Ray II

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If you have seen review articles, he picked simplified versions of slides to give general overview of apoptosis. If you want to read in detail, the information is for people who want to go into area. Otherwise you do not have to go into detail. Generating interest.
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Most caspases are initiators, executors, or inflammatory caspases. Inflammation not involved in apoptosis. Caspase 1 is related to ICE. Difference initiator and executor : Inititators are autoactivated after receiving signal. Autoactivation requires close proximity of more than one molecule. Clustering required. Receptor mediated pathways allow initiator caspases to form a complex, acting as modulators. 8 and 9 are major players in almost all cell types. 3,6,7 execute. No turning back. If 3,6,7 are inhibited, prevent cell death. If massive insult to the cell occurs with executor caspase inhibition, cells undergo necrosis.Mild condition- during a short window of tiime, you can prevent caspase 3 activation if you remove the insult.
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Caspase structure and regulation:30 kD protein. Have prodomain, large, small domain. Removal prodomain results in release of large and small fragments. Complex forms active caspase. Must be processing at 2 sites for activation. Need amplifying signal to complete.
Caspases differ according to structure of domains. Variations affect binding. Any caspase with CARD domain does not act alone.Must recruit proteins to get activated.
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List of caspases having CARD required for protein-protein interactions. Sequence is well-conserved.
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DFMO inhibits enzyme to synthesize polyamines and inhibits apoptosis.
Add put, restore function.
With TNF alpha, which increases apoptosis in dose-dependent manner, active form appears of caspase3. Add compound back- get active Caspase 3. Western blot, but you could use ELISA to see it as well.
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Caspase regulation: Cytochrome C released from mitochondrion as result of insult. It is released into cytoplasm. Binds APAF 1 and forms complex to recruit caspase 9. More recruited, more formed. Caspase 3 and all executor caspases need other caspase upstream in hierarchy.
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Process of apoptosis is regulated like rheostat. Upregulation and downregulation.
Inhibitors can bind APAF complex or bind caspases directly.
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Role of mitochondrion. Signal from outside can lead to release cytochrome C from periplasmic space. Extensive damage from insult- cytochrome C. formation of apopotosome complex requires ATP to enhance formation. Can be inhibited by IAP- inhibitor of apoptosis protein. AIF directly causes DNA fragmentation. Caspase independent.
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Mitochondrion membrane permeability transition pore complex (PTP) regulated by several proteins. Bcl2 1st identified from B cell lymphoma cancers. Bcl-2 was anti-apoptoic. Accumulation of cells by increasing number or preventing decreasing number can cause cancer. There are different forms in different cell types. But function is same.Bcl-2 can form homodimer. Can form heterodimer. Bcl-2-Bax is balanced. Bax-Bax allows formation PTP pore. In cell where Bcl-2 expression is high, it inhibits Bax
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Bcl-2 can regulate severity of any of these proteins.
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Mcl is Bcl-2. All antiapoptosis proteins have all 4 homology domains. Pro-apoptosis lack one or more domains.
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Transmembrane protein is required to insert protein in membrane. It has a membrane localization signal.
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How are these proteins regulated?
Bid cleaved to t-Bid which can enter mitochondrial membrane. Bad is present in cytosol sequestered by other proteins like 14-3-3. When 14-3-3 is phosphorylated, releases Bad and Bad can translocate. Also phosphorylated for inactivation .. Regulated by signalling mechanism.Proteins subjected to additional level regulation. Amplification of signal from mitochondrion increases activation of caspase 3,6,7.
Process is complex.
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Tip of villus, cells slough off and apoptosis. Nobody knows which comes first.
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In animal experiment, Irradiated animal had increased caspase 3. Inhibition does not completely block caspase. Can reduce severity of response. Radiation increases Bax. DFMO inhibits ornithine carboxylase. Activates anti-apoptotic singnalling pathways.
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Survival induction
Erk can phosphorylate Bad.Akt can also phosphorylate Bad. Under normal circumstances growth factors cause prolifreraton through upregulation of expression of genes required for cell proliferation. Induce stress- activates Jnk kinase. Jun kinase is apoptotic or anti-apoptotic. Must determine which way it goes.With TNF-alpha- gets phosphorylated. Compound to inhibit Kinase, Establish JNK can be blocked by inhibition or stimulated by TNF alpha.
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Saw caspase 9 activation. Sig indicating mit damage. Assessed mit damage by mitosensor dye. Monomer fl green, multimer red. In mit fl red b/c it aggregates . Green indicates apoptosis.JNk inh decreased mit damage. JNk is pro-apoptosis in the cells illustrated.
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more evidence of above. Caspase 3 same pattern b/c is downsream of caspase 9.
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Okadaic acid is inhibiting protein phosphatase. PP2a inhibited. Prevents dephosphorylation. Accumulation of phosphorylated proteins results. Fostreicin does same thing. Inhibiting phosphatases increases phosphorylated protein, or survival proteins.Ser-Thr phosphatases amplifies signalling in apoptosis process
PP2a regulates Jnk and a wide variety of other proteins.
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What pathway leads to protection of cells- found increased ERK activity. ERK involved in proliferation. These cells were serum starved, so they would not transfer into proliferation. Increased Bad dephosphorylation increases ERK. Phosphroylation of BAD increases apoptosis resistance.
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Bcl-2 practical example with rat tumors. P53 knocked out- got some tumor resistance to cell death, so p53 was necessary for apoptosis. All animals treated with cyclophosphamide. With p53 mutation, chemoresistance resulted.
High level Bcl-2, chemoresistance and animals did not respond. P53 mutant had reduced response to chemotherapy.
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Bcl-2 is not part of apoptosis machine but part of mitochondrial homeostasis and membrane maintenance.
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p53 was characterized a long time ago as tumor suppressor protein. S to G1-p53 goes up. It is a transcription Factor that can also bind to apoptosis proteins.Induced by list on left. Can lead to apoptosis or cell cycle arrest depending on context. Transcibes p21, which negatively regulates CDK activity. Prevents phosphorylation CDK2 to block cell cycle progression.
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IAPs
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Bir1,2,3 bacculovirus repeats. Conserved domains bind caspases to prevent access of substrate. Activity is blocked by protein. Upstream process is fine, but do not see effect of caspase 3.
These are from different organismss or different compartments. All do same job.
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All growth factors activate pathways to phosphorylate Bad. Bad has 7-Ser phosphorylation site to be phosphor¥lated by different kinases.
Ser, Thr, Tyr can be switches to adapt a protein for a different job.
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AKT activates nfkappaB. When there is a growth factor signal, converges to nucleus to increase transcription to put cells in proliferative or survival mode.
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Everything is linked in chain.
RAC1 is part of GTPase and NADPH complex in mitochondrion for respiration. Activates or inactivates causing changes in redox state of th mitochondrion, which is also involved in apoptosis.
NFkB negatively regulates apoptosis by leading to increased transcription. IAP proteins to block process. Does not totally block- changes the balance toward survival.
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Jnk is rate modulator, not direct effector. Depends on conditions in model.
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Death receptor showing another pathway.
Read the articles.
NFkB inc transcription Mcl1. Has NLS sequence. STAT3 also translocates to nucleus, binds promoter region, activates transcription. Other cytokines activate transcription other genes.