Programmed cell death
Fundamnetals
Individual Cells sacrifice to support the multicellular organism.Gluconeogenesis
Urea Cycle
Cell Death
Concept: #3 Apoptosis
General Information:
Characteristics: initiated, regulated, controlled, monitored
Requires energy (nucleolytic and proteolytic) derived from mitochondria destruction
Function: programmed cell death, remove unwanted/unnecessary cells/maintain cell numbers
a)Adult – Homeostaisis: adult tissues balance cell division with cell death
b)Developing Organisms – major structuring
PHASES
1)Signal Initiation
1a)Extrinsic: originate from the environment
1b)Intrinsic: originate from cell interior
2)Amplification
Details:
1a) Extrinsic Signal - 8
Growth factor withdrawal
Death Ligands activate Death Receptor (TNF-alpha activation)
Ligands: 1)TNF-alpha, 2)FASL
Receptors: 1)TNFR, 2) FAS
Death receptor activation
FADD (FAS associated Protein w/ Death Domain): adaptor proteins
Binding of adaptor proteins
Formation of Death Inducing Signaling Complex (DISC)
FADD
Recruits Pro-caspase 8
Activation of Caspase 8
Pro-caspase 8 -(autocatalyzes )- Caspase 8
ONE peptide bond
Activation of Caspase 3
1b) Intrinsic Signal - 9
Activation of P53/Accumulation of pro-apoptotic proteins (BAD,BAX)
P53: activated during DNA damage, aids in DNA restoration/arrest cycle by providing time
Tumor Suppressor gene – that regulates cell cycle, induction of apoptosis, development, differentiation, gene amplification, DNA recombination, chromosomal segregateion and cellular senescence
Preventing inappropriate cell proliferation, maintining genome integirty
Activated P53 is phosphoryalted to prevent binding to MDM2
MDM2 regulates P53
BAD and BAX: puncture holes in mitochondria
BAX and BAK dimerize
Loss of voltage across mitochondrial membrane
Cytochrome C release
Cytochrome C interacts w/ apoptosome
Formation of apoptosome
Cytochrome C
APAF-1
Pro-Caspase 9
Activation of Caspase 9
Pro-caspase 9 -(autocatalyzes )- Caspase 9
ONE peptide bond
Activation of Caspase 3
Intrinsic Signal Regulation
BAX and BAK controlled by BCL-2 and BCL-XL
P53 is regulated by MDM2 (Murine double minute 2)
MDM2: promotes ubiquitination of P53, targeting it for destruction by the proteasome
Focus on P53
Before entering S phase, Myc and E2F are elevated.
In unfavorable S phase, Myc and E2 F INC ARF
ARF: blocks binding of MDM2 to P53
DEC MDM2 = INC P53
INC P53 leads to cell cycle arrest/apoptosis
INC ARF or phosphorylate P53 = APOP
Elevation of P53 in this manner can lead to cell cycle arrest OR apoptosis.
2) Caspase 3 initates the Caspase Cascade
Caspase Cascade: positive feedback event which rapidly increases the amount of active caspases
Caspase 3 cleaves Poly-ADP Ribose Polymerase (PARP)
PARP: repairs dSS breaks
Overall destruction:
Cytoskeleton
Organelles (Mitochondria first)
Nucleus
Clearance by macrophages
Other factors of apoptosis:
Activation of the specific MAPK, JNK, promotes apoptosis
MAPK (Mitogen Activated Protein Kinase)
1)Phosphorylates Bcl-2 – Deactivates Bcl-2
Disassociation from the mitochondrial membrane INC [BCL2]
2)Phosphorylate P53 – Activates P53 – Protects = INC [P53]
However, this signaling is cell type dependent – depending on the environemnt
Detecting Apoptosis
Inhibition of Proteasome – specifically what Dr Daniel
Activaiton of Caspase 3
Cleavage of PARP
Morphological Changes
Punctate DNA staining
Rounding up and release from substratum
Presentation of phosphatidyl serine
Phosphatidyl serine: usually in the inner leaflet, when it is on the outer leaflet = APOPTOSIS
Apoptosis is Clean
Inducing Apoptosis
Removal of growth factors from media.
Induction of DNA damage (why?)
Inhibition of proteasome activity (why?)
Activation of Caspases
Differenciate the descriptions of the stains
Punctate
PARP
Phosphatidyl serine
Which protein compromises the integrity of the mitochondrial membrane
Which protein protects the mitochondrial membrane
What protein complex does cytochrome c help form
What key protein does caspase 3 cleave (hallmark of apop)
Self perpetuating activation of caspases is named
Why does proteasome inhibition result in apopotsis
Upand down regulation
Methylation: locks on/off genes
Summary
Apoptosis is a highly regulated, organized, and controlled form of cellular suicide.
There are two key pathways in apoptosis
Extrinsic
Extrinsic apoptosis originates from signals outside of the cell.
Extrinsic apoptosis involves the formation of DISC and activation of Caspase-8
Intrinsic
Intrinsic apoptosis originates from signals inside the cell.
Intrinsic apoptosis begins with failure of the mitochondrial membrane.
Formation of the apoptosome (cytochrome-c, APAF1, and Caspase-9) leads to activation of Caspase-3
Apoptosis has several hallmarks
Activation of Caspase-3
Presentation of phosphotidyl serine
Cleavage of PARP
Apoptosis, as opposed to necrosis, is clean
