Protein Electrophoresis and Western Blotting
Download
Report
Transcript Protein Electrophoresis and Western Blotting
Intercalated BSc 2007-08
CELL DEATH
an overview
Dr Cathy Baker
22nd October 2007
How do cells die?
Killed by injurious agents
NECROSIS
Induced to commit suicide
APOPTOSIS
LEARNING OBJECTIVES
Understand, describe and illustrate …
Differences: necrosis vs. apoptosis
Morphological changes of apoptosis
Function of apoptosis
Principal biochemical mechanisms
Role of apoptosis in pathologies
Lecture overview
Necrosis
Function
Apoptosis
Morphological
changes
Biochemistry
Pathology
Necrosis
Mechanical injury & toxic agents
Cell groups
Membrane integrity destroyed
Cells and organelles swell, burst
and leak contents
Inflammatory response
Other cells and tissues damaged
Cell death by necrosis
John Kerr et al
Br.J.Cancer 26: 239-257, 1972
Apoptosis
Essential biological process
Cells have role in own death told or decide to commit suicide
Programmed cell death (PCD)
Apoptosis
Distinct form of single cell death
Tightly regulated
Very localised
Energy consuming process
Membranes intact (early stages)
Safe disposal of cell corpse
No inflammation
Necrosis
Apoptosis
Morphological
changes
Changes in cell morphology
Cells shrink and become detached
from adjoining cells
Cytoskeleton collapses
Mitochondria remain intact
Plasma membrane develops bubbles
(blebs) on surface
Membrane blebs during apoptosis
Nucleus and chromatin condense
Aggregates at periphery of nucleus
Nuclear envelope disintegrates
DNA fragmentation
Budding off and breakage into small
membrane wrapped fragments apoptotic bodies
Formation of apoptotic bodies
What happens to apoptotic cells
and apoptotic bodies?
Ingested & degraded by phagocytes
Macrophages and dendritic cells
Adjacent cells in tissue
High speed and efficiency
Histologically inconspicuous
No inflammation
Phagocytosis of apoptotic cells and bodies
Necrosis
Function
Apoptosis
Morphological
changes
Function of apoptosis?
Deliberate removal of specific,
unwanted cells
Organised and controlled manner
Without damaging other cells or
tissues
Circumstances?
Homeostasis
Constancy of internal environment
Tissue turnover
Cell numbers have to be maintained
Homeodynamics
Embryonic development
Removal of unwanted cells
Damage
Organ and tissue differentiation
Vestigial structures
Alteration of tissue form
5 weeks
8 weeks
Neurological development
Deletion of excess immature
neurons that have failed to establish
synaptic connections
Occurs in CNS and PNS
Prevents redundant cell in mature
nervous system
Involution of tissue
Endometrial breakdown prior to
menstruation
Regression of lactating breast tissue
after weaning
Cell damage
Internal cell damage
Inappropriate 3o protein structure
Cell Infection
Viral
Stress
Starvation
DNA damage
Ionizing radiation, ROS
Necrosis
Function
Apoptosis
Morphological
changes
Biochemistry
Biochemistry of apoptosis
Intense area of research
Complicated integrated mechanisms
Much more to be revealed!
Common core process
Underpins morphological changes
Four stage process
Stage 1 - The Death Signal
Stage 2 - Integration and Transduction
Stage 3 - Execution
Stage 4 - Cell Removal
Stage 1- The Death Signal
Absence or withdrawal of positive
survival factors
Presence of negative pro-apoptotic
factors
Survival or positive signals
Cell survival relies receiving
positive stimuli
Neuronal growth factor
Interleukin 2 for lymphocytes
Hormones
Withdrawal is a death signal
Default pathway for many cells
Death or negative signals
Signals to induce apoptosis
Damaged DNA
UV light and X rays
Chemotherapeutic drugs
Oxidants/free radicals
Oxidative stress
Death activators or receptor
ligands
What are Death Activators?
Molecules that bind to specific
receptors on cell surface
Tumour necrosis factor alpha
Lymphotoxin TNF beta
Fas ligand (CD95)
Binding of death activator to its
specific receptor is a pro-apoptotic
signal
Stage 2 - Integration and Transduction
Signals linked to execution phase
through an integration stage
Uses positive and negative
regulatory molecules
Inhibit, stimulate or forestall
apoptosis
To die or not to die?
Integrated balance between positive
survival factors and negative death
signals decides fate of cell
Common intracellular machinery
for apoptosis
The three main players
Family of enzymes - Caspases
Protein family - Bcl-2 proteins
Regulating gene - p53 gene
Caspases
Family of protease enzymes
14 isoforms identified
Have Cysteine at active site
Synthesised as inactive precursors procaspase
Not all involved in apoptosis
Procaspase structure
prodomain
large subunits
small subunits
cleavage
sites
Procaspase are activated through cleavage
Re-association of large and small subunits
Activated caspase has proteolytic activity
Initiator
caspases
Effector
caspases
Initiator
caspases
Effector
caspases
• Activate other caspases
• Amplify caspase activity
Apoptosis
execution
Bcl-2 proteins
Large family of proteins
Named from B cell lymphoma
Some are pro-apoptotic some are
anti-apoptotic
Bcl-2 proteins and apoptosis
Main mechanism is regulation of
mitochondrial permeability
Cell survival stimuli induce the
expression of anti-apoptotic Bcl
proteins
Death signals induce pro-apoptotic
Bcl proteins
p53 gene and p53 protein
p53 is tumour suppressor gene
Active gene product p53 produced in
response to DNA and cell damage
Prevents cell completing cell cycle
If damage is minor - allows repair
If major - induces apoptosis
Complex mechanisms
Apoptotic transduction pathways
Mitochondrial or intrinsic pathway
Death activator or extrinsic pathway
Intrinsic or mitochondrial pathway
Cell and DNA
damage –
Active p53
Bcl-2
Bax
●Changes in trans-membrane potential
●Pores form in (outer) membrane
●Inner & outer membrane proteins involved
Bcl-2
Bax
Irreversible
cell death
Bcl-2
Bax
Cyt C
Cyt C
Apaf-1
Apoptosis
activating factor -1
Aggregation of Cyt C/Apaf 1 complexes
Binding of Procaspase - 9
ATP
Auto-activation of Procaspase - 9
ATP
Formation of Active Caspase -9
ADP
Death receptor or extrinsic
pathway
Molecules that bind to specific
receptors on cell surface
Tumour necrosis factor alpha (TNF)
Lymphotoxin TNF beta
Fas ligand (CD95)
Binding of death activator to specific
receptor is pro-apoptotic signal caspase activation
Cell membrane with specific death receptors
Binding sites for death activators
Death domains extending into cytosol
Death receptors bind Death Activators
Clustering of death domains
Binding of adaptor protein(s)
Binding of caspase-8
Release of activated caspase-8
3. Execution
Achieved through activation and
deactivation of target proteins
by effector caspases
Activated effector caspases lead to …
Digestion of cytoskeleton proteins
Nucleus and chromatin degradation
Plasma membrane changes
Cytoskeleton degradation
Chromatin
degradation
Caspase-9 enlarges nuclear
pores
Allows entry of Caspase-3 and 7
Activation of nucleases
Caspase Activated DNAase - CAD
ICAD
CAD
Nucleosome
cleavage
CAD
Linker DNA
Nucleosome bead
8 histone molecules +
146 nucleotide pairs of DNA
mw ladder
DNA from
apoptotic cell
Other nuclear
changes
Structural proteins - Lamins
degraded by caspase-6
DNA repair enzymes inactivated
Nuclear membrane degraded
4. Cell removal
What is the eat me signal?
Enzyme system keeps PS on
inner surface
Inhibited during apoptosis
PS redistributed to extra-cellular
surface
Macrophage receptors recognise
and bind PS
Phagocytosis of apoptosome
Release of anti-inflammatory
substances
Necrosis
Function
Apoptosis
Morphological
changes
Biochemistry
Pathology
Homeostasis
Cell numbers have to be maintained
Cell formation
Cell death
Uncontrolled growth of cells
Insufficient apoptosis
Diseases featuring insufficient
apoptosis
Many cancers
Autoimmune Lymphoproliferative
Syndrome (ALPS)
Excessive apoptosis
Uncontrolled cell loss
Diseases featuring excessive
apoptosis
Neurodegenerative
Parkinson’s disease
Alzheimer's disease
Amyotrophic lateral sclerosis (ALS)
Huntingdon’s disease
Diseases featuring excessive
apoptosis
AIDS
Excessive apoptosis of T helper cells
Ischaemia
Cerebral caused by stroke
Cardiac caused by MI
You should now be able to …
Understand, describe and illustrate …
Differences: necrosis vs. apoptosis
Morphological changes of apoptosis
Function of apoptosis
Principal biochemical mechanisms
Role of apoptosis in pathologies