Question

You are culturing fibroblasts derived from human tissue. By using a genetic technique called CRISPR-Cas9, you knockout the gene in these cells that expresses the fumarase enzyme. To your surprise, the cells continue to respire normally. You discover that after knocking out fumarase, the cells start excreting large concentrations of the metabolite fumarate into the culture medium. The only carbon source in the culture medium that is available to feed into the TCA cycle is glucose.

a) If you enrich all of the glucose carbons in the media with 13C, do you expect to detect 13C labels in the excreted fumarate? If so, please draw the structure of fumarate and clearly indicate where the 13C carbons will be located. You may assume that the cells have been labeled with glucose for 4 days.

b) You learn that these cells require a large amount of aspartate to synthesize nucleotides when they are dividing. Yet, the knockout of fumarase does not greatly affect the growth rate of the cells. Notably, you find that there is no aspartate in the culture medium. Please provide a rationale for the observation that the cells continue to grow normally

c) For this part, all glucose carbons in the medium are labeled with 13C. Imagine that you isolate a molecule of aspartate from the cells. How many 13C-labels will it have?

d) You do two experiments for part D. First, you culture the cells in the same conditions as part C, but glucose carbon is labeled with 14C instead of 13C. Second, you culture the cells in 14C-labeled glucose (i.e., all six carbons are 14C) and add fatty acids (palmitate and oleate) to the culture medium. You then isolate aspartate from each sample and measure its radioactivity. Do you expect the radioactivity to be higher, lower, or the same in the first sample relative to the second sample?

Answer 1

A. The glucose is converted into pyruvate and subsequently passes through Tca cycle producing different metabolites. Due to mutation, the cells cannot convert fumarate into malate, so they release the excess fumarate into media. If the all the carbon atoms in glucose is labeled with C13, we can track its movement in different pathways. The labeled carbon will be found in ten excreted fumarate. Since the cells are labeled with glucose for 4 days, most of the carbon containing metabolises will have C13 label. The pyruvate will pass on to produce fumarate with 50% 13 label, but as the tcs continues, due to the mutation the oxaloacetic acid concentration decreases. Now it is directly produced by pyruvate by addition of unlabelled CO2. Since all the molecules of pyruvate are labels, 3/4 of oxaloacetate also get C13 labelling. It goes onto form new metabolites with 3/4 labeled C13. When fumarate is formed, it looses an unlabelled CO2 and thus becomes a 4 carbon molecule with all the carbon labeled with C13.

B. Aspartate is formed from oxaloacetate. Since oxaloacetate is being produced directly from pyruvate, there is no shortage of precursors for Aspartate. Hence cell DNA grow normally even in the absence of Aspartate in the media.

C. The oxaloacetate has 4 carbon atoms in which 3 are labelled. The Aspartate shares the same 4 carbon atoms, instead of O and NH2 group binds to the unlabelled carbon atom. Hence Aspartate has 3 out of 4 carbons labels with C13.

D. The first will have most of its carbon atoms radioactive, because it is grown in C14 labeled carbon. The second was supplemented with non labels fatty acids, which can also be used to form Aspartate via beta oxidation and tca cycle. Hence it will have less radiolabeld Aspartate compared to first one. So the first one will have higher radioactivity compared to second.