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19 Cards in this Set
- Front
- Back
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What are the mechanisms (pathways) initiating apoptosis? |
- Intrinsic pathway (mitochondrial) - extrinsic (receptor mediated) - granzyme B mediated |
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What initiates the extrinsic pathway and what cells does that kill> |
- Members of the tumour necrosis factor (TNF) binding to their receptors - kills cells targeted by the immune system - cancer cells, viral cells, excess lymphocytes after immune response |
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What are the general features of the TNF family of ligands and their receptors? |
- Ligands known as "death ligands" - produced by or present on the cells of the immune system such as cytotoxic T-lymphocytes, activated macrophages and natural killer (NK) cells - mediator of inflammation - produced by CTLs and activated macrophages in response to infection |
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Talk about the structure of TNFs |
- bind receptors as homotrimers causing trimerization of cognate receptors - a and b forms- but a is most important |
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Give example of members of the TNF family? |
- FasL - transmembrane protein present on the surface of CTL (cytotoxic T-lymphocytes) - binds to FASr on surface of viral cells, cancer cells and other lymphocytes - Tumor necrosis factor-related apoptosis-inducing ligand - TRAIL - induced apoptosis in cancer cells; expressed on surface of CTLs and NKs; 4 receptors of TRAIl found on target cell surface |
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The death receptors have associated adaptor proteins. Describe the features of both: |
- Receptors - extracellular domain, transmembrane domain and intracellular region containing "death domains" - Adaptors - death domain - interacts with death domain on receptor; Death effector domains (DED) - interacts with DED domain on initiator pro-caspases |
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What is the term for the whole receptor, adaptor and pro-caspase complex? |
- DISC - death inducing signalling complex |
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What events does TNF induce in the extrinsic pathway? |
- trimaric ligand causes receptor trimerisation - 2 cytosolic adaptor proteins FADD and TRADD and pro-caspase-8 are recruited by TNF to form DISC - 2 pro-caspase-8 activate each other to initiate caspase cascade |
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What events does TNF induce in the extrinsic pathway (image) |
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What happens in extrinsic pathway events induced by FAS engagement? |
- FasL on CTLs binds and activates Fas on target cells - Fas forms DISC with FADD and pro-caspase-8 to initiate caspase cascade |
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Extrinsic pathway events induced by FAS |
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What is the role of FLIP? |
- Binds to DISC and prevents activation of pro-caspase 8 - modulates death receptor activity |
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What ligand in Drosophila is a homologue of TNF? |
- Eiger - binds to Wengen receptor - stimulates transcription of HID - HID binds DIAPs to promote apoptosis |
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What are the features of the Granzyme B pathway? |
- contained with granules in CTLs and NK cells - enter target cell via perforin pores - similar action to caspases - can process caspase-3 and caspase-7 - can activate ICAD (inhibitor of caspase DNAse) independently of caspases resulting in activation of CAD and DNA cleavage - can cleave BID, which promotes activation of the intrinsic route of apoptosis |
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What are the similarities of the three pathways> |
- they communicate between each other - all converge at BID |
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How can apoptotic cells be identified? |
- cell membrane changes - DNA breakdown - caspase activity |
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What are some ways to detect cell membrane changes associated with apoptosis? |
- Annexin V binds to phosphotidylserine on the outside of early apoptotic cells - tag annexin with fluorescent dye and measure fluorescence - increased membrane permeability in late apoptosis - allows iodide to enter cells and bind DNA |
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What are some ways to identify DNA breakdown associated with apoptosis? |
- DNA laddering by gel - TUNEL assay |
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How do you detect caspase activity? |
- immunohistochemical identification using a specific antibody |
