CaspasesCysteine-dependent aspartate specific proteasesHave a cysteine at the active siteCleave target just after aspartic acid residuesSubstrate specificity is determined by the 4 residues upstream ofcleavage site( towards N-terminal)Exist in cytosol as single chain proenzymes (procaspases) whichare activated when cleaved by other caspasesOnce activated, cleave other caspases – results in proteolyticcascadeAlso cleave key proteins in the cell, causing the characteristicmorphology 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 death14 CASPASES identified in humans.
AntiapoptoticProapoptoticBcl-2 family membersA very large family with 30 members identified and belongs to both:BidBimBikBadBmfHrkNoxaPumaBlkBNIP3SpikeBH1, BH2,BH3,BH4BH3BH1, BH2,BH3
THE BCL-2 FAMILYRaf-1calcineurinLiganddomainNPoreformationMembraneanchorphosphorylation Receptor domainC
Regulatory interactions between Bcl-2 family members
Youle and Strasser (2008) The BCL-2 protein family: opposing activities that mediate cell death. NatureReviews Molecular Cell Biology, 9, 47-59BH3 only protein binding specificity for BCL-2 homologuesBIM and PUMA bind to all BCL-2 family members tested; by contrastNOXA only binds to A1 and MCL1.These binding specificities recapitulate the ability of these proteins toactivate apoptosis e.g. BIM et al can induce apoptosis alone whereasa 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 degradationInhibited by SMAC/DIABLO.Salvesen and Duckett Nat rev mol cell biol (2002)
Kinchen andRavichandran (2007)Fig1Stages ofengulfment ofapoptotic cellscan be dividedinto 4 stagesBindingRecognitionPhagocytosisInternalization
Lauber et al.(2004) Fig 2 Lack of “don’t eat me” signals on thesurface of apoptotic cells
Anterior chamber of the eye and testes fail to elicit an immune response becausethese cells produce lots of FasL, so kill immune cells when they enter thesesites.Possible therapy by inducing production of FasL in other tissues – loweringneed for immune rejection drugsORGAN TRANSPLANTS
(HIV deactivates Bcl-2)(
1. p53 mediates apoptosis in response to DNA damage, oncogeneexpression (adenovirus E1A, myc etc.), or withdrawal of growthfactors2. Overexpression wild-type of p53 leads to apoptosis3. p53 induces apoptosis through transcriptional activation ofproapoptotic 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 crelease.p53 in apoptosis
p53 binds to Bcl-xL and releases Bax.Bax is sequestered byBcl-xL and inactiveBax is released by p53from Bcl-xL and formsoligomers, leading toapoptosis. p53 can release both proapoptotic multidomain proteinsand BH3-only proteins
p53 and Puma inapoptosisRef: K.Vousden, Science,2005
p53 in DNA repairand apoptosisRef: Bensaad &Vousden, NatureMed, 2005ROS: reactive oxygen speciesG1- S
Role of P53 in DNA damage induced Apoptosis• DNA damage activateATM & Chk2 proteinkinases• Phosphorylation of P53• Activate Cdk inhibitorP21, which inhibitsCdk2/cyclin Ecomplexes, halting cellcycle progression in G1NOXA
The p53-MDM2 feedback loop1. MDM2 binds to p53 N-terminal transactivationdomain and inhibits p53-dependent transcription.2. MDM2 is a transcription target of p53.3. MDM2 is an E3 ubiquitin ligase of p53, thustargeting p53 for proteolytic degradation.4. MDM2 knockout is lethal for mouse embryonicdevelopment, but simultaneous deletion of p53and MDM2 genes rescues MDM2-KO.
The p53 pathways
Ref: Nature 408, 307 - 310 (2000)Viral oncogenes and the p53 network
Genetic Control of "GeneticallyProgrammed Cell Death"Cellular Location ofProtein ProductMitochondrial MembraneNuclear EnvelopeEndoplasmic ReticulumNucleusNucleusCell MembraneEffect on ApoptosisBlocksStimulatesWild-type StimulatesMutant BlocksStimulatesApoGenesbcl-2mycp53p53*APO-1/FASApoptosisGene
Tumor-derived mutations affectingapoptosisProtein Role in apoptosis RefATM Mutated in ataxia-talangiectasia syndrome. Senses DNAdouble strand breaks and stabilizes p53. Deficienciesincrease risk of developing haematological malignanciesand breast cancerKhanna andJackson, 2001Bax (p53 targetgene)Mutated or decreased expression in some tumors.Mediates mitochondrial membrane damage. Sufficientbut not necessary for drug-induced apoptosis.Rampino et al.,1997Bak Mutated or decreased expression in some tumors.Mediates mitochondrial membrane damage. Sufficientbut not necessary for drug-induced apoptosis.Kondo et al., 2000PTEN (p53target gene)Mutated or altered expression in cancers. Regulates Aktactivation and subsequent phosphorylation of Bad. Lossof PTEN results in resistance to many apoptotic stimuli.Di Cristofano andPandolfi, 2000Ref: Cell, 2002, 108:153-164
Tumor-derived mutations affectingapoptosisProtein Role in apoptosis RefApaf-1 (p53target gene)Mutated and transcriptionally silenced in melanomaand leukemia cell lines. Necessary for activation ofcaspase-9 following cytochrome c release. Apaf-1-/-cells are chemoresistant.Soengas et al., 2001CD-95/Fas Mutated and down-regulated in lymphoid and solidtumors. Initiates the extrinsic apoptotic pathway. Lossof function is associated with resistance to drug-induced cell death.Muschen et al., 2000TRAIL-R1/R2Mutated in metastatic breast cancers. Initiate theextrinsic apoptotic pathway. Mutations lead tosuppression of death receptor-mediated apoptosis.Shin et al., 2001Caspase-8 Gene silenced in neuroblastomas. Activates bothextrinsic and intrinsic apoptotic pathways. Silencingresults in resistance to drug-induced apoptosis.Teitz et al., 2000Ref: Cell, 2002, 108:153-164
Tumor-derived mutations affectingapoptosisProtein Role in apoptosis RefBcl2 Frequently overexpressed in many tumors. AntagonisesBax and/or Bak and inhibits mitochondrial membranedisruption. Inhibits drug-induced apoptosis.Reed, 1999MDM2 Overexpressed in some tumors. Negative regulator ofp53. Inhibits drug-induced p53 activation.Sherr and Weber,2000IAPs Frequently overexpressed in cancer. Down regulationof XIAP induces apoptosis in chemoresistant tumors.Deveraux and Reed,1999NF B Deregulated activity in many cancers. Transcriptionallyactivates expression of anti-apoptotic members of theBcl-2 and IAP families. Can inhibit both the extrinsicand intrinsic death pathways and induce drug-resistance.Baldwin, 2001Ref: Cell, 2002, 108:153-164
Tumor-derived mutations affectingapoptosisProtein Role in apoptosis Refp53 Mutated or altered expression in many cancers.Initiates the intrinsic apoptotic pathway. p53-/-cells are resistant to drug induced apoptosis.Vogelstein et al., 2000p19ARF Mutated or altered expression in many cancers.Blocks MDM2 inhibition of p53. Enhances drug-induced apoptosis by p53.Sherr and Weber, 2000Rb Mutated in some cancers, and functionallydisrupted in many cancers. Inhibits E2F-medidated transcription. Loss of Rb functioninduces p53-dependent and independent apoptosis.Harbour and Dean,2000Chk2 Mutated in Li-Fraumeni syndrome. Senses DNAdouble strand breaks and phosphorylates andstabilizes p53.Khanna and Jackson,2001Ref: Cell, 2002, 108:153-164
Tumor-derived mutations affectingapoptosisProtein Role in apoptosis RefMyc Deregulated expression in many cancers. Inducesproliferation in the presence of survival factors, such asBcl-2, and apoptosis in the absence of survival factors.Can sensitise cells to drug-induced apoptosis.Evan and Vousden,2001Akt Frequently amplified in solid tumors. PhosphorylatesBad. Hyperactivation induces resistance to a range ofapoptotic stimuli including drugs.Datta et al., 1999PI3K Overexpressed or deregulated in some cancers.Responsible for activation of Akt and downstreamphosphorylation of Bad. Inhibition of PI3K enhanceschemotherapeutic drug-induced apoptosis.Roymans andSlegers, 2001Ras Mutated or deregulated in many cancers. Activates PI3Kand downstream pathways. Induces proliferation andinhibits c-myc and drug-induced apoptosis.el-Deiry, 1997Ref: Cell, 2002, 108:153-164
Tumor-derived mutations affectingapoptosisProtein Role in apoptosis RefFLIP Overexpressed in some cancers. Prevents activation ofcaspase-8 and apoptosis induced by somechemotherapeutic drugs.Tepper and Seldin,1999Ref: Cell, 2002, 108:153-164
Tumor advantages following p53mutations1. Cell cycle - mutant cells are able to progressthrough the cell cycle and divide, passing onmutations.2. Apoptosis - these cells ignore signals to commitcell suicide.3. Genetic instability - continued division withoutcheckpoints leads to chromosomalaberrations, incorrect rejoining of chromosomesactivation of oncogenes and inactivation of tumorsuppressor proteins
Apoptosis in the treatment of cancerAn important goal of cancer drug development should be tofacilitate apoptosis in neoplastic cells. Drugs that restore apoptosismight selectively kill cancer cells that have triggered a death signaland have become dependent on the deregulation of apoptosispathways.Strategies already used:• Administration of death ligand• Bcl-2 family inhibitors• XIAP inhibitorsFesik Nat Rev Cancer (2005)
Ribozyme Inhibition of Bcl-2 Expression• Apoptosis removes damaged cells from the body. Thebcl-2 gene prevents this.• The role of bcl-2 in oral cancer and glioblastoma isunexplored.• We constructed a hammerhead ribozyme that woulddigest the bcl-2 mRNA message and delivered it to oralcancer and glioblastoma cells with an adenovirusvector.
Ref: Nature, 412, 865 - 866 (2001)AAV kills cancer cells
APOPTOSIS: Role in DiseaseAGINGAging --> both too much and too little apoptosis(evidence for both)Too much (accumulated oxidative damage?)---> tissue degenerationToo little (defective sensors, signals?---> dysfunctional cells accumulatehyperplasia (precancerous lesions)
„Dont think of death as an ending. Think of it as a really effective way ofcutting down your expenses.” Woody Allen