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Lecture 33

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Biology (Sci)
BIOL 300
Siegfried Hekimi

th BIOL 300 November 26 2012 Lecture 33 Dr. Shock Scaffolds, as we said, have two main functions: pathway separation and speeding up pathways. We will continue to discuss the mating mechanism in yeast, mediated by 3 kinases binding to a scaffold protein • In this system is a positive regulator (Ste50) which further enhances the pathway’s activity, and a negative regulator which inhibits it • In this system, they test the “readout” of this pathway by GFP tagging the target gene of this pathway to directly measure the activity of the pathway • The end result of this pathway is a transcription factor binding to the promoter of this target gene, activating it This pathway is so well understood that scientists have engineered methods of making this pathway more efficient • Given that the positive regulator protein is cytoplasmic, they made the hypothesis that by localizing the activator to the location of the scaffold protein, they could make this pathway happen much more efficiently • Using a leucine zipper dimerization domain, they were able to create an artificial binding site on a modified scaffolding protein which was able to bind a target protein tag • They then attached this protein tag to Ste50 so that it would be able to bind to this scaffold protein, thus localizing it By having the positive regulator close by, we can see that the pathway will be active more than usual, resulting in a stronger GFP fluorescence reading • We can compare the grey bars in the graph that the unrecruited (cytoplasmic) Ste50 is less efficient, and produces a lower GFP reading than the recruited (scaffold-bound Ste50) has a higher GFP reading • On a timeline, we can see that the, while both of them represent a positive feedback mechanism (quick initial response to reach a max value), the wild-type (black) response is not a quick, and does not have as high a maximum output as the recruited (blue) Ste50 1 th BIOL 300 November 26 2012 Lecture 33 Dr. Shock They then further tinkered with this system by changing the types of leucine zippers they used as recruiters for Ste50, with different levels of affinity • As expected, the higher the affinity of the recruiter for Ste50, the higher the output (and the quicker the ascent to a max value) They also played with the promoter strength of the gene they want to express; if they express a lot of the positive regulator, they would get more of a response (and less Ste50 would produce a lower response) This shows that by fully understanding a pathway, the between we are able to play with the mechanism, and improve it • Yeast are useful organisms as they can be used to produce a variety of chemicals, and it’s useful to tinker these pathways to maximize output and modify it to your own purposes Termination of the EGFR signal can be applied in general to most RTKs • The receptor is synthesized in the ER, and goes through the Golgi to be sorted into the plasma membrane • Then, though bulk flow of endocytosis, they are by chance endocytosis from the PM where they can either be stored, degraded, or recycled back to the membrane • Receptor endocytosis (in particular degradation) is one of the cell’s way of downregulating a response in a given pathway Another pathway of termination is through phosphatases • Phosphatases, unlike their counterpart kinases, are not very well regulated in the cell, they tend to be constitutively on • They also have very little specificity compared to kinase, which is why there are much fewer of them • The phosphatases will eventually end up dephosphorylating all target proteins in the pathway, resulting in termination of the signal There are other mechanisms which are specific to certain pathways; one important one is a specific endocytosis mechanism for the active EFGR 2 th BIOL 300 November 26 2012 Lecture 33 Dr. Shock • Usually, bulk membrane flow will be responsible for random endocytosis of EGFR (inactive or active) • However, upon EGFR activation, an E3 ligase enzyme called Cbl will recognize and ubuitylate the cross-phosphorylated EGFR, resulting in its endocytosis; this is SPECIFIC to activated EGFRs only • In contrast to proteasomal degradation, a single ubquitin, or a different linkage of ubuquitins are sufficient to induce sorting to the internal membranes of multivesicular bodies • Single ubuiquitination is usually to lysosome mediated degradation while polyubquitination is usually for proteasomal degradation • This is usually done in the event of adaptation mechanisms in which you have a lot of ligand, so you downregulate the amount of receptors on the cell membrane to dampen the response Another interesting termination mechanism for EGFR signaling works through a secreted factor which inhibits the EGF ligand • Say in this diagram that the orange cell make EGF; through paracrine signaling, the green cell would receive very high levels of EGF • The green cells would then activate and secrete an inhibitory factor; this inhibitor will then bind and inactivate EGF in the extracellular space and prevent it from binding other receptors • This is a mechanism of paracrine signaling which limits the range of signaling • Naturally, through diffusion, there will be a gradient of ligand, in this case EGF, as you move away from the secreting cell • The inhibitory factors act to limit the range of signaling by making this gradient steeper (i.e. the concentration drops faster); therefore, fewer cell rows beyond the secreting cell will actually receive the active ligand • Without this inhibitory signal, the paracrine signal would just act over a larger range 3 th BIOL 300 November 26 2012 Lecture 33 Dr. Shock Many EGF receptors, when over activated, cause cancer (the examples are not important to remember) • We see here instances of lung and brain cancer; whenever the mutations are sequences, we see that Ras and other components of the EGF pathway are almost always found Hereceptin is a drug developed from cancer research of the EGFR pathway which induces ERBB2 endocytosis (which is the mouse analog to EGFR) • This, in addition to other drugs which inhibit mitosis, work in about 30% of cases Most cells in our body can fall within to classes: • Epithelial, a lining of cells all adhered to a surface, called a
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