L5: Apoptosis II Flashcards
How is apoptosis induced?
Apoptosis is induced in two fundamentally different ways:
* Most cells require signals (trophic factors) (things like growth factors, nutrients, food, amino acids-in extracellular fluid) to stay alive and will undergo apoptosis in the absence of these signals
* Some cells are triggered to undergo apoptosis by specific signals
Apoptotic pathways?
Apoptotic pathways:
1. Intrinsic pathway – growth factor withdrawal (aka removing trophic factors), DNA damage, other stress signals
2. Extrinsic pathway - signalling through cell surface ‘death’ receptors by death ligands on surface of surveilling immune cells which directly induces apoptosis after noticing a cell is not functioning properly.
Apoptotic control in cells?
Apoptotic control in cells: Many different ways to trigger apoptosis but Final pathway and features the same. Once initiated, the process is irreversible – needs tight control. Features such as activation of enzymes- caspases that chop up substrates inside cells to destroy cells architecture and protein function is the same.
Cell membrane - death receptors (extrinsic pathway) - but can also involve other membranes
* Cytoplasm – caspases (initiator and effector caspases) - effectors of apoptosis are cytosolic proteins that diffuse around and find substrates throughout the cytoplasm.
* Mitochondria – cytochrome c release and formation of apoptosome - important for apoptotic induction.
* Nucleus – endonuclease activation- fragmentation of dna by CAD, held inactive by binding to a substrate inhibitor- ICAD.
Why are worms used as models for apoptosis?
Worms can be used as a model for apoptosis due to their smaller genomes, less complex pathways and simpler systems. More genetically manipulable than humans and easier to see down microscopes- may be transparent. Can see the early stages of cell division after their eggs are fertalised. Egg has pro nuclei- haploid nuclei. Nuclear envelope around haploid nuclei breaks down and mitotic spindle forms. Condensed chromatids are released into the cytoplasm. Asymmetric division- can partition diff amounts and types of proteins into each daughter cells. After many divisions enough cells make an entire worm.
Adult males- 1039 cells
Hermaphrodites- 959 cells
Have a defined somatic lineage.
(not m and f, male and hermaphrodites)
Apoptosis in developing c.elegans embryo?
transparency allows scientists to see which cells are being destroyed by apoptosis.
*3 phases of death:
* Specification (instruction to die): genes required for apoptotic instruction: genes involved in chopping proteins and genes in engulfment
* Killing (dying)
* Execution (engulfment)
siRNA or mutagenic visual screens for apoptosis
conserved components in the regulatory cascade?
Regulator: genes that act early
Adapter: genes that acted in the middle
Effector: genes that act late. Allow the process to be amplified and allow the execution for the protein cutting pathway.
Activation of the caspases leads to death.
A human gene could be inserted into a worm lacking the worm gene and it would still function showing how conserved the proteins involved in eliciting apoptosis are.
c.elegans regulator: ced-9– adaptor: ced-4—> effector: ced-3–> death
vertebrates: regulator bcl-2–Apaf-1–>casp9–>casp3–> death
human bcl-2 could suppress apoptosis in the worm
caspases?
Caspases are multi-domain proteins with a large subunit and a small subunit, which is common to all caspases.
They are usually inactive and exist as monomers.
Activation occurs through cleavage and conformational changes.
Control of apoptosis: caspases
Cells are executed by intracellular proteases - Caspases (Cysteine-Aspartic Acid-Proteases) - cleave aspartic acid and have cysteine in their active sites
* Caspases are normally inactive (pro-caspases) until activated during apoptosis
* Caspases are activated by death receptors (extrinsic pathway) (TNF receptor, Fas) and Cytochrome C release (intrinsic acid)
* Caspases cleave proteins after aspartic acid (Asp = D) residues * Eg, Caspase 3 recognises D-X-X-D sequences and cleaves after final D. X= amino acid e.g: Dead- e and a are amino acids.
Examples of Caspase substrates:
* A regulator of DNase (CAD) which cleaves DNA
* Nuclear lamina: releases the nuclear membrane
* ROCK1: induces blebbing
initiator and executioner caspases?
Initiator and executioner caspases: 14 caspases in humans (initiators and effectors)
* Initiator caspases: start the apoptotic commitment
* Effector caspases: do the business of degrading substrates in cells 14 caspases in humans (initiators and effectors)
* Initiator caspases: Caspase 8, Caspase 9 (also Caspase 2 & 10)
* Effector caspases: Caspase 3, Caspase 6, Caspase 7
- Some caspases are involved in signalling pathways of inflammation not apoptosis.
Initiator caspases: start the apoptotic commitment - Effector caspases: do the business of degrading substrates in cells
caspase-3 activation?
active caspase: hetero-tetramer from 2 x large 2 x small. 12-stranded beta sheet. catalytic residues (his and cys) contributed by large subunit.
The active version of casperases dimerises to make an active site between the large 2 subunits. The small and large subunits come together in one monomer and another monomer binds- 6 stranded beta sheet assembles to make a 12 stranded beta sheet. Process is irreversible- if only active when cleaved they cant come back together and become inactive again so this is a good way of making sure apoptotic commitment is absolute.
caspase targets in cells?
Caspase Targets:
Activated caspases are in the cytoplasm and cause the destruction of cellular structure and function.
Kinase Activation:
ROCK1 kinase has its regulatory domain cleaved, becoming active and phosphorylating MLCK.
This phosphorylation leads to cytoskeleton contraction and membrane blebbing.
DNA Fragmentation:
Caspases cleave the inhibitor of caspases-associated DNase (ICAD), releasing CAD.
CAD enters the nucleus, chopping DNA between nucleosomes, causing DNA fragmentation.
Nuclear Lamina Disruption:
Caspases can directly act on the nuclear lamina, cleaving it.
They also cleave the regulatory subunit of MST1 kinase, which enters the nucleus to phosphorylate lamins and nucleosomes, leading to nuclear structure disassembly.
Golgi Disruption:
Caspases cleave GRASP proteins, which hold the Golgi structure together, causing it to disassemble.
*MST1 is s er/thr kinase activated by caspase 3 cleavage
