TEST 3 I Apoptosis Topic 20 a Apoptosis Programmed cell death distinct from necrosis i Necrosis occurs as a result of tissue damage or cell lysis which exposes the extracellular medium to toxic conditions lysosomal enzymes in particular results in peripheral damage and inflammation as a result of the immune system ii Apoptosis 1 A mechanism by which the cell systematically kills itself in a controlled fashion 2 as to avoid the effects of nercrosis that would result otherwise The cell when signaled to go through apoptosis sections off into small vesicles surrounded by plasma membrane that are than taken up by phagocytes via phagocytosis where they are subjected to lysosomes b Apoptosis during normal development normal development i Occurs constantly in the body to get rid of cells that have gotten old or damaged part of ii The mechanism for controlling multicellular development roles in pattern formation differentiation morphogenesis and motility iii There are far more neurons that are produced in the brain during development that do not make contact or synapse anywhere it is critical for normal development that these neural cells die off or else normal development cannot occur apoptosis and phagocytosis must work without hindrance at this crucial stage c Trophic factors ii Results i Chicken embryo experiment removal of a developing limb of one embryo and placement of removed limb on another embryo 1 Under normal circumstances where the limb bud development went 2 3 unperturbed there were a number of neurons present during the motor neuron generation stage but about half after motor neuron apoptosis a normal attrition of 50 of the motor neurons If the leg tissue is removed only 10 of the motor neurons survive If the leg tissue is reattached to another normal developing embryo 75 of the motor neurons survive there is something about this tissue that is inhibiting apoptosis the effect is a result of the signaling processes occurring during development that promote the survival of more neurons iii Further investigation A series of factors extracellular signals released by signaling processes is what was causing stimulated neural activity Trophic factors nerve growth factors d Cell survival and apoptosis i There are a number of pathways that control this mechanism of apoptosis our focus is on the pathway that gets deactivated as a result of trophic factors the trophic factor is always INHIBITING apoptosis when it is present its absence is what signals apoptosis ii Presence of trophic factor inhibition of apoptosis 1 2 3 4 1 2 The receptor for the trophic factor is a single transmembrane domain protein that is analogous to the tyrosine receptor kinase system The presence of the trophic factor activates PI 3 Kinase which activates protein kinase B PKB PKB targets BAD BAD when phosphorylated is inactive by binding to 14 3 3 inhibitor protein This results in the inhibition of apoptosis In the absence of a trophic factor PI 3 Kinase does not activate PKB which does not phosphorylate BAD This results in BAD being uninhibited unphosphorylated BAD is what signals the cell suicide pathway iii Absence of trophic factor 3 Active BAD binds to a protein BCL 2 BCL 2 is an anti apoptosis protein that blocks the downstream activity of BAD when active 4 Active BAD blocks the inhibitory activity of the BCL 2 which allows a protein BAX to become active 5 Active BAX forms a channel in the outer mitochondrial membrane that allows for the release of cytochrome C CytC from the intermembrane space CytC is part of the electron transport chain for the generation of ATP 6 CytC becomes a signaling molecule in the cytoplasm it interacts with a protein called Apaf 1 and activates its activity 7 Apaf 1 is a protein that when activated starts to form a very large complex of 8 subunits Apaf 1 is a dimer four of these dimers come together to make a 8 subunit complex that binds 8 CytC molecules this complex is called an apoptosome the Apaf 1 complex recruits a class of proteins called caspases 8 A caspase is a protease it has a very targeted activity that turns on an inactive protein by clipping off an inhibitor domain There are many proteins that are created and maintained in an 9 inhibited form Proforms until needed active caspases the result of a caspase binding to an apoptosome can cleave off residues from the C terminal end of prorform proteins to activate them 10 This process is what occurs when the apoptosome binds procaspase in this case procaspase 9 which self activates it via proteolyzing its C terminal to aspartate residues cuts a very specific bond forming active caspase 9 11 The target of caspase 9 is another procaspase procaspase 3 it activates procaspase 3 to caspase 3 which is a protease a protease cascade that involves amplification of the signal 12 Caspase 3 clips a lot of different substrates that results in the deformation of the cell s structural integrity cleavage of things in the cytoskeleton filaments and the nuclear lamina degradation of the superstructure also activates other proteins that aid in the managed destruction of the cell by creating tiny vesicles surrounded by plasma membranes that can be phagocytosed II Protein Synthesis and Targeting RNA transcription and translation a Eukaryotic gene structure i Entails a protein coding region and a control region ii The control region is where regulatory proteins transcription factors bind to promote the recruitment of RNA polymerase and induce transcription of the region of DNA the control region mediates iii The protein coding region is composed of DNA divided into two different categories of information 1 2 Exons the bases that code for functional proteins Introns do not encode a protein but very important in terms of what the final protein looks like b c Transcription via RNA polymerase yields primary mRNA transcript of gene composed of both exons and introns pre mRNA Pre mRNA gets processed before it is translated by ribosomes into a protein i Transcription into pre mRNA occurs in the nucleus of the cell where the DNA is located ii Addition of 5 cap to pre mRNA iii Addition of poly A tail to 3 end of pre mRNA via enzymatic activity important for stability of mRNA and how efficiently the mRNA is translated in the nucleus iv Removal of introns and splicing together the ends of the exons complexes carry out this splicing activity can be done differentially off a single type of mRNA sequence from a single gene yielding inherently different