damage p53 TNF Cell death.ppt

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DNA Damage Signaling DNA Damage Signaling

DNA damage sensors DNA repair Cell cycle arrest (checkpoints activation) Apoptosis DNA damage sensors DNA repair Cell cycle arrest (checkpoints activation) Apoptosis

PI 3 K-related protein kinases in DNA damage signaling ATM DNA-PK ATR PI 3 K-related protein kinases in DNA damage signaling ATM DNA-PK ATR

DNA damage sensing and signaling to cell cycle DNA damage sensing and signaling to cell cycle

DNA repair pathways by iniciation - Global Genomic Repair (GGR), including Replication-coupled repair (connected DNA repair pathways by iniciation - Global Genomic Repair (GGR), including Replication-coupled repair (connected to stalled replication fork signaling, replicative stress signaling) - Transcription-coupled repair - Diferentiation-associated repair

DNA repair pathways by type of DNA damage Single-strand damage repair Double-strand damage repair DNA repair pathways by type of DNA damage Single-strand damage repair Double-strand damage repair

DNA repair pathways by type of DNA damage Double-strand damage repair Single-strand damage repair DNA repair pathways by type of DNA damage Double-strand damage repair Single-strand damage repair Mismatch repair Direct reversal Gap ligation Base excision repair Nucleotide excision repair

Base vs nucleotide excision repair Base vs nucleotide excision repair

Excision repair pathways Excision repair pathways

Depurination and deamination Depurination and deamination

UV-induced pyrimidine dimers UV-induced pyrimidine dimers

Mammalian DNA damage sensors Mammalian DNA damage sensors

Mammalian UV-induced DNA damage recognition Mammalian UV-induced DNA damage recognition

PARP in single-strand breaks repair PARP in single-strand breaks repair

DNA damage sensing and signaling to cell cycle DNA damage sensing and signaling to cell cycle

ATR activation ATRIP — esssential regulatory subunit For activation reguires RPA (binds to ss. ATR activation ATRIP — esssential regulatory subunit For activation reguires RPA (binds to ss. DNA) and TOPBP 1 (usually binds to Rad 9 -Rad 1 -Hus 1 complex detecting ss-ds. DNA border) Timeless and Tipin (Circadian clock proteins) and BRCA 1 needed for ATR activation or signaling. Activated in responce to: - NER - Stalled replication fork - Fanconi anemia pathway - ATM activation or strand resection in DSB (in S/G 2) - Mismatch repair (via Msh 2) Has functions in normal replication

ATM and ATR activation ATM and ATR activation

ATR functions - cell cycle arrest (S, G 1, G 2) - preventing firing ATR functions - cell cycle arrest (S, G 1, G 2) - preventing firing of late replication origins (Cdc 45 binding) 12 in S - ? prevention of lycenzing in G 1 (Cdt 1 degradation) - stabilizing stressed replication forks in S - promoting DNA repair and restart of replication - apoptosos induction - functions in normal replication

ATR substrates Rad 17, Claspin — for Chk 1 binding Chk 1 — for ATR substrates Rad 17, Claspin — for Chk 1 binding Chk 1 — for cell cycle arrest p 53 — for cell cycle arrest, apoptosis, repair Siah 1 — for HIPK 2 stabilisation to p 53 Hus 1, ATRIP, RPA, Top. BP 1 — feedback E 2 F 1 — for cell cycle arrest Cdc 7 -DBF 4, Mll 1 — blocking firing of later origins (also via Cdc 25) BRCA 1, Mdc 1, H 2 AX — usually by ATM for HR PLK (via Claspin and Mcm 2, for Chk 1 inactivation)

p. S/p. T recognision domains and proteins in DNA damage response BRCT domains (in p. S/p. T recognision domains and proteins in DNA damage response BRCT domains (in BRCA 1, Top. BP 1, Mdc 1, Nbs 1, PARP 1, 53 BP 1) FHA domains (in Chk 2, Mdc 1, RNF 8) 14 -3 -3 proteins SCF Ub-ligases

HIPK 1 to p 53 HIPK 1 to p 53

Chk 1 substrates Cdc 25 A (degradation) — for cell cycle arrest Cdc 25 Chk 1 substrates Cdc 25 A (degradation) — for cell cycle arrest Cdc 25 C (inactivation) — for cell cycle arrest Wee 1 p 53 — for cell cycle arrest, apoptosis, repair BRCA 2, Rad 51 — for HR

Transcription-coupled repair To NER — via CSA, CSB, XAB 2 To MMR — via Transcription-coupled repair To NER — via CSA, CSB, XAB 2 To MMR — via Mlh 1, Msh 2 To BER — via CSA, CSB, BRCA 1

DNA repair pathways by type of DNA damage Single-strand damage Double-strand repair damage repair DNA repair pathways by type of DNA damage Single-strand damage Double-strand repair damage repair Classical non-homologous end jojning (c. NHEJ) Alternative Repair by non-homologous end jojning (a. NHEJ, MMEJ) recombination (HRR)

Double strain breaks repair Double strain breaks repair

Double-strand damage - Double-strand breaks (competing pathways: Ku-DNA-PK (c. NHEJ) and PARP-ATM (HR, a. Double-strand damage - Double-strand breaks (competing pathways: Ku-DNA-PK (c. NHEJ) and PARP-ATM (HR, a. NHEJ) - RAG-induced breaks (i. e, V(D)J recombination) — c. NHEJ via DNA-PK - Stalled-replication fork-induced breaks - Single-strand to double-strand break conversion - Interstrand crosslinks (detection by Fanconi anemia pathway — first to ATR)

Pathways of DSB repair Pathways of DSB repair

c-NHEJ and DNA-PK Ku c-NHEJ and DNA-PK Ku

HRR and PARP HRR and PARP

ATM activation Mre 11 -Rad 50 -Nbs 1 complex binds DSB and activates ATM. ATM activation Mre 11 -Rad 50 -Nbs 1 complex binds DSB and activates ATM. Inactive dimeric ATM → trans-autophosphorylation → active monomeric ATM Activated in responce to double-strand breaks γH 2 AX, BRCA 1, Mdc 1, 53 BP 1 help in activation (positive feedback)

ATM and ATR activation ATM and ATR activation

ATM substrates H 2 AX — for Mdc 1 and other binding Mdc 1 ATM substrates H 2 AX — for Mdc 1 and other binding Mdc 1 — for γH 2 AX spreading (interacts with Nbs 1 in MRN) and further complex formation Chk 2 — for cell cycle arrest (dimerisation via FHAdomain and autophosphorylation) 53 BP 1 — for p 53 binding (via γH 2 AX) Mdm 2, Mdm. X — for p 53 stabilisation p 53 — for cell cycle arrest, apoptosis, repair Siah 1 — for HIPK 2 stabilisation to p 53 BRCA 1, 2 — for HR (via γH 2 AX) Nbs 1 (MRN complex) Smc 1 (cohesin)

Signaling from γH 2 AX to BRCA 1 and BRCA 2 z Signaling from γH 2 AX to BRCA 1 and BRCA 2 z

Chk 2 substrates Cdc 25 A (degradation) — for cell cycle arrest Cdc 25 Chk 2 substrates Cdc 25 A (degradation) — for cell cycle arrest Cdc 25 C (inactivation) — for cell cycle arrest p 53 — for cell cycle arrest, apoptosis, repair Mdm. X — for p 53 stabilisation (Chk 2) BRCA 1 — for HR Fox. M 1, E 2 F 1

DNA damage sensing and signaling to cell cycle DNA damage sensing and signaling to cell cycle

p 53 p 53

DNA damage ROS, UV, hyperosmolarity ATM, ATR, DNA-PK, HIPK 2 JNK, p 38, MK DNA damage ROS, UV, hyperosmolarity ATM, ATR, DNA-PK, HIPK 2 JNK, p 38, MK 2 Too strong proliferation Mdm 2 p 53 stabilisation and activation Apoptosis Autophagy Cell cycle arrest Senescence Other, including DNA repair

p 53 response to DNA damage p 53 response to DNA damage

Phosphorylation of p 53 Phosphorylation of p 53

Phosphorylation of p 53 Phosphorylation of p 53

Phosphorylation of p 53 (continued) Phosphorylation of p 53 (continued)

HIPK 1 to p 53 HIPK 1 to p 53

Acetylation of p 53 Acetylation of p 53

Other regulators of p 53 Other regulators of p 53

Cell cycle dependent p 53 induction E 2 F-1 p 19 ARF mdm 2 Cell cycle dependent p 53 induction E 2 F-1 p 19 ARF mdm 2 p 53 p 21 waf Cy/cdk

p 53/MDM-2/p 19 ARF feedback loop p 53/MDM-2/p 19 ARF feedback loop

DNA excision repair proteins induced by p 53 - p 48/DDB 2 (XPE/UV-DDB component) DNA excision repair proteins induced by p 53 - p 48/DDB 2 (XPE/UV-DDB component) - XPC - GADD 45

p 53 and cell cycle regulation p 53 and cell cycle regulation

p 53 and apoptosis p 53 and apoptosis

p 53 and apoptosis p 53 and apoptosis

p 53, oncogenic transformation and the decision between life and death p 53, oncogenic transformation and the decision between life and death

Other functions - Inhibits angiogenesis (thrombospondin expression) - Inhibits glycolysis (TIGAR), promotes oxidative phosphorylation Other functions - Inhibits angiogenesis (thrombospondin expression) - Inhibits glycolysis (TIGAR), promotes oxidative phosphorylation (SCO 2) - Stimulates embrio implantation (LIF expression) -Pro- or antioxidative features

Pro- and antioxidant functions of p 53 regulated by acetylation Pro- and antioxidant functions of p 53 regulated by acetylation

c-Abl and p 73 c-Abl and p 73

TNFR-family and IL-1 R-family receptors TNFR-family and IL-1 R-family receptors

TNFR-family Death Domaincontaining (Fas, TNFR 1, DR 3, DR 4, DR 5) Activation of TNFR-family Death Domaincontaining (Fas, TNFR 1, DR 3, DR 4, DR 5) Activation of caspase 8 (and 10), pro-apoptotic IL-1 R-family (IL-1, IL-18, TLR) Without Death Domains (TNFR 2, CD 27, CD 30, CD 40, Ox 40, LTβR) Activation of NFκB and JNK, anti-apoptotic

TNF family TNF family

Fas. L, TNF and Apo 3 L/TWEAK signaling Fas. L, TNF and Apo 3 L/TWEAK signaling

TRIAL signaling and decoy receptors TRIAL signaling and decoy receptors

Direct interactions between TRAFs and TNF R-family receptors Direct interactions between TRAFs and TNF R-family receptors

Interactions between TRAFs and other intracellular proteins Interactions between TRAFs and other intracellular proteins

NF B activation TAK TNF-fam. , IL-1 fam. , stress factors NIK MEKK 1 NF B activation TAK TNF-fam. , IL-1 fam. , stress factors NIK MEKK 1 (NF B-inducing kinase) Ik. B NLS NF B

NFk. B activation NFk. B activation

NFk. B activation NFk. B activation

IL 1 R family IL 1 R family

Toll-like receptors Toll-like receptors

Apoptosis Apoptosis

He. La, etoposide 2. 5 mk. M Blue – DAPI Green – cytochrome c He. La, etoposide 2. 5 mk. M Blue – DAPI Green – cytochrome c Red – active caspase 3

Caspases Caspases

Apoptotic caspases Initiatory – caspases 8 (and 10), 9, 2, 12(? ) Executive – Apoptotic caspases Initiatory – caspases 8 (and 10), 9, 2, 12(? ) Executive – caspases 3, 7 and 6

The two pathways for caspase activation The two pathways for caspase activation

The two pathways for caspase activation The two pathways for caspase activation

Bcl-2 family proteins in the regulation of cytochrom c release Bcl-2 family proteins in the regulation of cytochrom c release

Bcl-2 family Bcl-2 family

Mitochondrial permeability transition Mitochondrial permeability transition

Mitochondrial permeability transition Mitochondrial permeability transition

Mitochondrial intermembrane protein release Mitochondrial intermembrane protein release

Mitochondrial intermembrane protein release Mitochondrial intermembrane protein release

Some inhibitors of caspase activation - IAPs (IAP 1, 2, survivin, XIAP) (for caspase Some inhibitors of caspase activation - IAPs (IAP 1, 2, survivin, XIAP) (for caspase 9) -FLIP, BAR, ARC (for caspase 8)

Some more important apoptosis inducers -Caspase 2 -Nuclear Daxx -Nur 77 -p 73 -Ceramide Some more important apoptosis inducers -Caspase 2 -Nuclear Daxx -Nur 77 -p 73 -Ceramide and sphingosine

Caspase 2 in apoptosis p 53 TNFR-family PIDD RIP, TRADD RAIDD Pro-caspase 2 Caspase Caspase 2 in apoptosis p 53 TNFR-family PIDD RIP, TRADD RAIDD Pro-caspase 2 Caspase 2

Some more apoptosis inducers -Caspase 2 -Nuclear Daxx -Nur 77 -p 73 -Ceramide and Some more apoptosis inducers -Caspase 2 -Nuclear Daxx -Nur 77 -p 73 -Ceramide and sphingosine

Ceramide and sphingosine Ceramide and sphingosine

Ceramide and sphingosine Ceramide and sphingosine

Ceramide in apoptosis Ceramide in apoptosis

Apoptotic ceramide targets Apoptotic ceramide targets

Non-apoptotic cell death Non-apoptotic cell death

Non-apoptotic cell death variants - Caspase-independent apoptosis-like processes - Autophagy (type II programmed cell Non-apoptotic cell death variants - Caspase-independent apoptosis-like processes - Autophagy (type II programmed cell death) - Necrosis - Mitotic catastrophe

Autophagy Autophagy

Autophagy and apoptosis Autophagy and apoptosis

Signaling to autophagy Signaling to autophagy

Signaling to autophagy Signaling to autophagy

Signaling to autophagy -(PKR) e. IF 2α -GAIP (GAP for Gαi 3) -BNIP 3 Signaling to autophagy -(PKR) e. IF 2α -GAIP (GAP for Gαi 3) -BNIP 3 both apoptosis and autophagy -DAPK, DRP 1 - PI 3 K-PKB-m. Tor inhibits - AMPK (inhibits m. TOR) - Beclin 1 (activates Class III PI 3 K, inhibited by Bcl-2/Blc. XL)

NO and apoptosis-to-necrosis switch NO and apoptosis-to-necrosis switch

Mitotic catastrophe U 1810, etoposide 2. 5 Blue – DAPI Green – Ran Mitotic catastrophe U 1810, etoposide 2. 5 Blue – DAPI Green – Ran

He. La, etoposide 2. 5 mk. M Blue – DAPI Green – cytochrome c He. La, etoposide 2. 5 mk. M Blue – DAPI Green – cytochrome c Red – active caspase 3