Passage 3 from Blueprint MCAT (formerly Next Step Test Prep) full-length 10 covers an interesting discussion on unfolded protein response and endoplasmic reticulum homeostasis. We’re joined by Clara from Blueprint MCAT (formerly Next Step Test Prep) every week. Please also take the time to listen to all our other podcasts on MedEd Media Network.
[01:45] Back With Some More Biochem Work
Our passage today is a good example that looks really dense. It strikes fear into the hearts of some premeds. But Clara says we don’t need a whole lot of background information here. Here, we’d be talking about a lot of processes and pathways so we need to just pay attention to use the passage when we tackle the questions. Today, we talk about protein cascades and secretory protein processing.
[02:45] Passage 3
Changes in the extracellular environment of a cell can adversely affect the homeostasis of the endoplasmic reticulum (ER) disrupting the folding and processing of secretory pathway proteins. The resulting accumulation of unfolded proteins in the ER increases the demand from molecular chaperones and folding enzymes and activates the signal transduction cascade known as the unfolded protein response (UPR). This signal transduction pathway is largely cytoprotective, serving the decrease to detrimental effects of accumulated unfolded proteins by decreasing overall protein synthesis and increasing the capacity of the cell to eliminate unfolded proteins.
In mammalian cells, the UPR is controlled in part by IRE1, a protein that sense ER stress and activates signals to downstream elements. IRE1 is an ER transmembrane protein that has a kinase and endoribonuclease domain in its cytosolic tail. When the ER is stressed, IRE1 is phosphorylated which in turn activates its endonuclease domain leading to the excision of 26 bases from the X-box binding protein, XB1 transcript. The resulting frameshift encodes the transcription factor that upregulates the expression of proteins that contribute to the folding or degradation of unfolded or misfolded proteins.
Clara’s Notes: Highlight as you go through the passage so this doesn’t just look like a wall of text. But we see a lot of familiar stuff.
Angiogenesis refers to the sprouting, migration, and remodeling of blood vessels and plays an important role in both normal and pathological physiological processes. In cancer cells, proangiogenic factors such as vascular endothelial growth factor or VEGFA, angiogenin, and interleukin-8 (IL-8) are released in response to decreased oxygen in nutrient supplies. A cellular stress pathway turned the hypoxia-inducible factor (HIF) pathway, has been shown to promote the upregulation of proangiogenic factors in response to inadequate oxygen delivery. Recently, UPR activity has also been associated with abnormal levels of these factors. Researchers wish to assess the relationship between the UPR and the induction of proangiogenic factors. Cells from a human medulla blastoma cell line were treated with three known UPR inducers – tunicamycin, thapsigargin, and no glucose. And with two different inducers of the HIF pathway CoCl2 and 1% oxygen for 24 hours and the induction of VEGFA, angiogenin, and IL-8 was measured.
[06:00] Question 10
The experimenters concluded that UPR activation significantly increases the level of VEGFA, angiogenin, and IL-8 transcription. What additional finding would most undermine this conclusion?
- (A) Angiogenin transcript levels were increased significantly more by hypoxic conditions than by treatment with thapsigargin or no glucose media.
- (B) Both tunicamycin and thapsigargin treatment resulted in significantly higher levels of IL-8 than exposure to no glucose conditions.
- (C) Cells treated with tunicamycin, thapsigargin, and no glucose conditions were found to contain lower levels of unfolded proteins than untreated cells.
- (D) In addition to being potent UPR inducers, tunicamycin, thapsigargin, and no glucose conditions also act to induce the HIF pathway.
Basically, what they’re saying is that the experimenters came to this conclusion, but what additional finding would most undermine this conclusion. So which of these answer choices, if we found this out in addition to what we know now, will undermine what the first sentence of the question says.
We found out that UPR activation decreases these things. Alternatively, we also found out that the increase is not caused by UPR activation.
If this question seems too much for you, you can flag the question and then ten minutes left to the end of the exam, you can just come back to it. So if you think this question could take you too long then save your time for the easy points.
To help you understand the answer to this question, there are a couple of keywords that can help you. So what we’re trying to find out here which of these will undermine this idea that UPR activation increased these things. And we’re trying to prove that it’s not true essentially.
A is talking about the difference between hypoxic conditions, thapsigargin, and no glucose media. Looking at the last paragraph of the passage, you would then know that hypoxic means low oxygen. So hypoxic conditions increase angiogenin more than thapsigargin or no glucose. But this sort of compare two unlike things since hypoxic conditions were parted as HIF pathway. And thapsigargin is part of this UPR pathway. So there are two different pathways and we have to figure out how they relate to each other.
Now, second to the last paragraph, you see that the HIF pathway has been shown to upregulate proangiogenic factors. And all these factors we’re talking about are proangiogenic factors like VGFA and angiogenin. So essentially, what it’s saying is that the UPR is what we’re actually studying in this passage, the unfolded protein pathway. But the HIF pathway is another pathway that also does the same thing as what we suspect the UPR pathway does. So let’s just leave choice A.
[13:20] Dissecting Answer Choice B and C
B is talking about tunicamycin and thapsigargin treatment resulted in significantly higher levels of IL-8 than exposure to no glucose conditions. And this looks similar to A since it’s saying these are the new treatments used are causing higher levels than this other treatment. So leave this choice as well.
C says that cells treated with tunicamycin, thapsigargin, and no glucose conditions were found to contain lower levels of unfolded proteins than untreated cells. The UPR is the unfolded protein response, which is meant to save ourselves from unfolded proteins. So if cells treated with all these things that are part of the UPR, we found they contain lower levels of unfolded proteins, that makes total sense. But that doesn’t undermine the conclusion of the question stem.
[14:25] Dissecting Choice D
This is where a phrase should stand out to you right away, which is “in addition to” and the “also act to induce.” They’re saying: In addition to being potent UPR inducers, tunicamycin, thapsigargin, and no glucose conditions also act to induce the HIF pathway.
As soon as you see these things we’re using in this experiment and treatments also do something else essentially that is related to this pathway, that is a confounding factor. So if tunicamycin and thapsigargin also act as part of the HIF pathway, maybe it’s just the HIF pathway that’s doing everything. Maybe it’s just the pathway that’s increasing the level of these angiogenic factors. And the UPR factor doesn’t have anything to do with it. There’s no way now for us to distinguish between UPR and HIF because the treatment used was badly chosen. It does both things at once.
[16:53] Question 12
Which of the following statements about IRE-1 is least supported by passage information?
- (A) If IRE-1 promoted the excision of one additional consecutive base from XBP1, the transcription factor typically produced as the result of this excision would not be encoded.
- (B) A regional cytosolic tail of IRE-1 catalyzes the cleavage of bases one at a time from the end of the XBP-1 transcript.
- (C) Phosphorylation of IRE-1 results in posttranscriptional modification of XBP-1.
- (D) The endoribonuclease domain of IRE-1 is more likely to be rich in KND residues than in LNF residues.
Remember this is a “least” question so you always have to understand the question first. Posttranscriptional just means that it happens after the act of transcription. So essentially when transcription happens, that’s what makes the transcript. And any changes we make to the transcript after that are post-transcriptional because it happened after transcription.
Actually, C is supported by the passage information so we can get rid of this.
D mentions amino acids. Even if the passage doesn’t mention amino acids, they could still come up because one key factor you’re supposed to know is some amino acids are polar and others are nonpolar. The polar ones tend to be found on the outside of proteins, facing the cytosol. And the nonpolar ones tend to be found buried in the inside of the proteins. And if we look at D, it’s talking about the endoribonuclease domain of the protein which is in its cytosolic tail. In that case, it’s in the cytosol and cytosol is polar and is likely to have a lot of polar amino acids. Then we have to know that KND are polar and LNF are nonpolar amino acids. So D is saying that this polar domain is more likely to be rich in these two polar amino acids than these nonpolar ones. And so this is true.
For the MCAT, just know the amino acids, whether they’re polar and nonpolar, and what their abbreviations are. These are information you need to know as early as possible in your prep. And everything else is just piled on top of that.
The right answer here is B as it talks about the cytosolic tail of IRE-1 catalyzing the cleavage of bases one at a time from the end of the XBP-1 transcript. And if you go back to the second paragraph, they talk about being an endonuclease, which doesn’t cleave from the end of the transcript, but somewhere in the middle. So it’s exonuclease.
[26:00] Question 13
The unfolded proteins that accumulate in response to ER homeostatic disruption most likely:
- (A) Exhibit disrupted tertiary structure due to a lack of proper covalent bond formation.
- (B) Exhibit disrupted secondary structure due to a lack of proper disulfide bond formation.
- (C) Exhibit disrupted secondary structure due to a lack of proper hydrophobic interactions.
- (D) Exhibit disrupted primary structure due to a lack of proper peptide and bond formations.
D is tempting since we know proteins have peptide bonds. But peptide bonds are not involved in folding, but only in the primary structure of protein which is the linear sequence of amino acids. So get rid of D.
Tertiary structures talk about high level protein folding but secondary structure in Choices B and C, are talking about protein folding too. So at this point, we need to use this type of bond to find our answer.
Starting with C, proper hydrophobic interactions are involved in folding but they’re involved in tertiary structure which is that large scale folding. They’re actually not involved in secondary structure. Secondary structure is really basic repetitive folding. And it involves hydrogen bonds only. So B and C are out because they don’t talk about hydrogen bonds.
Hence, the correct answer here is A since one of the key components of tertiary structure are disulfide bonds which are a type of covalent bond. So secondary structure means folding and hydrogen bonding is something you need to remember!
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