Stress and Iron: The Role of Mitochondrial Iron-Sulfur Clusters in p38 Pathway Regulation

Paper: 

The p38 Pathway Regulates Oxidative Stress Tolerance by Phosphorylation of Mitochondrial Protein IscU

In this paper, the authors examined the role of the P38-mitogen activated protein kinase pathway (MAPK), an eukaryotic evolutionary conserved pathway which mediates responses to abiotic stress stimuli. The specific role of oxidative stress tolerance is conserved within this pathway, and was the authors predominant target of inquiry.

The role of Iron-Sulfur Clusters (ISC) within the pathway, cofactors which play major roles in promoting electron transport and redox reactions as well as acting as an upstream protein in the formation of Aconitase, was further examined with the understanding that such cofactor formation takes place via a highly complex and catalyzed process[1].  The protein component IscU was specifically analyzed, as it acts as a scaffolding protein for overall ISC biosynthesis and eventual transfer to acceptor proteins. While there exist two isoforms of IscU within human populations, Drosophilla melanogaster possesses a single isoform which makes it an attractive experimental population for analysis of ISC biosynthesis, specifically as it pertains to function.

The role of MAPK activated protein kinase 2 (MK2) was another element of significant interest within the research, with the understanding that it is known to regulate gene transcription, cytoskeleton architecture formation, and play a role in  inflammatory response within mammalian populations.[2] The Drosophila specific variant, dMK2, had a corresponding lack of detailed information regarding its function. To resolve this, analytical techniques including RT-PCR, In-vitro kinase and activity assays, oxidative stress assays, and immunoblotting were utilized. The authors plan was to perform a wide-spectrum analysis within likely parameters ; The impact upon oxidative stress as resulting from successful ISC formation, the mechanistic details thereof, and the ramifications felt  on aconitase activity in-vitro.
From these disparate analytical techniques, several functional factors were identified. It was determined that dMK2 knockout flies as generated by p-mediated genomic excisions, a method which reduces disruption outside the excision cite[3], have shorter lifespans under oxidative stressor conditions as effected by H­2O2 or Paraquat (a redox-active heterocyclic compound) addition.  This effect was maintained across both genders, and proper genomic modification verified by PCR analysis.

In order to examine downstream effects of the dIscU gene product as mediated by dMK2, in-vitro kinase assays and co-immunoprecipitation techniques were utilized using E. coli generated dMK2 recombinant proteins as a generative source. The likely possible phosphorylation sites on dIscU were analyzed by alignment with human and mouse sequences of IscU, and individual mutation of the residues performed in order to discern any effect upon phosphorylation by dMK2. The Ser-20 site  mutation was found to be the only site which  fully inhibited phosphorylation, painting it as the most likely target of dMK2. This was borne out by kinase assay.

In order to perform analysis regarding in-vivo effects, isoelectric focusing[4] followed by western blotting was performed upon tissue harvested from dIscU-overexpressed eyes of the transgenic population. From this analysis, there was evidenced a decrease in phosphorylation of dIscU which while significant, did not wholly exclude the possibility of other kinases acting to phosphorylate dIscU. This was enough data however to demonstrate that dMK2 phosphorylates dIscU at Ser20 both in vitro and in vivo within the fly population. Measurements of aconitase activities in vivo revealed that there was no significant control exerted by Ser-20 phosphorylation. Analysis of dMK2 further revealed a lack of control over aconitase activities. However, mitochondrial respiratory complex 1 was determined to be negatively regulated by phosphorylation of Ser-20, as mediated by dMK2.

Oxidative stress within the fly population was the subject of further analysis, as the transgenic flies were exposed to oxidative stressors and their lifespans measured. The researchers were able to determine a quantitative decrease in both the homozygous and heterozygous transgenic populations, and thus helping to confirm the role of the p38 pathway (as mediated by dMK2 phosphorylation) in managing oxidative stress.
The point which likely makes this paper so significant as to be a hallmark in JBC lies in the final experimentation, where the phosphorylation of IscU by MK2 was evidenced to occur in mammalian cells. The authors embarked on this line of reasoning with the knowledge that IscU  (and the phosphorylation site on Serine) are conserved in mammals, and subsequently demonstrated in HeLa cells hMK2 (a human isoform of MK2)phosphorylates IscU.  As a result of this analysis, the authors were able to determine that phosphorylation of IscU by way of MK2 mediates key elements of the TCA cycle, specifically complex 1 activity. While the increased activity thereof possibly harbors responsibility for reduced tolerance to oxidative stress (as seen with fly populations) more investigation is admitted by the authors as necessary to solidify any claims thereof. The authors likely aim to carry on research to elucidate the precise cause for oxidative stress tolerance within this system, and how it carries over to human populations, as oxidative stress management is directly pertinent in regards to cancer treatments.

[1] http://www.annualreviews.org/doi/pdf/10.1146/annurev.biochem.74.082803.133518

[2] http://www.uniprot.org/uniprot/P49137

[3] https://www.jstage.jst.go.jp/article/jjg/67/4/67_4_291/_pdf

[4] http://www.aesociety.org/areas/pdfs/Garfin_IEF_WebArticle9-07.pdf

23 Replies to “Stress and Iron: The Role of Mitochondrial Iron-Sulfur Clusters in p38 Pathway Regulation”

  1. I believe the authors were very thorough in their experimentation, and merited credit for their work based on their ability to reproduce their results both in vivo and in vitro.

    However, I also feel as though the authors were first searching for a niche to fill. The driving force for this study was simply wanting to learn about the unkown dIscU of d. melanogaster. While the discovery of a novel substrate of MK2 and MK2 phosphorylation of dIscU is significant, I think that the real impact of the study came in when the fly results were able to be reproduced in mammalian cells. What else do you think could have been done to up the ante in the study at hand?

    1. Hey Ryan, great question!

      As you noted, the mammalian results came in at the very end of the research and certainly prove slightly more pertinent to the overall theme of cancer metabolism than do the fly studies. I feel that if additional work was done on the HeLa cell cultures in order to examine the effects of mitochondrial complex 1 on oxidative stress tolerance, they could have published in a higher impact journal. Until such research is codified, these results (as far as cancer treatment is concerned) are of most pertinence to fly populations.

  2. Thanks for the analysis, Anthony! The findings in this paper demonstrated that phosphorylation of IscU (an iron-sulfur complex of the p38 signaling pathway) negatively affected the ability of Drosophila to regulate oxidative stress tolerance. We have learned in class that iron complexes act as strong oxidizing agents that are essential for performing free radical chemistry of unactivated alkanes. I’m curious as to how the phosphorylation affects the ability of IscU to perform oxidative chemistry. Do you know how the oxidation state of the iron complex changes as a result of phosphorylation? Does phosphorylation make the iron complex a weaker oxidizing agent, thus making it less able to regulate oxidative stress?

    1. Thanks for the very nice chemistry questions, Zach! As far as the oxidation states of Iron-Sulfur clusters go, there appears to be a degree of variance between Fe+3 and Fe+2 sulfur complexes. Upon being phosphorylated, the iron-sulfur cluster (Fe4S4) will shift from a +3 charge to a +2 charge, thus forming a less effective oxidizing agent. Depending on the specific Iron-suflur cluster altered (some have oxidation states as low as +1) this could very well inhibit any oxidative stress relief that might otherwise exist.

      1. A good question, and Anthony is right that Fe-S clusters can have different Fe oxidation states. But IscU is a scaffolding protein used to build the clusters and then transfer them to another protein. It is not performing the redox chemistry itself.

  3. Anthony – Cool article! The last time I considered drosophila science was in Bio III, so it’s really interesting to see publication quality research on that model. In investigating the figure you posted above, I was struck by something odd in the paper. They have a discussion section titled “The Phosphorylation of IscU by MK2 Also Occurs in Mammalian Cells”. Originally, I appreciated the novel result – translating the drosophila observations to something more complex. But the authors included some brashly candid talk of a failed experiment. They wrote, “Unfortunately, the phospho-hIscU2 antibody fail to detect endogenous mIscU in MEF cells…” In Experimental Biochem, we learned that extra information, especially info pointing out what failed, indicating something important (in a good or bad way). So my question is this: What does their sudden experimental shift from drosophila to MEF to HeLa cells mean in terms of assessing the strength of the paper? Was it experimentally acceptable to switch from MEF to HeLa cells. As I understand it, HeLa cells can act a little crazy, so is observing a certain result (or, perhaps in this case, a desired result) in HeLa cells significant?

    1. Hey Zach, glad to know I was able to form part of a CUE experience! As far as I was able to discern, they had intended to approach the prospect of human oxidative stress management in gradations, and hoped to use mouse models first. As their experimentation failed to yield a significant result within an in vivo model, and the fact that the mammalian model was primarily in situ regarding HeLa cells, it may suggest a more sober appraisal of the human-centric significance of the paper. As it stands, without further in vivo mammalian models, there is a lack of motive force behind making claims regarding oxidative stress management in humans.

      1. Also, if you read closely – you see their antibody was raised against the human phospho-IscU, and then they tried to use that antibody to detect murine/mouse phospho-IscU in a murine cell (MEF), and it didn’t work (although it did work in vitro, which means their AB did cross species, just not super well). This is probably due to crossing species. When they went back to HeLa cells (a human cell), they could find it. Since generating antibodies is so expensive and human applications are always more sexy, I’m not surprised that they didn’t have an antibody against the mouse version.

  4. Hi Anthony! I was wondering if you could offer your opinion on the potential thought process for this paper. Drosophilia melanogaster is known to be a model system of study. Discoveries made in this system are translated into other organisms. Because of this, the research trajectory often moves from flies to mammals. However, this paper pointed out a deficiency in knowledge of dMk2 in Drosophilia, while having a fairly decent understanding of MK2 in mammals. Now, I don’t mean to be too “humancentric” here, but what do you think the field stood to gain from this discovery, if MK2 is already understood outside of a model system? The authors end their paper by saying that this work is a foundation for future studies, but don’t elaborate on what that work might include. Do you have any ideas about what direction the authors might be headed? What makes this work, or future work in this vein, impactful enough to warrant a “working backwards” approach?

    1. Hello Kelly,

      While it certainly provokes some curiosity as to why one would move from a known, more advanced system (mammalian) to a more simple system (drosophilla), it presents an easier target in regards to gene identification, especially given conserved nature of the enzymes and cofactors involved. The ability to create transgenic species and test in vivo for certain traits and responses can certainly prove invaluable, as it allows for a more robust viewing of the outcomes specific polymorphisms would play upon an organism. Given the lack of information about their mice trials, I am led to believe that they encountered unforeseen roadblocks when attempting to duplicate their results. This can be further implied by their use of in vitro analytic techniques in order to verify any mammalian correspondence to their drosophilla observations.

      I would expect the authors to work on duplicating their results in transgenic mammalian models, so as to examine the specific nature of oxidative stress management within a complex system. This would open up the possibility of drug treatments to inhibit oxidative stress, and aid in recovery of many maladies.

      1. Good point Kelly! I think these authors just got lucky. I think they started out with a fairly boring Drosophila project, and then hit the jackpot when the downstream target they identified was also in human. Lucky for them.

  5. Hi Anthony,

    As I read through the study, one of the things that I was most surprised by was the simple lack of applications for the results of the study. The authors fail to really mention why they specifically wanted to learn more about the relationship between p38 and iron-sulfur clusters. I understand that pursuing knowledge for the sake of knowledge can be useful in some cases, but in today’s world where scientific funding is becoming increasingly difficult to obtain, how do you think the authors justified the costs associated with their study? Even if it is something minor, do you see any “real-world” applications for their results? Do you think that the lab could follow up on this study to design a way to modulate stress response in flies? Laboratory animals are likely stressed by cramped spaces and other unnatural conditions, so could this new information about the role of MK2 potentially serve as a way to modulate stress levels in Drosophila? It’s previously been shown that p38 has a role in modulating both inflammation and cell death, so perhaps decreasing stress could lead to longer life for animals in captivity (Ono and Han 1999). Or do you think that the authors had another type of application in mind when designing their experiment and applying for funding?

    1. Hey Michael, is the paper you cited in your comment?

      Its a very interesting slant on the whole topic, and one which frankly I hadn’t considered at all!

      While there is some role of environmental stressors on p38, the sense that I got was that such stressors were more to do with physical stimuli applied to the organism, rather than that triggered by neurological means. That being said, it certainly seems plausible that an attempt to bolster oxidative stress relief would ultimately benefit captive experimental populations, given the role of oxidative stress in cell death.

      As to the main purpose of the authors experiment, I believe that given the roles of oxidative stress in many cancers, finding a way to inhibit said stress in a mammalian population could have profound therapeutic effects. To this end, the authors seem to have attempted to move from a more simple organism with conserved genes (Drosophilla) to organisms more approximating the human condition. Unfortunately, they seem to have had some issue with the in vivo mouse experimentation, and as such will likely attempt to move further in that specific venue. Finally, considering the Chinese state laboratory states on their their intent to work within mouse models with an ultimate goal of understanding oxidative stress as it relates to cancer, I feel that the research is predominantly human-centric.

  6. Hey Anthony,
    I have three questions in all, the first two dealing with the iron-sulfur cluster. Since effects on the Fe-S cluster are a significant part of the paper, I had some questions about their methods. The yeast-cornmeal-agar medium looks like it is a staple for growth of fruit flies (and, it seems, fungi when I googled it). I couldn’t, however, find anywhere in the methods a source of iron for the bred drosophila, so I was wondering how they made sure the flies had enough iron and sulfur for their needs (namely, enough for proper circulation and obviously iron-sulfur cluster formation)? I imagine if it’s acquired through the parents somehow, there would be a limited supply spread among >200 offspring, which seems problematic. Similarly, how did they know (or did they know) that they gave ample time for the drosophila to fully develop the Fe-S clusters? 3-5 days is a long time for drosophila, but nonetheless I didn’t see them mention that it was ample time to form these clusters at the required locations. Lastly (and least importantly), is all of this oxidative stress talk the reason Dr. C has said in class “We all die because we breathe air”? From a cursory Wikipedia search it looks like oxidative stress causes/exacerbates a lot of diseases, including cancer.
    Thanks!

    1. Those are some really insightful questions بحشر!

      p>As far as dietary concerns go regarding the iron-sulfur complexes, I believe that is less of an issue within the bounds of the experiment, given that according to very little Fe and S are required to bisynthesize such complexes, owing in part to the natural biological toxicity of such elements. That being the case, and the fact that all transgenic organisms were tested against wild type’s under the same environmental conditions, I would expect deviation to be rather low due to Iron-Sulfur complex variation.On the same note, I would expect the Iron-Sulfur complexes (due to their critical nature regarding oxidative stressors) to exist in at least some capacity early on in life. In fact, there exists theories which state that life ultimately evolved around these iron-sulfur complexes, a brief summary being found here

      Regarding the role of oxidative stress and our own mortality, there is definitely the handiwork of oxidative stress among the many diseases and cancers that affect organisms. The capacity for cellular and genetic damage abounds from these charged particles in motion, and as result it can be hypothesized that working to reduce oxidative stressors (or otherwise inhibit their effects upon the body) would lead to less incidences of those conditions.

      1. Great question! Generally, yeast based medias (which are basically dead yeast…ew) are replete with iron, but you are right…iron can vary from media to media. It is something they should have controlled for!!

  7. Hi Anthony- Thanks for your work here. I have a couple questions/thoughts. First, is there a reason that dIscU was not that well-understood in D. melanogaster in the first place? As Kelly said, D. melanogaster has been a commonly used model organism for quite a while now- why do you think the authors were so interested now? Also, I was thinking about the potential significance of the paper. The authors showed that knocking out the Drosophila equivalent of MK2 led to shorter fly lifespans in the presence of oxidative stress. In the past, I have heard of oxidative stress being associated with cancer and other diseases- do you think that it’s possible that a study like this could lead to further study of human individuals that have a family history of cancer/short life spans? Perhaps they have a polymorphism in MK2 that could lead to an inefficient reaction to oxidative stres. I just think that would be cool, and potentially useful, but I do feel that there would need to be more work that would support MK2 as a theraputive target.

    1. Hello Gabby,

      You hit the nail right on the head! As far as the authors ultimate goals attend regarding oxidative stress and mitigation thereof, they hope (according to their Chinese state laboratory website, and inferred through other publications) to characterize the role of MK2 and other components of oxidative stress reduction pathways. While the authors wish eventually to move into a mammalian system to better approximate human biochemistry (and the resulting diseases), the necessity of transgenic mutations to solidify the role of various elements and proteins leads more towards the use of model populations with highly conserved genes of interest. To this end, the authors seem to have chosen Drosophilla as a model population.

      The unexplored failure to complete additional trials in a mammalian (mouse) population may be part of the reason why so little definitive statements in regards to mammalian oxidative stress are forthcoming in the paper. That being said, with effective characterization of in vivo mammalian populations (more work, as you said) this line of research could be highly pertinent (and impactful) in a therapeutic sense.

  8. Vos,

    First of all, all of this was just awful. The review, awful. The paper awful. Even the writing, awful ;).

    Second and more seriously, I had a couple thoughts about this paper. The most interesting part of the paper to me is that the authors briefly address apoptosis in the discussion. They suggest that the P38 pathway is important in mediating apoptosis under stressful conditions. They then narrow their approach and suggest that the P38 pathway is important for down regulating complex 1 in the mitochondria, and that in mutant Drosophila flies, the absence of portions of this pathway result in up regulation of complex 1 and flies with decreased life span. There are a few connections I could see playing out from this information. For one, I think I am remembering correctly in recalling that the ability to disregard apoptosis is a common trait of cancer cells. I also would imagine that under oxidative stress, an overactive complex 1 might produce an excess amount of free radicals that could be potentially mutagenic and lead to cancer. I was wondering if you think that this pathway might play a significant role in cancer development? For example, do you think its possible that polymorphisms in some of the specific genes, like MK2 or IscU, of the pathway that result in an individual less capable of down regulating complex 1 or effectively controlling apoptosis might result in a higher susceptibility of developing cancer?

    1. My good Mr. McCarthy Hoffmann, your kind words swell my self-esteem to all new heights. As far as your question goes regarding cancer development, I can definitely see this pathway paying a key role in oxidative stress mediation, as oxidative stress has been previously suggested to lead to the cellular damage that forms a major role in causing cancer.

      Certainly within the tested population, your scenario of MK2 or IscU polymorphism could lead to subsequent lack of regulation regarding apoptosis, and thus cancer.

      Without definitive results in HeLa cells however there certainly exists the capacity for differences, the difference in Serine phosphorylation sites (Ser-20 vs Ser-29) being an example. Until the authors (likely) publish follow-up research, I feel that there exists the possibility of external factors.

      1. There is a known IscU enzymopathy in humans – http://www.omim.org/entry/255125 – as we might expect, the afflicted individuals exhibit “lactic acidosis” – basically, overactive glycolysis, likely because their ETS is malfunctioning due to insufficient iron-sulfur clusters, and they get really, really tired with any physical exertion.

        While a decoupled or out of control ETS can spew free radicals (spoiler alert !!! Unit 3!!) and shorten life span, just an IscU enzymopathy does not seem to cause that particular effect.

  9. Hi Anthony. The authors were able to prove that MAPK knockout flies had significantly shroter lifespans under conditions of oxidative stress, but I was wondering if there could have been more effects seen than simply a down regulation of complex 1 in the mitochondria. Since there can be oxidative stress that comes from multiple sources do you think there would be any effect on other metabolic functions that contributes to the shorter lifespan of these flies?

    On a slightly different note, the authors’ data suggested that MK-2 mediated phosphorylation of IscU can negatively affect complex 1 in the mitochondria, but what would be the benefit of that? Or, in other words, how does down regulating the activity of complex 1 help combat the effects of oxidative stress?

    1. Hey Matt,

      Good question on both accounts! As far as competing variables leading to transgenic mortality, there certainly exists the likelihood for unknown downstream effects secondary to the oxidative stress relief mechanism. However, due to the highly localized polymorphism, the pronounced effects of oxidative stressors, and the survival of transgenic populations past birth, the role of oxidative stressors is the most likely significant cause as to the shortened lifespans. Oxidative stress causes much harm within an organisms body, and Drosophilla are no exception. The free radicals involved therein have numerous detrimental effects, including neurological, musculoskeletal, and immunological destruction of key mechanisms.

      In regards to regulating mitochondrial complex 1, it is known to produce the majority of reactive oxygen species which are major effectors of oxidative stress in organisms. As a result, regulating Complex 1 activity could decrease those same reactive oxygen species, and resultingly the oxidative stress applied upon the organism (and the dangers that come with that).

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