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apotosis part 2
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RECENT ADVANCES IN APOPTOTIC PATHWAYS
DEPARTMENT OF BIOCHEMISTRY
BY: Dr JYOTI
continued
Vedio
Initiators of Apoptosis
Caspases
Cysteine-dependent aspartate specific proteases
Have a cysteine at the active site
Cleave target just after aspartic acid residues Substrate specificity is determined by the 4 residues upstream of
cleavage site( towards N-terminal)
Exist in cytosol as single chain proenzymes (procaspases) which are activated when cleaved by other caspases
Once activated, cleave other caspases – results in proteolytic cascade
Also cleave key proteins in the cell, causing the characteristic morphology and biochemistry of apoptosis.
3 Types of Caspases
• Inflammatory Caspases: -1, -4, and -5 • Initiator Caspases: -2, -8, -9, -10& -12
– Long N-terminal domain– Interact with effector caspases
• Effector Caspases: -3, -6, and -7– Little to no N-terminal domain– Initiate cell death
14 CASPASES identified in humans.
Antiapoptotic Proapoptotic
Bcl-2 family members
A very large family with 30 members identified and belongs to both:
Bcl -2Bcl-XL
Bcl-W
A1Mcl-1
BaxBakBok
BidBimBikBadBmfHrk
NoxaPumaBlkBNIP3Spike
BH1, BH2,BH3,BH4
BH3
BH1, BH2,BH3
THE BCL-2 FAMILY
Raf-1calcineurin
Liganddomain
N
Poreformation
Membraneanchor
phosphorylation Receptor domain
C
Regulatory interactions between Bcl-2 family members
Youle and Strasser (2008) The BCL-2 protein family: opposing activities that mediate cell death. Nature Reviews Molecular Cell Biology, 9, 47-59
BH3 only protein binding specificity for BCL-2 homologues
BIM and PUMA bind to all BCL-2 family members tested; by contrast NOXA only binds to A1 and MCL1.
These binding specificities recapitulate the ability of these proteins to activate apoptosis e.g. BIM et al can induce apoptosis alone whereas a combination of NOXA and BAD is required.
IAPs (Inhibitors of Apoptosis)
These proteins act inhibiting caspase activity in 2 different ways:• Direct binding inhibiting the proteolytic activity of caspases• Marking caspases for ubiquitination and so degradation
Inhibited by SMAC/DIABLO.
Salvesen and Duckett Nat rev mol cell biol (2002)
Kinchen and Ravichandran (2007) Fig1
Stages of engulfment of apoptotic cells can be divided into 4 stages
Binding
Recognition
Phagocytosis
Internalization
Lauber et al.(2004) Fig 2 Lack of “don’t eat me” signals on the surface of apoptotic cells
Anterior chamber of the eye and testes fail to elicit an immune response because these cells produce lots of FasL, so kill immune cells when they enter these sites.
Possible therapy by inducing production of FasL in other tissues – lowering need for immune rejection drugs
ORGAN TRANSPLANTS
(HIV deactivates Bcl-2)
(
1. p53 mediates apoptosis in response to DNA damage, oncogene expression (adenovirus E1A, myc etc.), or withdrawal of growth factors
2. Overexpression wild-type of p53 leads to apoptosis
3. p53 induces apoptosis through transcriptional activation of proapoptotic genes, such as Puma, Noxa, p53AIP1, Bax, Apaf-1 etc.
4. DNA-damage leads to mitochondrial translocation of p53.
4.p53 binds to Bcl-2 family protein Bcl-xL to influence cytochrome c release.
p53 in apoptosis
p53 binds to Bcl-xL and releases Bax
.
Bax is sequestered by Bcl-xL and inactive
Bax is released by p53 from Bcl-xL and forms oligomers, leading to apoptosis
. p53 can release both proapoptotic multidomain proteins and BH3-only proteins
p53 and Puma in apoptosis
Ref: K. Vousden, Science, 2005
p53 in DNA repair and apoptosis
Ref: Bensaad & Vousden, Nature Med, 2005
ROS: reactive oxygen species
G1- S
Role of P53 in DNA damage induced Apoptosis
• DNA damage activate ATM & Chk2 protein kinases
• Phosphorylation of P53
• Activate Cdk inhibitor P21, which inhibits Cdk2/cyclin E complexes, halting cell cycle progression in G1 NOXA
The p53-MDM2 feedback loop
1. MDM2 binds to p53 N-terminal transactivation domain and inhibits p53-dependent transcription.
2. MDM2 is a transcription target of p53.
3. MDM2 is an E3 ubiquitin ligase of p53, thus targeting p53 for proteolytic degradation.
4. MDM2 knockout is lethal for mouse embryonic development, but simultaneous deletion of p53 and MDM2 genes rescues MDM2-KO.
The p53 pathways
Ref: Nature 408, 307 - 310 (2000)
Viral oncogenes and the p53 network
Genetic Control of "Genetically Programmed Cell Death"
Cellular Location of Protein Product
Mitochondrial Membrane
Nuclear Envelope
Endoplasmic Reticulum
Nucleus
Nucleus
Cell Membrane
Effect on Apoptosis Blocks
Stimulates
Wild-type StimulatesMutant Blocks
Stimulates
ApoGenes
bcl-2
myc
p53
p53*
APO-1/
FAS
Apoptosis Gene
Tumor-derived mutations affecting apoptosisProtein Role in apoptosis Ref
ATM Mutated in ataxia-talangiectasia syndrome. Senses DNA double strand breaks and stabilizes p53. Deficiencies increase risk of developing haematological malignancies and breast cancer
Khanna and Jackson, 2001
Bax (p53 target gene)
Mutated or decreased expression in some tumors. Mediates mitochondrial membrane damage. Sufficient but not necessary for drug-induced apoptosis.
Rampino et al., 1997
Bak Mutated or decreased expression in some tumors. Mediates mitochondrial membrane damage. Sufficient but not necessary for drug-induced apoptosis.
Kondo et al., 2000
PTEN (p53 target gene)
Mutated or altered expression in cancers. Regulates Akt activation and subsequent phosphorylation of Bad. Loss of PTEN results in resistance to many apoptotic stimuli.
Di Cristofano and Pandolfi, 2000
Ref: Cell, 2002, 108:153-164
Tumor-derived mutations affecting apoptosisProtein Role in apoptosis Ref
Apaf-1 (p53 target gene)
Mutated and transcriptionally silenced in melanoma and leukemia cell lines. Necessary for activation of caspase-9 following cytochrome c release. Apaf-1-/- cells are chemoresistant.
Soengas et al., 2001
CD-95/Fas Mutated and down-regulated in lymphoid and solid tumors. Initiates the extrinsic apoptotic pathway. Loss of function is associated with resistance to drug-induced cell death.
Muschen et al., 2000
TRAIL-R1/R2
Mutated in metastatic breast cancers. Initiate the extrinsic apoptotic pathway. Mutations lead to suppression of death receptor-mediated apoptosis.
Shin et al., 2001
Caspase-8 Gene silenced in neuroblastomas. Activates both extrinsic and intrinsic apoptotic pathways. Silencing results in resistance to drug-induced apoptosis.
Teitz et al., 2000
Ref: Cell, 2002, 108:153-164
Tumor-derived mutations affecting apoptosisProtein Role in apoptosis Ref
Bcl2 Frequently overexpressed in many tumors. Antagonises Bax and/or Bak and inhibits mitochondrial membrane disruption. Inhibits drug-induced apoptosis.
Reed, 1999
MDM2 Overexpressed in some tumors. Negative regulator of p53. Inhibits drug-induced p53 activation.
Sherr and Weber, 2000
IAPs Frequently overexpressed in cancer. Down regulation of XIAP induces apoptosis in chemoresistant tumors.
Deveraux and Reed, 1999
NFkB Deregulated activity in many cancers. Transcriptionally activates expression of anti-apoptotic members of the Bcl-2 and IAP families. Can inhibit both the extrinsic and intrinsic death pathways and induce drug-resistance.
Baldwin, 2001
Ref: Cell, 2002, 108:153-164
Tumor-derived mutations affecting apoptosisProtein Role in apoptosis Ref
p53 Mutated or altered expression in many cancers. Initiates the intrinsic apoptotic pathway. p53-/- cells are resistant to drug induced apoptosis.
Vogelstein et al., 2000
p19ARF Mutated or altered expression in many cancers. Blocks MDM2 inhibition of p53. Enhances drug-induced apoptosis by p53.
Sherr and Weber, 2000
Rb Mutated in some cancers, and functionally disrupted in many cancers. Inhibits E2F-medidated transcription. Loss of Rb function induces p53-dependent and independent apoptosis.
Harbour and Dean, 2000
Chk2 Mutated in Li-Fraumeni syndrome. Senses DNA double strand breaks and phosphorylates and stabilizes p53.
Khanna and Jackson, 2001
Ref: Cell, 2002, 108:153-164
Tumor-derived mutations affecting apoptosisProtein Role in apoptosis Ref
Myc Deregulated expression in many cancers. Induces proliferation in the presence of survival factors, such as Bcl-2, and apoptosis in the absence of survival factors. Can sensitise cells to drug-induced apoptosis.
Evan and Vousden, 2001
Akt Frequently amplified in solid tumors. Phosphorylates Bad. Hyperactivation induces resistance to a range of apoptotic stimuli including drugs.
Datta et al., 1999
PI3K Overexpressed or deregulated in some cancers. Responsible for activation of Akt and downstream phosphorylation of Bad. Inhibition of PI3K enhances chemotherapeutic drug-induced apoptosis.
Roymans and Slegers, 2001
Ras Mutated or deregulated in many cancers. Activates PI3K and downstream pathways. Induces proliferation and inhibits c-myc and drug-induced apoptosis.
el-Deiry, 1997
Ref: Cell, 2002, 108:153-164
Tumor-derived mutations affecting apoptosis
Protein Role in apoptosis Ref
FLIP Overexpressed in some cancers. Prevents activation of caspase-8 and apoptosis induced by some chemotherapeutic drugs.
Tepper and Seldin, 1999
Ref: Cell, 2002, 108:153-164
Tumor advantages following p53 mutations
1. Cell cycle - mutant cells are able to progress through the cell cycle and divide, passing on mutations.
2. Apoptosis - these cells ignore signals to commit cell suicide.
3. Genetic instability - continued division without checkpoints leads to chromosomal aberrations, incorrect rejoining of chromosomes activation of oncogenes and inactivation of tumor suppressor proteins
Therapeutic applications of regulating apoptosis
Promote apoptosis in cancer cells:
Lymphoma/leukemia
Oral cancer
Brain tumors
Prostate
Colon
etc.
Prevent apoptosis in certain disorders and degenerative diseases:
AIDS
Ischemia
Alzheimer's/Parkinson's
etc.
Apoptosis in the treatment of cancer
An important goal of cancer drug development should be to facilitate apoptosis in neoplastic cells. Drugs that restore apoptosis might selectively kill cancer cells that have triggered a death signal and have become dependent on the deregulation of apoptosis pathways.
Strategies already used:• Administration of death ligand• Bcl-2 family inhibitors• XIAP inhibitors
Fesik Nat Rev Cancer (2005)
Ribozyme Inhibition of Bcl-2 Expression
• Apoptosis removes damaged cells from the body. The bcl-2 gene prevents this.
• The role of bcl-2 in oral cancer and glioblastoma is unexplored.
• We constructed a hammerhead ribozyme that would digest the bcl-2 mRNA message and delivered it to oral cancer and glioblastoma cells with an adenovirus vector.
Ref: Nature, 412, 865 - 866 (2001)
AAV kills cancer cells
APOPTOSIS: Role in DiseaseAGING
Aging --> both too much and too little apoptosis(evidence for both)
Too much (accumulated oxidative damage?)---> tissue degeneration
Too little (defective sensors, signals?---> dysfunctional cells accumulatehyperplasia (precancerous lesions)
„Don't think of death as an ending. Think of it as a really effective way of cutting down your expenses.” Woody Allen
THANK YOU