Ramesh Ray 1

Ramesh Ray
Overview Apoptosis
Contact him for more info.His office at 521 Nash next to Dr. Rao.
Main aim of science is to increase life expectancy and quality of life.
During embryonic development life and death coexist. Cell death forms interdigital spaces of fingers.
Last decade apoptosis was discovered.
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Word “apoptosis” is “falling off” -natural process by which normal cell dies.Also called programmed cell death.
Adult human body- every day 10 billion cells made, so same number cells continuously eliminated.
Before 1972- most cell death seemed to be by necrosis. Cells die randomly, rupture, and contents of cell relesed. Apoptosis is a lot cleaner.
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2 processes contrasted. How to differentiate? Know these hallmarks. Apoptosis- all intracellular organelles intact. Cell forms blebs with small vesicles. Those vesicles are engulfed by neighboring or phagocytic cells. Necrosis is opposite. Cell ruptures and contents released.
All necrotic processes lead to inflammatory response. Apoptosis does not cause inflammatory response.
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See Lodish for diagram. SEM shows blebbing.
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TNF alpha
How to quantify cells dying of apoptosis? When you induce apoptosis in cell culture- See DNA fragments in gel. Nucleosomes- eukaryotic DNA wrapped around histones.Each element of Dna and histone is nucleosome. Specific endonucleases are activated to cut open area. Each histone has about 150 kbp. See multiples of 150 on gel. Ladder shows up because endonucleases are activated sequentially. ELISA done in top graph. Antibody against histone used. Can use quantitatively.TNF alpha induces apoptosis in many systems.Cyclohexamide is an antibiotic that is inhibiting protein synthesis by inhibiting ribosomes. Combination causes apoptosis. In presence of TNF alpha- cells synthesizes survivor proteins. The antibiotic keeps these proteins from being synthesized and promotes apoptosis in a dose-dependent way.
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Development of the immune system is dependent on proper apoptosis. Not-RA, diabetes, other autimmune disease.
Adulthood- fixed number neurons maintained by regulation of apoptosis.
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To understand, must have a model to study. C. Elegans had fixed number cells. Fixed number undergoes apoptosis at different stages of development. Bcl-2 prevents apoptosis in adult life.
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Knocking out certain developmental genes in c. elegans is not fatal.GFP tags can be used to follow proteins in a spatiotemporal way in these animals.
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We should be able to see relationships between model and human. The information from elegans translates in a conserved way so far.
CED means Cell death or c.elegans death genes. APAF- apoptosis activating factor. Caspases kill cell. There is also caspase independent-death. It is a Failsafe mechanism with more than one alternative pathway.
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If we know components, we can put together picture of how cellular machinery works
Adapters are same component in 2 different systems. Higher level organization, more complexity. Vertebrates have more components.
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2 mechanisms cell death. Threat can be from outside or inside. One is extrinsic other intrinsic. Threat outside- extrinsic pathway. Threat sensed by death receptors on membrane. Signal transmitted inside for response. Fas and other receptors recognize ligand. Receptors have multimeric components. Binding opens intracellular domains and induces binding of interior proteins. Apoptosis proteins not active until start signal given. Regulator binds receptor, adaptors bind,DD is death domain. Cell tries to correct damage or meet threat first. Halts cell cycle (cell cycle arrest) first. Then brings processes to basal level and assesses damage. Then tries to repair damage. If damage too much, apoptosis commitment starts. DISC involves lots of proteins.(5 or 6). TNF signal forms TNFADD. Death complex forms and activates Caspase 8, which activates caspase 3. Caspase 3 is commited step. Granzymes activate caspase 3 or cause caspase independentendent death.
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Intrinsic pathway
Internal threat includes damage to DNA. 1500 nucleotides added per second in replication. Most mistakes are repaired, but presence of certain chemicals or lack of cytokines in medium can cause damage or cell death. Caspase 1 originally known as interleukin converting enzyme (ICE).
Mitochondrion is important for respiration and for sensing internal and external trouble in cell. Low ATP in cell gives signal for apoptosis.
Deplete ATP-induce apoptosis.BCl-2 is a negative regulator of apoptosis, promoting cell survival. It regulates cytochrome-c. Cytochrome C is between inner and outer membrane. Released- out of place. Threat signal .Cytochrome c binds APAF-1 to form apoptosome. The apoptosome is made of a number of APAFs and recruits caspase 9 molecules. Other proteins also leak out from periplasmic space to bind inhibitors of apoptosis molecules to encourage apoptosis.
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Genotoxic damage can be caused by drugs.
Cytokine deprivation- withdraw IL-3. Initiates cell death signal.Checkpoint- decision is made.BCl-2 is anti-apoptosis. BAX is pro-apoptosis. Mitochondrion is on left. Once capsases activated, going toward commitment. Today about 250 known proteins are known targets for caspase cleavage.
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Pathway based on information from past 10 years. Slide illustrates classic TNF alpha induced apoptosis. TNF binds trimeric receptor, goes inside to TRADD or FADD. Those proteins bind inside to give signal that something is bound outside. Procaspase 8 binds to the bound FADD. Procaspase is like a zymogen or proenzyme. Activated by cleavage. Active fragment is caspase 8. When multiple molecules bind, one activates other and signal is amplified. Procaspase 3 forms caspases 3,6,7, which are executional caspases. Earlier ones are initiational caspases.
When caspase 8 activates, cleaves Bid (BCl family protein). Increases permeability of mitochondrion. Bad is pro-apoptosis protein. Pores form in the mitochondrial membrane to allow leakage of cytochrome C.Cytochrome-C with APAF activates caspase 9, caspase 3, apoptosis.
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Will stop here. Caspases cleave Cys after Asp in specific sequences. Cell chops up big proteins into small fragments into vesicles for neighboring cells to clear.