Biochemistry 3385A Lecture Notes - Lecture 3: Mcl1, Apoptosis, Phosphorylation
Apoptosis: “programmed cell death”
Active process that activates cell destruction
Process regulating cell number
- Essential during development
oFrog – regression of tail of tadpode during metamorphosis
oVertebrate – mouse paw/human digits: elimination of tissue between the developing digits.
- Control of organ and body size
oIntestinal epithelial cells are continuously being eliminated by apoptosis following their
migration from the crypts to the lumen.
oRegression of mammary gland following weaning of offspring – epithelial cells eliminated by
Apoptosis is activated by
- Cellular stress
oLack of nutrients, growth signals or oxygen
oDamage, or malfunction of organelles or alteration of cellular metabolic processes (Including,
but not restricted to: ER stress, excessive internal Ca+ concentrations, oxydative agents,
inhibition of protein synthesis / protein degradation)
- Signals from the immune system
- Apoptosis is often inhibited in cancer cells which allows them to survive despite the internal stress
signals and deregulated metabolism that occurs in these cells.
Characteristics of apoptosis
- Slow and active process
- Cells undergoing apoptosis exhibit a series of
- characteristic morphological changes:
oCollapse of cytoskeleton
oDegradation of nuclear envelope
oChromatin compaction and degradation
oCompaction plasma membrane blebbing, cell body shrinkage
oFormation of membrane- bound apoptotic bodies
oAlterations in cell surface attract macrophages that engulf apoptotic cells
- During apoptosis, intracellular contents are not released and potentially harmful inflammatory
responses are prevented.
By contrast: Necrosis is fast and passive traumatic cell death with leakage of intracellular contents
Caspases – effectors of apoptosis
Proteases that cleave an Asp-XXX bond
Caspases play an essential role in apoptosis
•Inhibition of caspases effectively abrogate apoptosis induced by diverse apoptotic stimuli.
•Caspases are responsible for most proteolytic cleavages that lead to apoptosis.
Two mains types of caspase:
Initiator: caspase-8 and 9
Effector (executioner): caspase 3, 6, 7
- Synthesized as inactive precursors called procaspases
- Need to be cleaved into active form
- Procaspase activation by cleavage:
oSchematic of pro-caspase: [Prodomain][Large subunit][Small subunit]
oTwo inactive pro-caspase molecules are cleaved
oProdomains leave and both large/small subunits remain
oTwo pairs of each (large and small) subunits form a mature caspase tetramer
Activation of the caspase cascade
- Apoptotic signal induces the oligomerization of procaspases, leading to their autoproteolytic activation
- Cleavage of procaspase into active tetramer
- Initiators are cleaved and activated, this will activate effector procaspase
- This follows the cleavage of substrates/cell structures/survival proteins leading to cell destruction
How is apoptosis activated?
Two distinct pathways:
Death receptor pathway = Extrinsic pathway
Activated by extracellular ligands binding to a transmembrane receptor called Death Receptor.
Initial event is activation of Death receptor through binding of its ligand
Mitochondrial pathway = Intrinsic pathway
Activated by intracellular signals
Central event is depolarization of mitochondria triggered by pro-apoptotic factors of the Bcl-2 family
Note: Executioner caspases and death of substrates are common to both pathways
Intrinsic/mitochondrial apoptotic pathway
Activated by intracellular signals:
- DNA damage
- Damage, or malfunction of organelles Alteration of cellular metabolic processes
- Bcl-2 family:
oPro-apoptotic: Bax, Bak, Bad, Bim
oAnti-apoptotic: Bcl-2, Bcl-X, Mcl-1
- Pro-apoptotic members promote mitochondrial membrane depolarization which releases Cytochrome C
in the cytoplasm.
- Cyt c forms a complex with APAF-1 and procaspase-9 called the “Apoptosome”. This complex serves
to convert procaspase-9 into active caspase-9.
Decision of activating apoptosis is determined by the balance between pro- and anti- apoptotic factors of the
Pro-apoptotic (2 families): Pore-forming and facilitator members (BH3 only)
- Domain (pore-forming): [BH3][BH1][BH2]
oThese will promote release of Cyt C
- Domain: [BH3]
oPromote antagonist apoptotic signal, no release of Cyt C
- Note: BH3 here is the ligand domain
Anti-apoptotic: Bind to and inhibit the pro-apoptotic members
- Domain: [BH4][BH3}[BH1][BH2]
- Note: BH3/BH1/BH2 are receptor domains
- When same or higher level of anti-apoptotic proteins and pro-apoptotic: “Status quo” = all the pro-
apoptotic members are neutralized, no apoptosis.
- When excess of pro-apoptotic members compared to anti-apoptotic members: apoptosis is activated.
To activate apoptosis:
- Bax multimerization form pore in the outer mitochondrial membrane, release Cyt C
Absence of apoptotic signalling
- Bax inactive no apoptosis
Activation of apoptotic signalling
- BH3/Bax Bcl-2 (neutralized) pore-forming members’ multimerize in mitochondria release Cyt C
Overview of the intrinsic apoptotic pathway
- Pro-apoptotic signals open up mitochondrial channels
- Cytochrome C released
- SMAC/DIABLO released which inhibit IAPs (IAPs inhibit caspases)
- Cytochrome C/Apaf-1/procaspase-9 forms “apoptosome” complex
oForms to cleave procaspase-9 into caspase-9
- Apoptosome complex cleaves procaspase executioner 3, 6, 7 into caspase 3, 6, 7
- Ultimately resulting in death of substrates
Regulation of pro- and anti-apoptotic factor activation
Mode of regulation:
Transcription: Activation of pro-apoptotic genes repression of anti-apoptotic genes
Major regulator of apoptosis: p53. Transcription is allowed, Bax/Noxa/Bad produced
•Activates Bax, and several BH3 protein genes.
•Represses Bcl-2 and IAPs
Phosphorylation: the BH3 protein Bad is activated by phosphorylation
Extrinsic/death receptor apoptotic pathway
Activated by extracellular signals:
- Activating signal is binding of ligand to extracellular portion of cell surface receptor – Death receptor.
- Binding of ligand induces conformational change of intracellular portion of the receptor.
- Induces binding of FADD (Fas-Associated protein with Death Domain) to the intracellular receptor
domain to form the DISC complex. (Death-Inducing Signaling Complex)
- The DISC recruits procaspase-8 into a complex where multiple copies of procaspase-8 associate and
activate themselves by self-cleavage to yield active caspase-8.
- 5 families – about 30 different receptors
•FAS, DR4, DR5 TNFR1, DR3
- Bind their specific ligands that are members of the Tumor necrosis factor (TNF) family.
- Most cells in a variety of tissues, including epithelial cells, express Fas
- Activation by p53:
•Activates transcription of the Fas and DR5 receptor genes