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)

http://www.immunityageing.com/content/3/1/5/figure/F1?highres=y

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