23rd Nov - Cell Death Flashcards
Describe accidental cell death
Cells exposed to extreme stimuli succumb in a completely uncontrollable manner reflecting the immediate loss of structural integrity
Describe regulated cell death
Initiated by genetically encoded machinery in response to an extracellular signal
Describe programmed cell death
Regulated cell death that occurs as part of the developmental program or to protect tissue homeostasis
What are the four types of cell death?
Necrosis
Apoptosis
Autophagy
Conification
What are the universally applicable criteria for assessing cell death?
Loss of plasma membrane integrity
Cell fragmentation
How can loss of plasma membrane integrity be detected?
IF microscopy
FACs to asses the exclusion of vital dyes e.g. tryphan blue
How can cell fragmentation be detected?
IF microscopy
FACs quantification of hypodiploid events
Outline the progression of regulated cell death
- Homeostatic Pertubation
- Signal Transduction
- Point of no return
- Signal transduction
- RCD Correlates
OR
- RCD causes
- Primary RCD
- Release of DAMPs
- Inflammatory reactions and initiation of secondary RCD
Give an example of a molecular criteria used in cell death
Massive activation of caspases - part of the classic apoptotic programme
Mitochondrial membrane potential dissipation - protracted MM potential dissipation usually proceeds MMP and cell death however transient dissipation is not always a lethal event
Mitochondrial transmembrane permeabilisation (MMP) - complete MMP results in the liberation of lethal catabolic enzymes or activators of such enzymes
Phosphoserine exposure - on the outer leaflet of the pm is often an early event of apoptosis but may be reversible by the upregulation of scramblases
How can massive activation of caspases by identified?
Immunoblotting
FACs quantification
How can mitochondrial membrane dissipation be identified?
FACs (flow cytometry) quantification with mitochondrial membrane sensitive probes and calcineurin
What is FACs?
Fluorescence activated cell sorting, a form of flow cytometry, in which a heteregeneous mixture of cells can be sorted one cell at a time using the specific light scattering and flourescence properties of each cell
How can mitochondrial transmembrane permeabilisaiton (MMP) be identified?
IF colacalisation studies
Immunoblotting after subcellular fractionation
How can phosphoserine exposure be identified?
FACs quantification of Annexin V binding
What are the morphological features of apoptosis?
Rounding up the cell
Retraction of pseudopodes (cytomplasmic projections)
Pyknosis
Karyorrhexis
Minor modification of cytoplasmic organelles
Plasma membrane blebbing
Engulfment by resultant phagocytes
What is pyknosis?
Reduction of cellular and nuclear volume
What is karyorrhexis?
Nuclear fragmentation
Who won the nobel prize in physiology or medicine in 2002?
Brenner, Honitz and Sulston for their work on programmed cell death in Caenorhabiditis elegans
How many initial somatic cells are eliminated by apoptosis?
131/1090
Outline the process of apoptosis in C. elegans
EGL1 –| Ced-9 –| Ced-4 –> Ced-3
What is the human homolog of CED-3?
Caspase
What is the human homolog of CED-4?
Apaf-1
In C. Elegans how is the apoptotic pathway regulated?
CED-9 binds and negatively regulates CED-4, inhibiting apoptosis. Death is activated by EGL-1 displacing CED-4.
Outline the mammalian apoptosis pathway
Bid, Bim –| Bcl-2, Bcl-XL –| Bax, Bak –> Apaf1 –> Caspase 8/9 –> Caspase 3
Which are the caspases that initiate cell death?
Caspase 10, 2, 9 and 8
Which are the caspases that execute cell death?
Caspase 3, Caspase 6 and Caspase 7
Which are the caspases that cause inflammation?
Caspase 1, Caspase 5 and Caspase 4
What is a DED domain?
Death effector domain - common in caspases
What is a CARD domain?
Caspase activation and recruitment domain -common in capsases
How are caspases activated?
They are synthesised in zymogen form thus activated by cleaving
Initator caspases (2,9,8,10) directly cleave Effector caspases (3,6,7)
What is the caspase catalytic triad?
Cys-His-Carbonyl
What does the additional interaction of Caspase 2 at residue S5 do?
Enhance catalysis at least 30 fold
What are the targets of caspases?
Caspases Bid DFF45/ICAD NDUF51 IAPs Nuclear Lamins Catenins Vimentin DNA-PK Rad 51 ATM p21 Wee1 Rb
What is CAD?
Caspase activated DNA fragmentation factor
How is CAD activated?
Caspase 3 cleaves it
What sets the threshold of intrinsic apoptotic pathway susceptibility and how?
The anti-apoptotic BCL-2 : pro-apoptotic BCL-2, as they regulate the mitochondrial outer membrane permeabilization
What is a BIR?
A Baculoviral IAP repeat found in apoptotic proteins, cytokine production proteins and proteins involved in chromosome segregation
What is RING?
Really interesting new gene, found in many IAPs and encodes a B3 ligase that presumably directs proteins to the ubiquitin-proteasome degradation system
What are the proteins that signal phosphatidyl serine exposure?
MFG E8 GasG Anxl Beta1-GP1 ProtS
How is the extrinsic apoptotic pathway activated?
Through cell surface receptors
Outline the three possible signalling pathways of the extrinsic apoptotic pathway
A. Death receptor signalling and activation of caspase 8 to caspase 3 cascade
B. Death receptor signalling and activation of caspase 8 to tBID to MOMP to caspase 8 and caspase 3
C. Ligand deprivation induced dependence on receptor signalling followed by activation of the caspase 9 to caspase 3 cascade
What are the key events of the intrinsic apoptotic pathway?
Generalized and irreversible dissipation of mitochondrial membrane permeability
Release of mitochondrial inner membrane space and proteins into the cytosol
Respiratory chain inhibition
What protein is a regulator of crosstalk between classically intrinsic and extrinsic apoptotic signalling pathways?
XIAP
What are the functions of XIAP?
Mediate cross-talk between the intrinsic and extrinsic apoptotic pathways
Rapidly induce caspase 8
Define cell type specific elements of FAS signalling
What are the main biochemical features of extrinsic apoptosis by death receptors?
Death receptor signaling caspase-8 activation BID cleavage Increase in mitochondrial outer membrane permeability Caspase 3 activation
What are the possible inhibitory interventions for extrinsic apoptosis triggered by death receptors?
CnmA expression
Genetic inhibition of caspases
Z-VAP-fmk administration
What are the main biochemical features of the extrinsic apoptotic pathway activated by dependence receptors?
Dependence receptor signalling PP2A activation DAPK1 activation Caspase-9 activation Caspase-3 activation
What are the possible inhibitory interventions of the extrinsic apoptotic pathway activated by dependence receptors?
Genetic inhibition of caspases
Genetic inhibition of PP2A
Z-VAD-fmk administration
What are the main biochemical features of caspase dependent intrinsic apoptosis?
Increase in mitochondrial outer membrane permeability
Irreversible mitochondrial membrane membrane potential dissipation
What are the possible inhibitory interventions for caspase dependent intrinsic apoptosis?
BCL2 OE
Z-VAD-fmk administration
What is Z-VAD-fmk?
Cell-Permeant Pan Caspase Inhibitor of Apoptosis
What are the biochemical markers of caspase independent intrinsic apoptosis?
Release of IMS proteins
Respiratory chain inhibition
What are the biochemical markers?
Release of IMS proteins
Respiratory chain inhibition
What is the possible inhibitory intervention for caspase dependent intrinsic apoptosis?
BCL-2 OE
What are the morphological features of necrosis?
Oncosis
Rupture of plasma membrane
Cytoplasmic organelle swelling
Moderate chromatin condensation
What is oncosis?
Cytoplasmic swelling
What are the biochemical features of necrosis?
Activation of calpains
Activation cathespins
What activates calpains and cathespins?
Massive increase in calcium which is disregulated
What is necroptosis?
Regulated necrosis
What can trigger necroptosis?
Alkylating DNA damage, excitotoxins and ligation of death receptors
What inhibits necroptosis?
necrostatin -1 a chemical RIPK1 inhibitor
What is used to identify necroptosis in cells?
necrostatin-1
What is MLKL?
Mixed-lineage kinase domain like protein that causes cell lysis by translocation to the membrane
What are the diseases that necroptosis plays a role in?
Artherosclerosis Ischemiareperfusion injury Transplantation Lung infection COPD Remote lung injury Hepatotoxicity Alcoholic and non-alcoholic steatiohepatitis Remote liver injury Kidney ischemiareperfusion injurt Bone marrow failure Crohns and ulcerative colitis Acute pancreatitits Traumatic brain injury Stroke
Outline the cycophilin D- mediated mitochondrial permeability transition
A caspase independent necrotic pathway which occurs in traumatic brain injury or stroke. Associated with calcium overload, oxidative stress and ATP depletion
Cyp-D binds the active target protein to form a pore traversing the mitochondrial membrane
What inhibits CypD-mediated mitochondrial membrane permeability transition?
Cyclosporinea
What is pyroptosis?
A form of regulated necrosis in which Osmotic cell lysis and release of intracellular pro-inflammatory molecules such as ATP and HMGB1
What typically initiates pyroptosis?
Toll like receptors (TLR) or NOD like receptors (NLR) binding to PAMPs/DAMPs
What is parthanatos?
A form of regulated necrosis induced through PARP overactivation
What is ferroptosis?
A form of regulated necrosis dependent on Iron and under the control of glutathione peroxidase 4 (GPX-4)
What inhibits ferroptosis?
Erastin which inhibits the Glu-Cys exchanger System Xc
What are the morphological features of cornification?
Elimination of cytosolic organelles
Modifications of the plasma membrane
Accumulation of lipids in F and L granules
Extrusion of lipids in the extracellular space
Desquamation by protease activation
What is cornification?
A form of active cell death in which the cornified epithelium is formed by the basal lamina dying
What are the biochemical features of cornification?
Activation of transglutaminases
Caspase-14 activation
What is autophagy?
consumption of the body’s own tissue as a metabolic process occurring in starvation and certain diseases
Does triggering the autophagic pathway always lead to cell death?
No sometimes this process promotes survival by removing dangerous protein aggregates
What are the morphological features of autophagy?
Lack of chromatin condensation
Massive vacuolization of the cytoplasm
Accumulation of bilameller autophagic vesicles
Litlle or no uptake of phagocytic cells
What are the classifications of autophagy?
Microautophagy
Chaperone mediated autophagy
Macroautophagy
What is microautophagy?
Molecules are sequestered at the level of the lysosome
What is chaperone mediated autophagy?
Entry is chaperone mediated, and molecules are sequestered at the level of the lysosome
What is macroautophagy?
Has a larger capacity in the pathway therefore can involve much larger protein complexes or organelles
Outline the process of macroautophagy
- A sequesteration crescent is formed, with LC3 and Atg5-12/16 free in the cytoplasm
- A phagophore forms with LC3 and atg in its membrane
- Autophagosome forms encapsulating the organelle and LC3-II
- Intermediate autophagosome forms through fusion with the endosome, containg the organelle, LC3-II and LAMP1/2
- The autolysosome forms through fusion with the lysosome encapsulating LAMP1/2, Acid phosphatases and cathespins
What are the biochemical features of autophagy?
LC3I –> II conversion
Depending on atg gene production
Beclin 1 dissociation from Bcl-2/Xl
p62 LCK degradation
What is the clinical relevance of autophagy?
Believed to be important for getting rid of protein aggregates such as those causing neurodegenerative diseases
