1. Name two first messengers that bind intracellular receptors (8 points).
2. A new bacterial disease is spreading unchecked in several parts of the world. Since you are now working as a researcher at the CDC, you are dispatched to the epicenter of the disease to understand how this bacterium causes severe gastrointestinal symptoms, such as diarrhea. You suspect that there’s a toxin secreted by the bacteria that is subverting a normal signaling pathway in the intestinal cells causing disease. You isolate the toxin and incubate increasing amount of the toxin with intestinal cells. You measure the amount of cyclic AMP (cAMP) produced by the cells (open circles in the graph below). Remembering this class, you suspect that a GTPase is involved in this process and measure cAMP production by the toxin after the addition of GTP (filled circles) or a non-hydrolysable version of GTP (Gpp(NH)p, filled triangles). You see the results as shown in the graph above.
a. Based on these data, which signaling pathway does the toxin subvert? Please mention specific proteins in the signaling pathway and how this pathway results in cAMP production. (6 points)
b. Why does GTP increase cAMP production? (6 points)
c. Why do you think Gpp(NH)p lead to a substantially greater production of cAMP? (6 points)
3. As a researcher studying actin function in mammalian cells, you discover a new actin binding protein that you name Dobby. To understand its function, you decide to use an in vitro actin polymerization assay. In the presence of Dobby, you get the following result:
a. From these data, what would be your hypothesis on mechanism of action of Dobby (5 points)?
b. Explain why the data support your hypothesis regarding the function of Dobby. Be sure to explain what you would expect the curve to look like in the absence of Dobby (10 points).
4. Researchers interested in understanding the signaling pathway for a growth factor worked engineered a FRET reporter for Ras activity. This FRET reporter consisted of a fusion protein (called the reporter) where Ras was fused to Raf (MAP kinase kinase kinase), each flanked by Yellow Fluorescence protein (YFP) and Cyan fluorescence protein (CFP) as shown in the schematic below:
The researchers measured the FRET emission ratio (YFP emission at 527nm/CFP emission at 475nm) of the reporter. They measured the ratio after addition of the growth factor to cells expressing the reporter by itself or the reporter together with the effector proteins (p21 or NS4a). Based on the data below, what is the function of p21 and NS4a in this growth factor pathway? Explain why the data supports your conclusion (16 points)
5. A rare type of hereditary blindness is often caused by mutations in genes expressed in rod cells. Scientists identified a family that had several members who suffered from blindness. To understand where the mutation could be, the scientists measured cGMP levels in the blood of several family members, including those that did not suffer from blindness, along with healthy controls not from the family and got the data below:
a. Based on these data, what three genes expressed in Rod cells would you sequence to determine if they have a blindness-causing mutation (15 points)?
b. Explain why you think a mutation in the genes named in previous question would lead to blindness. Be sure to mention the effect of the mutation on protein function (18 points).
6. To understand Factor X Growth Factor (FxGF) signaling pathway, scientists studied the localization of the FxGF receptor (FxGFR) using immunofluorescence microscopy. Human cells shown below were exposed to FxGF for the times indicated and then stained with antibodies for FxGFR.
0’= 0 mins 30’= 30 mins 120’= 120 mins
Based on the data what is happening to the FxGFR? Please explain, in detail, why the data supports your hypothesis (15 points).
7. A key pathway in metazoan cells is the survival signal, which ensures that cells do not undergo apoptosis. This pathway requires the function of the serine/threonine kinase, Akt. In a lot of cancer cells, this pathway is mutated to ensure survival of cancerous cells even in the absence of the survival signal. Scientists suspected that Akt works by phosphorylating a protein known as BAD. Therefore, they studied how Akt phosphorylates BAD.
a. BAD has two potential serine residues that could be phosphorylated (S1 and S2). Researchers expressed wildtype BAD (WT) and mutants where the serine residues were changed to alanine either individually (S1-A, and S2-A) or both together (2S-A) in cells along with inactive Akt or constitutively active Akt (active Akt). Using a Western blot, they monitored the phosphorylation of BAD using an antibody that ONLY recognizes phosphor-serine residues on BAD. The cells expressing inactive or active Akt along with wildtype or BAD mutants were incubated with Growth Factor X and then lysates were loaded in each lane as shown below the blot. Based on this data, which serine on BAD is phosphorylated by Akt? Explain in detail how Growth Factor X leads to phosphorylation of BAD (8 points).
b. Researchers then measured cell death (apoptosis) of cells expressing normal Akt along with either wildtype BAD or mutant that were treated with Growth Factor X. Given these data, which serine needs to be phosphorylated by Akt for cell survival? Explain why the data supports your conclusion (6 points).
c. Given the data above and your knowledge of the Akt pathway, name two genes that tumor cells will mutate in this pathway. Explain the effect of these mutations on the Akt pathway and how this will lead to tumor survival (6 points).
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