Short Answer Questions MCB_3010

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Feb 20, 2024

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Quiz #1 Notes 1. A single amino acid change in RAS eliminates its ability to hydrolyze GTP, even in the presence of a GTPase-activating protein (GAP). Roughly 30% of human cancers have this change in RAS. You have just identified a small molecule that prevents the dimerization of a receptor tyrosine kinase that signals via RAS. Would you expect this molecule to be effective in the treatment of cancers that express this common, mutant form of RAS? Why or why not? Only one of these two outcomes is possible This treatment should be effective in this type of cancer. Ras being unable to hydrolyze its GTP means that once it is activated via RTK signalling, it remains active and its downstream pathways and genes will be consecutively active as well. If RTK is unable to dimerize, then Ras will never activate in the first place, meaning the downstream pathways are usually involved in growth, this should prevent cancer cells from growing. 2. A ligand binds to the extracellular domain of its target receptor tyrosine kinase (RTK) which promotes proliferation. What are the two processes that must occur for the signal to be transmitted to the other side of the plasma membrane? (ie. What leads to the formation of the phosphor-tyrosine residues) 1. Receptor dimerization 2. Trans-auto phosphorylation High-affinity docking sites (phosphor-tyrosine residues) for intracellular signalling proteins are created on the intracellular domain of the RTK. What type of domain must these intracellular proteins contain to dock at an activated RTK? SH2 domain Not all intracellular proteins that bind to the high-affinity docking sites of RTKs act to relay the signal onward. The c-Cbl protein causes receptor recycling by targeting the RTK to lysosomes. What post-translational modification does c-Cbl perform on the RTK to signal its targeting to lysosomes? Monoubiquitylation promotes endocytosis and degradation of RTKs by targeting clathrin-coated vesicles and, ultimately, lysosomes. What would you expect to happen to a person who acquired an inactivating mutation in c-Cbl?

Mutations that inactivate c-Cbl-dependant RTK down-regulation cause prolonged RTK signaling leading to cancer. 3. *Lateral inhibition is critical for maintaining proliferative progenitor populations in many tissues. This involves cross-talk between the Notch and WNT signalling pathways. Describe how activation of the notch-delta pathway leads to lateral inhibition in the development of neural cells in drosophila Notch is processed/cleaved at the Golgi, cleavage event causes notch to be inserted into the plasma membrane. It binds delta from neighbouring cells which causes the release of the extracellular domain and it gets taken up into delta expression cell. On the cell originally expressing notch 2 more cleavage events happen which release the cytoplasmic tail, translocated to the nucleus which acts as a transcription factor. It will activate the transcription of genes that will ensure the cells remain undifferentiated. 4. Describe how a mutation in the APC subunit of the GSK3 Beta complex can lead to colon cancer. Begin with describing the normal signalling pathway and then how APC mutation impacts its activity. (hint this is the WNT pathway) When Wnt is present, it binds to a frizzled receptor which recruits LRP and then we get a receptor complex at the surface that will activate Dischevelled. Dishevelled will then dissociate the APC complex by pulling Axin away. CK1 and GSK3 phosphorylate LRP. Beta cetenin goes to the nucleus and displaces Groucho, allowing transcription of Wnt genes. Any mutation in the APC complex would result in this pathway always being on and leading to cancer. 5. Insulin binds to its RTK activating the PI3-K/AKT pathway to promote growth. Explain how the activation of the RTK leads to the activation of AKT and how AKT promotes cell growth . The binding of insulin to RTK causes dimerization leading to the formation of phospho-tyrosine redisues. Then an adaptor protein that contains PI3-K which has a kinase domain that will phosphorylate PI generating PIP3 will act as a docking site for other proteins. PDK1 and Akt get recruited to the PM and bind to PIP3 sites, PDK1 will phosphorylate and activate Akt, then mTOR will further phosphorylate Akt at another site and will prompt Akt to dissociate from the PIP3 docking site. Akt will then phosphorylate Bad and release an apoptosis inhibitor that will inhibit apoptosis and promote cell growth. 6. What are the 4 mechanisms through which i–Smads negatively regulate the Smad pathway? a) Competing with Smads for binding sites on the receptor b) Recruiting the ubiqutin ligase Smurf, leading to receptor degradation c) Binding to co-Smad, Smad4, inhibiting it

d) Recruiting protein phosphatases, leading to receptor inactivation Short Answer Questions #2 1. The critical concentration of actin in a test tube is less than 1 μM. However, the concentration of actin in cells is 50 100 times greater than the critical concentration observed for pure actin in a test tube How is this possible? Why isn’t actin always polymerizing in cells? Specialized proteins such as thymosin lock up actin, allowing it to remain soluble without polymerizing. Why is this advantageous? It is advantageous because it allows for fast responses since it’ll always be over the critical concentration. 2. Dynamic instability causes microtubules either to grow or to shrink rapidly. Consider an individual microtubule that is in its shrinking phase. What is this shrinking phase called? Catastrophe What must happen at the end of a microtubule in order for it to stop shrinking and start growing? GTP-loaded subunits are added quickly enough to cover up the GDP-containing tubulin subunits at the microtubule end, a new GTP cap can form and regrowth is favored. How would an increase in the tubulin concentration affect this switch from shrinking to growing? The rate of addition of GTP-tubulin will increase as tubulin concentration increases. Therefore, the shrinking microtubules will start to grow with an increase in tubulin concentration. What would happen if GDP, but no GTP, were present in the solution? Microtubules would continue to shrink and eventually disappear. 3. In the polymerization of in vitro actin filaments from their subunits, What does the “lag phase” correspond to? The lag phase corresponds to nucleation.

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How do you eliminate the lag phase? If we add oligomers at the beginning, you get the same rate of growth but we reach the critical concentration in less time, so if you start with oligomers you can build a fibril faster, you bypass the nucleation step. Your actin filaments have an elongation rate shown as a dashed line. Which one of the other five lines (A to E) would you think better shows what happens when a plus-end capping protein such as CapZ is present? Explain. CapZ is responsible for binding and stabilizing the plus end of actin. Line A is the best example. Name a protein that you could add to your actin filaments to aid in their disassembly. How does it function? Cofilin destabilizes actin filaments by binding and forcing the filament to twist more tightly. 4. *A muscle contraction relies on calcium released from 2 sources, explain how a muscle contraction occurs, start with an action potential that triggers the small influx of calcium and explain how this results in sarcomere shortening. (be sure to explain how Calcium facilitates myosin binding to the actin filament and the resulting contraction, do not go into details about the structure and phosphorylation of myosin Action potential triggers the opening of voltage-gated ion channels on the plasma membrane allowing a small amount of calcium into the cell. This small amount of calcium binds to calcium release channels on the sarcoplasmic reticulum causing a massive release of calcium. This calcium binds to troponin c which allows for the dissociation of tropomyosin from the actin filament revealing the myosin binding sites.

ADP and Pi remain bound to myosin, forming a weak bond with the actin filament. That causes a release of Pi which leads to a tight binding to the actin filament. Which then leads to myosin being tightly bound to actin in rigour. 5. The picture below shows a kinesin molecule binding to an MT Label the leading and lagging heads, and indicate with an arrow which way the kinesin travelling. The lagging head is next to the ATP. Kinesin is in the middle. The leading head is under ADP. Explain how the kinesin protein walks along the microtubule through ATP hydrolysis. The lagging head is tightly bound to MT and ADP, meanwhile, the leading head is loosely bound to MT and ADP. ATP in the lagging head is hydrolyzed to ADP + Pi, which drives the rear head displaced. ATP replaces ADP on the front head and Pi is released causing a change from rear pointing to forward pointing by shifting the neck. This pulls the rear head forward where it binds MTs and completes the step. If the cargo needed to be moved towards the minus end of the microtubule which motor protein would be used? Dynein MC Questions (Might be in Quiz): From Review Lecture: 1. Which of the following proteins lacks a PH domain (bind phospholipids)? a) Sos (a Ras-GEF) b) Grb2 (an RTK signaling adaptor) c) Pleckstrin (a protein kinase C substrate) d) Akt (protein kinase B) e) PDK1 (phosphoinositide-dependant kinase) 2. PI 3-kinase a) Is a membrane-associated tyrosine kinase. b) Activates PDK1 by phosphorylating a serine residue on the protein

c) Is counteracted by PTEN phosphatase d) It is only known to be activated by receptor kinases e) Inhibits Akt 3. The actin-nucleating protein formin has flexible “whiskers” containing binding sites that help recruit actin subunits to enhance polymerization by this protein. What protein would you expect to bind to these sites? a) Thymosin b) Profilin c) Cofilin d) Gelsolin e) Tropomodulin 4. Regulation of the Ci (Cubitus interruptus) protein in the Hedgehog signalling pathway is reminiscent of that of Beta-catenin in the Wnt pathway. Which of the following features is shared among these two proteins? a) Both proteins are ubiquitylated when the signal (Hedgehog or Wnt) is present b) Both proteins can enter the nucleus only in the absence of the signal c) Both proteins can be phosphorylated by GSK3 and CK1 in the absence of the signal d) None of the above From Notes: 5. Signaling between cells usually results in the activation of protein _______. A) Lipases B) Kinases C) Proteases D) Nucleases 6. What does a SH2 domain bind to? A) Phosphorylated serines B) Complementary SH2 domains on other proteins C) Unphosphorylated transmembrane receptors D) Phosphorylated tyrosines 7. For the receptor tyrosine kinase (RTK)-Ras-MAP kinase pathway important for Drosophila eye development, what would be a direct functional consequence of mutating the SH3 domain of Drk? A) Drk would no longer bind and recruit Ras-GEF. B) Drk would no longer bind to phosphorylated tyrosine residues on activated

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receptor tyrosine kinases. C) The receptor tyrosine kinases would no longer be phosphorylated upon binding to signaling molecules. D) The receptor tyrosine kinases would no longer dimerize upon binding to signaling molecules. E) The receptor tyrosine kinases would no longer bind to signaling molecules 8. Mutations in the Apc gene occur in 80% of human colon cancers. Normal APC increases the affinity of the degradation complex for β-catenin, which in excess can enter the nucleus and promote transcription of key target genes for cell proliferation. Given this information, which category – oncogene or tumor suppressor – would you expect the Apc gene to belong to? Tumour suppressor 9. Which of the following mechanisms is NOT employed by I-Smads to negatively regulate Smad signaling? A. Competing with Smads for binding sites on the receptor B. Recruiting the ubiquitin ligase Smurf, leading to receptor degradation. C. Binding to nuclear pores, blocking Smads from entering. D. Binding to the co-Smad, Smad4, inhibiting it. E. Recruiting protein phosphatases, leading to receptor inactivation 10. The role of ATP hydrolysis in actin polymerization is similar to the role of GTP hydrolysis in tubulin polymerization: both serve to weaken the bonds in the polymer and thereby promote depolymerization. True or False? True 11. The property of sudden conversion from growth to shrinkage, of a microtubule or an actin filament is known as what? Catastrophe 12. The drugs taxol and nocodazole have opposite effects. Taxol binds tightly to microtubules and stabilizes them. In contrast, nocodazole prevents microtubule formation. Both are toxic and used as anticancer drugs. How can drugs with opposite effects on microtubule formation both be toxic? - If it is too stable it cannot depolymerize - If not stable enough it cannot form structures

- If you cannot depolymerize and polymerize microtubules you cannot divide so anything that blocks this prevents cell proliferation 13. How does the centrosome “know” when it has found the center of the cell? Microtubules come out of the centrosome in all directions and they can extend to the edge, so it finds the middle based on that. 14. How are the γ-tubulin ring complex and the ARP complex similar, and how are they different? Similarities: both bind to and stabilize the minus end of a cytoskeletal element and promote nucleation. Difference – ARP is associated with branched actin given it interacts with ‘older’ actin filaments at a 70-degree angle, gamma-tubulin builds microtubules which are not branched 15. How does cofilin distinguish between old and new actin filaments? Cofilin has a higher affinity for ADP actin, which is the old actin, compared to the new actin which is ATP-bound. 16. There are no known motor proteins that move on intermediate filaments. Suggest an explanation for this observation. Intermediate filaments are just for strength, they're not used for transporting anything. They also have no polarity, so there is no way of having something directionally travel down it. 17. Which one of the following changes takes place when a skeletal muscle contracts? A) Z discs move farther apart B) Actin filaments contract C) Myosin filaments move away from the disc D) Sarcomeres become shorter 18. True or False? Motor neurons trigger action potentials in muscle cell membranes that open voltage-sensitive Ca 2+ channels in T-tubules, allowing extracellular Ca 2+ to enter the cytosol, this Ca 2+ binds to troponin C, and initiates rapid muscle contraction. False

Short Answer Questions MCB_3010

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