Identification of the Major Prostaglandin Glycerol Ester Hydrolase in Human Cancer Cells

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Zach Zimmerman, Spotlight Week 2

Paper:  Identification of the Major Prostaglandin Glycerol Ester Hydrolase in Human Cancer Cells

http://www.jbc.org/content/289/49/33741.full

Enzyme in a hay stack (if hay were hydrolytic…)

Endocannabinoids are lipid molecules implicated in a number of important physiological processes across many body systems. They are metabolized by several enzymes and converted into prostaglandin glyecerol esters (PG-Gs), which are implicated in myriad more processes including neuroinflammation, neural functioning, and high pain sensitivity. Understanding these pathways fully has been challenging historically because of the tendency of PG-Gs to be easily hydrolyzed to prostaglandins (PGs) in vivo. Experiments performed in vitro have shown at least 5 enzymes that will hydrolyze the PG-Gs, 2 of which show activity in vivo as well. A number of these enzymes also accept a broad range of substrates. All of these hydrolytic enzymes are members of the serine hydrolase superfamily, which classifies its members based on the use of an activated serine nucleophile for hydrolysis. Manna et. al. sought to shed light on the notion that a number of low-specific enzymes haphazardly hydrolyze PG-Gs in vivo. They theorized that a previously unidentified serine hydrolase was responsible for the hydrolysis of PGE2G (a specific PG-G) into the prostaglandin PGE2, a reaction that was shown to occur frequently in cancer cells, and attempted to find it.

By feeding human breast cancer cells PGE2G and analyzing the cell culture by LC-MS/MS, the authors show that PGE2G hydrolysis occurs in cancer cells. They demonstrate serine hydrolase activity by selectively inhibiting serine hydrolases and observing eradication of hydrolysis. An interrogation of serine hydrolase proteomics work done by Nomura et. al. against data from five cancer cell line experiments implicates a single protein as the source of PGE2G hydrolysis: lysophospholipase A2 (LYPLA2). siRNA experiments that reduce LYPLA2 gene expression show that without natural levels of LYPLA2, PGE2G hydrolysis is significantly reduced (Figure above, β-actin controlled). cDNA experiments then show PGE2G hydrolytic activity introduced in HEK293 cells. Substrate selectivity of LYPLA2 reveals itself as repeat experiments on the isoform lysophospholipase A1 fail to produce similar activity levels on PGE2G. In the presence of serum protein, LYPLA2 preferentially acts on PGE2G compared to 5 other substrates. By inhibiting LYPLA2 in vivo and observing increases in PG-G levels, the authors establish the importance of LYPLA2 in cells.

The sheer number of hydrolytic enzymes in cells makes it difficult to study the substrates of hydrolysis reactions. Manna et. al. use clever literature analysis to identify LYPLA2 as an important enzyme in cancer cells and then interrogate its role in the cell with numerous techniques. While the identification is exciting, the impact of the study should not be overstated. With so many enzymes performing similar functions and added complications of substrate isomerization, more work will need to affirm the unique relationship between LYPLA2 and PGE2G. If that happens, the true relevance of LYPLA2 as an enzyme in cancerous prostaglandin biosynthesis will emerge.

 

 

For Michael Chase:

LYPLA align

22 Replies to “Identification of the Major Prostaglandin Glycerol Ester Hydrolase in Human Cancer Cells”

  1. Hi Zach, thank you for this analysis. Are endocannabinoids NECESSARILY converted into glycerol esters? I.e. are all endocannbinoids subject to this conversion, and if so, why?

    1. Dr. C – It appears that the hydrolysis of endocannabinoids renders them inactive. The products are used in other pathways. If left unhydrolized, which is to say in the active form, then they are used for various organism level processes that were mentioned at the top of the review. This paper – http://molpharm.aspetjournals.org/content/86/5/522.full* – has a more robust introduction that talks about the metabolic fates of two endocannabinoids, and this KEGG pathway – http://www.genome.jp/kegg-bin/show_pathway?map04723+C13856 – sheds light on endocannbinoids at large (I have
      2-Arachidonoylglycerol highlighted in the linked chart). Does that answer your question?

      *note that to see the full text of the Savinainen paper published in Molecular Pharmacology (the first one I included in this response), you should click the Manna in text citation (it’s number 23). It appears that Muhlenberg doesn’t subscribe to Mol. Pharm., but JBC will allow you to link out to it.

  2. Hi Zach! After reading your spotlight and the corresponding paper, I have a question regarding PG-G substrate specificity. Your review states that the literature provides evidence for a number of low specificity enzymes that hydrolyze PG-Gs in vivo. This seems to raise specificity as an important question for the authors, so I read the paper through this lens. Therefore, when I read these sentences in the discussion, they caught my attention. “However, the inclusion of albumin in the assay buffer led to a reversal of substrate specificity such that PGE2-G was hydrolyzed more rapidly than other substrates, including lysophospholipids. Among the PG-Gs tested, PGE2-G was the preferred substrate, followed by PGF2α-G and PGD2-G.” (Manna et al, 2014). If the authors of this paper are attempting to understand the function and specificity of this enzyme, especially for a process that is suspected to include a number of low specificity enzymes, why did they use a method that impacted substrate specificity? They admit that the inclusion of the buffer created conditions where PGE2-G was hydrolyzed more quickly, but and then still state that it was the preferred substrate. Does this alter how seriously we can take their conclusions and the potential impact of their study? Do you believe that any of the other experiments they performed helped to compensate for this and bolster their claims?

    1. Kelly – that is a phenomenal question! I was wondering about it myself, but I didn’t find the answer until I set for the answer your question. Their use of BSA is attributed to one lonely article, a paper focused on the human monoacylglycerol lipase (MAGL) published by Jarmo T. Laitinen’s group in Molecular Pharmacology last August (http://molpharm.aspetjournals.org/content/86/5/522.full). In that paper, they show a discrepancy in results similar to the one you pointed out. Ultimately, they conclude that BSA is necessary in these lipid metabolism assays because it acts as a shuttle for lipophilic substrates (which are basically all of them!). They write “We argue that inclusion of BSA is fully justified, clearly facilitating availability of substrates like 1-DG for enzymatic hydrolysis.” They do go on to discuss how the use of BSA may alter the assay, but in the end they go with it, and they even suggest the omission of BSA as en explanation for previously published negative results. I have not been able to follow the literature debate that thoroughly, but it seems like here Manna and colleagues use BSA as a standard lipid assay technique. So to answer your questions, I don’t think that this was a major weak point in their paper. Based on their citing only one paper in regards to the BSA, I think they confidently used it without feeling the need to clarify. If anything, the experiment in the Laitinen paper provide the reasoning. Ultimately, I am going to agree with them – there were other weak points in the paper, but I think they were justified in including BSA. Again, you asked a really insightful question!

  3. Great analysis, Zach! I’m very curious as to why it was important for PG-G hydrolysis to be studied in cancer cells. I know you mentioned that the hydrolysis of PGE2G was shown to occur frequently in cancer cells, but do you know why? Could there possibly be a correlation between cell proliferation and serine hydrolase activity? Maybe an increased expression of serine hydrolase in cancer cells? Thanks! ☺

    1. Zack – That is a very sensible question. They originally report that they were testing PG-G hydrolysis in cancer cells because “of the high PGE2-G hydrolytic activity detected in preliminary experiments, the ease of cell maintenance, and the potential for straightforward biochemical and genetic manipulation”. This makes it sound like cancer cells were a convenient way to up their impact factor. But if we dig a little bit, we see that there is more of an informed reason. As you suggested, it looks like the lipid hydrolytic enzymes (from the serin hydrolase superfamily) play a big role in human disease. Manna mentions two studies by Daniel Nomura (in the citations but also in the main body of the paper!) that monoacylglycerol lipase is involved in spreading prostate cancer. Ultimately, they published a paper in 2010 (http://www.sciencedirect.com/science/article/pii/S0092867409014391) with the beautifully informative title “Monoacylglycerol Lipase Regulates a Fatty Acid Network that Promotes Cancer Pathogenesis”. They indicate that cancer cells display a lipogenic phenotype (meaning lots of lipid processing) and use the bioactive lipid derivatives as signaling molecules for cancer propagation. Additionally, Ben Cravatt (the last author in the Nomura papers) also published a 42 page review article (http://pubs.acs.org/doi/pdf/10.1021/cr200075y) detailing the role of serine hydrolases in human disease.

      1. Ha! We actually read the Nomura et al paper in BCM 441 last year. Small world. (I am a Cravatt fan…so perhaps not that surprising 😉

  4. Hi Zach,
    Good job with your analysis! I noticed that the authors tested both LYPLA1 and LYPLA2 to determine their importance in PGE2-G hydrolysis, yet the authors found that only LYPLA2 is able to successfully increase PGE2-G hydrolysis. The authors later go on to say that LYPLA1 and LYPLA2 are “related,” both in name and in sequence similarity, and that a single selective inhibitor is able to decrease hydrolase function in both; in the Discussion, the authors also note that LYPLA2 is able to hydrolyze a wide variety of PG-Gs, implying that the enzyme can act on several related substrates. Why do you think that LYPLA2 is able to hydrolyze PGE2-G, but the closely related LYPLA1 is not? Do you think it is as simple as a change in the active site that prevents the binding of substrate, and if so, why? Would there be a biological reason to limit the amount of PGE2-G hydrolysis in the cell? Prostaglandins have previously been shown to be involved in promoting colon cancer invasion (Pai 2003), so maybe limiting their hydrolysis is a genetic adaptation to allow for cancer cell survival?

    1. Michael – Thank you for the bioinformatics challenge! I set out to do an alignment of LYPLA1 and LYPLA2, but LYPLA2 was not readily available on the NCBI. I have no idea why that would be. Anyway, I went onto KEGG and was able to pull a sequence off of LYPLA2’s KEGG Gene page, which is what I used for the alignment. I’ve added an image of the alignment at the end of my post – click the blurry thumbnail to see a higher res. image. Broadly, it’s easy to see that the two proteins are related. But there is also enough difference to reasonably believe that they would have different active site architectures. To your other point, I think that the variety of destinies of the products of the hydrolysis (or not hydrolysis) explain why you would want to have a fine control over the reaction. The uses for molecules on both sides of the reaction are so diverse (from pain signaling to inflammation to all sorts of other complex neruochemical processes) that it is important (and literally awesome) to have controls over the metabolism. As we saw in the Science paper we read in class, losing control over enzymes in the inflammation pathways can have disastrous results (to name one example…).

  5. Good evening Sir Zimmerman,

    First, you did a fantastic job dissecting the argument and content of this paper. After going through the evidence and some of the other questions asked, I would like to build on the ideas Sir Shuler. I too find the description of the role of PGE2-G in cancer activity to be vague. A correlation has been noted between hydrolysis of PGE2-G and cancer cells, but is this to say that the hydrolysis of PGE2-G CAUSES cancer? This question resonates with me because it puts the findings into context. If PGE2-G hydrolysis is significant in the onset of cancer, and LYPLA2 is the enzyme responsible for its hydrolysis, can cancer be treated by inhibition of LYPLA2? I guess I am searching for some speculation as to application. Let me know what you think.

    1. Herr Ferrie – First of all, Sir Zimmerman is my father. You can call me Mister. I included a bunch of helpful links in my response to Sir Shuler’s question above. I encourage you to look at that response and follow those links. I would amend your theories only to say that it looks like the literature implicates LYPLA2 in the spread of cancer, not the cause. Moreover, I do not think LYPLA2 is a good therapeutic candidate because it strikes me as a rather important enzyme in our healthy nervous system tissue. Messing around with it in cancer cells could have very negative effects on patient health. Also, we would have to figure out a way to inhibit the enzyme that doesn’t act on any of its superfamily-shared traits. In other words, the serine hydrolase domains are probably off limits because they, too, are seemingly ubiquitous. I empathize with your desire to put the results in context. I would call this paper a step forward, but I think we are still in the early discovery phase of studying these enzymes. If you look at the response to Kelly’s question, you will see that there is even some debate on how we should even assay these enzymes of interest. This is all to say that I think you will have to wait a few more years before a landmark paper is published in Science that will present a clear application for all of this. But your question is valid and very perceptive. Not bad for a chemistry major 🙂

  6. Hey Zach,
    I liked your analysis, and not only because it had fewer abbreviations. I have short questions about the cellular environment of interest, namely the cytosol, and one mechanistic question. 1) Like Kelly, I noted the low specificity introduced early in the paper and in your analysis, and it seems that the hydrolases discussed are all cytosolic, do you think this match between “low specificity” enzymes and a “low specificity” environment like the cyotosol (without compartments) is important? 2) Part of the contextual problem is that in vivo studies of this mechanism are difficult because of the rapid hydrolysis, so they performed in vitro studies – but they are studying cytosolic enzymes, so does that give the benchtop experimentation more credibility because they are emulating (with their cultures, buffers, etc) a slightly simpler cellular environment? Lastly, the FP-TAMRA “probe” has been used historically as an irreversible serine hydrolase inhibitor. I was wondering if you had an idea if the fluorine substitution is a necessity (does it make the phosphate a worse leaving group, which would make it a reversible inhibitor?), or if a simple phosphate group could do the same job?

    1. Besher – Very astute observations. I would not necessarily call the cytosol “low specificity”. You are right in seeing that there are a number of substrates that “work” with the enzymes. But the researches were seeking which one works best. I would say that because the cytosol has so much “stuff” in it, the enzymes there need to be particularly sensitive so that we don’t have just random metabolic chaos occurring at all times. To that end, Manna concluded in this paper that LYPLA2 kinetically favors 1(3)-glycerol esters. I think the “specificity” that you seek in the cytoplasm can be attributed to the energetics; even though there are probably a number of molecules LYPLA can hydrolyze at any given moment, it will prefer (in an almost selective way) the 1(3)-glycerol esters. Your second point hits the “impact” nail right on the head. Obviously, studies in vitro will not fully capture the complexity of the intracellular environment. While they do try an emulate the cytosol, the benchtop experiments are much more scaled back in complexity than a real cell. In fact, I would say that the extent to which they relied on in vitro studies in this paper is probably why it was appropriate for publishing in the JBC. To your question about the FP-TAMRA, the short answer is that I don’t know. The probe that they used was from Thermo Scientific, who sells them as proprietary, which means that they are not forthcoming (as far as I can tell) with high level mechanistic information. I can say from looking at the product page on Thermo Sci’s website that it looks like the probes take advantage of the catalytic triad and an ethoxy group to covalently attach the probe to the serine while the fluorine gets kicked out. I invite you to look at that page too (http://www.piercenet.com/product/active-serine-hydrolase-probes, scroll down to the first figure for mechanism) and let me know if you see anything that catches your eye. Great questions from the chemist!

      1. Your mechanism idea for the FP-TAMRA probe is on track. This probe is a version of the classic serine hydrolase probes of the old biochemical literature, and of the same variety that Ben Cravatt first used when doing proteomic profiles of serine hydrolases. In brief, the serine nucleophile attacks the phosphorous, which preferentially eliminates a fluorine leaving group – covalently labeling the active site nucleophile

  7. Nice succinct analysis, Zach! So, I was thinking a bit about the significance of the paper. I know it was mentioned that the hydrolysis of PGE2G to PGE2 has been found to occur in cancer cells. I think of prostaglandins as being involved in the inflammatory response-is there a precedent in the literature for high prostaglandin levels to be associated with cancer? Perhaps is there a precedent for the hydrolysis of other PG-G’s being associated with cancer? I would like to hear more from the authors about the background and the context of the study, so I can better understand the significance of their findings.

    Right now, I see a correlation- what I would like to see in more detail from the authors is a study into the mechanistics of what PGE2 is actually doing in cancer cells. I mean, it is a prostaglandin, but is there anything about it that having it around in more bountiful qualities is advantageous to cancer cells? Is this a way that cancer could utilize the innate immune system as a tool to spread? That would be interesting. Again, nice work.

    1. Gabbie – Great ideas! If you scroll above, you will see some colleagues with similar questions. It looks like the advantage to cancer cells is that the bioactive lipid products are very useful in signaling and growing/metastasizing. These two papers (http://www.nature.com/nm/journal/v8/n3/abs/nm0302-289.html and http://cancerres.aacrjournals.org/content/55/12/2556.short) discuss the role of prostaglandins in colon cancer. Not quite related enough for Manna and colleagues to cite in this paper, but certainly your intuition is correct. The review article I posted in my response to Zack is also very thorough; you can see how all the lipase enzymes relate to a number of human diseases. I think your sense for seeking background information is a strong one – a better background section would have made for a better paper (in my humblest of opinions).

  8. Hey Zach,
    Thanks for breaking down a rather challenging paper into an easily digestible form! While the authors several times evidence the role of Bovine Serum Albumin (BSA) in dramatically increasing PGE2-G hydrolysis rates, they do not firmly elucidate the mechanistic and structural role as it applies to the overall process.

    In Savinainen et al (reference 23) BSA is seen as integral to the process of PGD2-G hydrolysis, to the point that such hydrolysis was not visualized in its absence. Given the facts that such a pronounced catalytic efficiency is dwarfed by the change in catalytic efficiency of PGE2-G, and that BSA primarily affects catalytic efficiency by way of acting as a carrier for lipophilic substrates, (Xie et al. 2010) it appears to imply that lipophilic substrates play a crucial role within the mechanism as a transporter. I am curious as to the structural details behind the favoring, which leads me to a question:

    What structural particulars of PGE2-G render it so capable of interfacing with BSA, as compared to PGD2-G?

    Savinainen et al. : http://molpharm.aspetjournals.org/content/86/5/522.full

    Xie et al:
    http://pubs.acs.org/doi/pdfplus/10.1021/tx1002194

    1. Anthony – Kudos to you for digging into the primary literature (and making my job easier!). This is a very complex question, and, unfortunately, it looks like the pros are still working out the answer. First of all, it does not surprise me at all that in reactions involving these lipid derivatives (read: long hydrocarbons), a lipid carrier would seem to play a role. It appears that in this case, BSA can act as a shuttle to increase the hydrolysis of 1-DG but can also decrease the conversion of other substrates like 2-AG. To confuse the issue even further, they report an insignificant difference between 15d-PGJ2-G reactions with and without BSA. In the discussion in the Savinainen paper, though, they strongly defend the merits of using BSA. I think this issue has to do with Besher’s question about accurately capturing a cytosolic environment in vitro. I would also not that in their discussion they say that the reason for any decreased catalysis is simply an affinity issue; it seems the substrates bind BSA too much but it doesn’t prevent the enzyme from acting on it. As to your specific question, I really don’t have an idea as to why the BSA binds with such variability. The structures of all the substrates are similar enough that nothing jumps out at me. And of course, BSA is a fair sized protein, which makes the idea of binding much more complex. I think you’re onto a really good experiment, though! I would love to model the binding of various lipid derivatives to BSA to assess the affinities and make a strong statement about the use of BSA in biochemical assays. I’m a bit biased, though, because my Experimental project was about molecular modeling. Culminating experiences for the win!

  9. Hi Zach, I thought that this paper was rather interesting and that you did a great job breaking it down for us. My question is regarding the PG-Gs and their tendency to hydrolyze. I noticed that in your description you talked about 5 different enzymes that have been known to hydrolyze PG-Gs, both in vitro and in vivo for some. Is there any chance that some of the PGE2-G molecules are being hydrolyzed by another enzyme that has broad specificity? Also, is the propensity to hydrolyze something that only happens within cancer cells? Or is it just that this process is mediated much better in healthy, mortal cells? Is there any evidence that certain serine hydrolases are expressed at higher levels in cancer cells than in healthy cells?

    1. Matt – I think that the broad enzyme specificity is part of what makes this all such an interesting puzzle. Lipid derivatives have so much similarity that it does not surprise me the involved enzymes can act on multiple substrates (after all, how many uniquely hydrophobic active sites can we have?) Broadly, I see authors trying to show a) a higher preference for a specific PG-G over the others and b) in vivo studies to back the first part up. Hydrolysis is definitely something that occurs in healthy cells – Figure 1 in the paper shows a very small snapshot of the diversity of reactions surrounding the PGE2-G. In a response to Zack’s question above, I detailed the role of serine hydrolases in cancer propagation. I encourage you to follow the links up there with specific attention to the Nomura papers.

  10. Hey Zach,

    Thanks for the paper. I like siRNAs so I thought that part was pretty cool. As for my question, I was wondering how you interpreted the portion regarding the 1(3) glycerol ester substrates versus the 2-glycerol ester substrates. The paper suggested that LYPLA2 only cleaves the 1(3) glycerol esters, and that in there experiments, the initially formed 2-glycerol esters isomerized into the 1(3) glycerol esters before being cleaved. I do recognize that it said that the LYPLA2 isoform is present throughout the body, but given its apparent high concentration in cancer cells, it might be an interesting route to maybe test and see if the lack of 2-glycerol esters in cells has any ramifications towards becoming a cancer cell. What are your thoughts on this matter?

  11. Thanks, Tommy! siRNAs are pretty nifty, huh? I thought that the work on the ester isomers, from a technical point of view, was very cool. I think your theory is very interesting, and given the way cancer research dollars are thrown around, it may be worth investigating. My only comment would be that the literature seems to indicate the relevant enzymes as propagators of cancers, not necessarily the causes. The papers I linked to in my response to Zack’s question detail the role of lipid signaling in metastasis, which can be achieved by these lipid-active enzymes. Speicifically, the 2010 Nomura paper shows how the monoacylglycerol lipase enzyme (MAGL) can turn nonagressive cancers protumorogenic, but it looks like the cells have to be cancerous before the lipolitic enzymes can wreak their havoc. Thanks for the question!

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