Apoptosis – mechanisms and role in

cancer therapy

TYPES OF CELL DEATH: Necrotic or apoptotic

APOPTOSIS

External signals

Normal development e.g. immune system

WHEN DOES APOPTOSIS OCCUR?

Disease states e.g. Alzheimer’s disease

WHEN DOES APOPTOSIS OCCUR?

Amyloid plaques in the brain

Caspases – key executioners of apoptosis

(cysteinyl aspartate specific proteases)

Highly conserved proteasesinactive zymogensCaspases divided into Group I

Inflammatory caspasesCaspases

1,4,5,11,12,13,14Group II

Initiator caspases Caspases 2,8,9,10

Group IIIEffector caspases:

caspases 3,6,7

Caspase structure

Properties of proteases

Irreversible -Autocatalytic: triggered by cofactor

binding or inhibitor removalProteases can regulate their own

activationprotease inhibitorsspecificity

Caspase structure3 domains 1) highly variable NH2 domain2) large subunit (p20; ~20kD)3) small subunit ( p10; ~10kD)

Highly specific absolute requirement for cleavage after aspartic acid

recognition of at least 4 amino acids NH2 terminals to the cleavage site

Caspase structure

2 key features: variable N domain regulates activation all domains derived from proenzyme precursor by cleavage specific sitesScheme of procaspase activation:Cleavage of the procaspase at the specific Asp-X bonds leads to the formation of the mature caspase, which comprises the heterotetramer p202–p102, and the release of the prodomain.

Structure of caspase-3 heterotetramerEach heterodimer is formed by hydrophobic interactions resulting in the formation of mostly parallel ß-sheets, composed of 6 antiparallel ß-strands.

Two heterodimers fit together with formation of a 12-stranded ß-sheet that is sandwiched by helices. N and C termini of the small and large protease subunits are indicated

Basic apoptotic machinery

DNA fragmentation,chromatin condensation,membrane blebbing, cell shrinkage & disassembly into apoptotic bodies engulfment

Initiator caspases inactivate proteins that protect cells from apoptosis Effector caspases are responsible for cellular changes associated with apoptosis.

How do caspases disassemble a cell? It slices, it dices!

Selective cleavage of specific proteins

eg bcl-2, or CAD/ICAD

e.g. nuclear lamins

eg. Gelsolin

What triggers apoptosis?• Growth factor withdrawal• Specific ‘death ligands • Loss of contact with surroundings• Irreparable internal damage• Conflicting signals for cell

division

How are caspases activated? Proteolytic cleavage

Cleavage of the procaspase at the specific Asp-X bonds leads to the formation of the mature caspase, which comprises the heterotetramer p202–p102, and the release of the prodomain.

2 key features: variable N domain regulates

activation all domains derived from

precursor by cleavage specific sites

How are caspases activated?

Induced proximity

aggregation of multiple procaspase-8 molecules into close proximity somehow results in cross-activation

How are caspases activated?

Holoenzyme formation

Activation of caspase-9 is mediated by means of conformational change, not proteolysis

nematode - C.elegansOne of the apoptotic pathways is triggered by internal signals- CEDCED-3 & 4 promote apoptosis CED-9 inhibits apoptosisApoptotic stimuli causes CED-9 dissociation by EGL-1 thereby activating CED-3.

Caspase signaling in Mammalian systems

IN Lavrik et al The Journal of Clinical Investigation 115(10):2665-72. (October 2005)

Mammalian systems Mammals

External signalsdriven by death receptors (DR) e.g. CD95 (or Fas/Apo)

Each CD95L trimer binds to 3 CD95 leading to DD clustering.

FADD ( Fas associated death domain/ Mort 1) binds via its own DD

Caspase –8 oligomerisation drives activation through self cleavage

Caspase –8 then activates downstream effector caspases like caspase –9 (CED-9 homolog)

Apoptosis initiation

Internal signals

BCL-2

DNA damageDeath receptorsGrowth factor

withdrawal

TRIGGERP53Bcl-2 familyCytochrome concogenes

REGULATOR

Apaf-1Caspases

EXECUTIONER

Green and Kroemer The Journal of Clinical Investigation 115(10):2610-17 (October 2005)

Chapter 12: Cellular & Mol Biology by Knowles and Selby

AND/OR Science (1998) Vol 281: No 5381; pgs 1298-1326

AND/OR J. Clin Invest (10 Oct 2005) 115(10):2665-72

AND/ORCancer Biology by RJB King pgs 160-167

AND/OR

References

NATURE | VOL 407 | 12 OCTOBER 2000 pp770-776The biochemistry of apoptosis by M.O. Hengartner

OptionalNATURE REVIEWS MOLECULAR CELL BIOLOGY Vol 5 | NOV 2004 | 897Molecular mechanisms of caspase regulation during apoptosisStefan J. Riedl and Yigong Shi(Only read it if you want to know more about caspase structure)