Investigating Changes in Protein Ubiquitylation Following Sleep Deprivation in Drosophila melanogaster

Student: Koral Kasnyik
Major: Biochemistry and Molecular Biology
Advisors: Dr. Seth Kelly, Dr. William Morgan

Koral KasnyikChronic sleep loss is a public health epidemic and is linked to an increased risk of metabolic diseases. Although several models attempt to explain the function of sleep, the molecular mechanisms that link sleep deprivation and disease are unclear. In order to better understand the biological changes that occur following sleep loss, our lab established a fly model of sleep deprivation. Our current data suggests that the ubiquitin proteasome pathway (UPP) may regulate sleep. The UPP functions to tag short-lived or defective proteins with ubiquitin and degrade these proteins via the proteasome complex. Flies were sleep deprived using a variety of methods. Western blot analysis showed that one method resulted in decreased levels of ubiquitylated proteins while alternative methods did not. These results suggest that different methods of sleep deprivation may cause different changes in protein abundance and question whether methods of sleep deprivation can be used interchangeably.

Koral will be online to field comments on May 8:
10am-noon EDT (Asia: late evening, PST 6am-8am, Africa/Europe: late afternoon)

36 thoughts on “Investigating Changes in Protein Ubiquitylation Following Sleep Deprivation in Drosophila melanogaster”

  1. Hi Koral,

    Do humans have any type of genes that activate wake-promoting or inhibit sleep-promoting neurons (similar to TrpA1 in fruit flies)? If yes, how are these genes activated (ex. are there some that are controlled on temperature as well)?

    Your conclusion is really interesting and I love your logic in trying to explain why you got results a bit opposite of what you expected. I’m sure that wasn’t very reassuring (and probably frustrating) when you were working on this. But in all, it really reemphasizes that sleep is so complex and we take it so so much much much for granted.

    Congratulations! Sounds like you had a great support group. What’s next after graduation?
    Thank you.

    1. Hi Brian!

      Humans, other mammals, and reptiles actually have Trp channels. These ion channels are found on cell membranes and help the organism sense environmental irritants like pain and cold temperatures. In this study, the channel was activated by increased temperatures which in turn activated those target neurons by sending the signal “it’s hot!” This stimulation kept the cells activated and the flies awake. Normally, flies do not have the TrpA1 channel, which makes its target extremely specific and well-cited. Humans do have wake-promoting and sleep-prompting circuits, but they are extremely complex and integrate hormones, external stimuli, etc. This is why sleep studies at the molecular level are best using models organisms with less complex neural circuitry like flies.

      The mixed results were a bit confusing, but intriguing enough for an undergraduate project. Hopefully the next senior project can dive into the results more. My plans after graduation are to go to medical school and specialize in infectious disease.

      Thanks for listening and your insightful responses.

  2. An excellent presentation. This information was helpful in determining that I should be getting more sleep. I am grateful for this study

  3. An awesome presentation with excellent use of posters/diagrams to further your explanations. Thank you for your effort!

  4. Very interesting study, Koral – and with excellent presentation of your question, approach, results, and conclusions (with an always-important take-home message as well!). Congratulations!

  5. Thank you for sharing your research. What is the similarity (if any) in the protein ubiquitylation pathways between fruit flies and humans? Have their been human studies on these pathways as well?

    Congratulations on all of your hard work. I hope you plan to continue work in science and research.

  6. Hi, Koral!

    Great presentation!

    In your current study, did you identify the peptides that were ubiquitylated? I’d be interested to see if certain peptides are targeted for degradation as a result of sleep deprivation.

    1. Hi Lexie,

      Unfortunately, that is a limitation of this study. The main aim was just to see if global ubiquitylation patterns were altered, but future work would definitely be interesting to determine if any specific peptides change in ubiquitylation. The antibody we used in the Westerns bound nonspecifically to ubiquitylated proteins. There have been other studies that indicate uniquitylation patterns of specific peptides change during sleep (which makes sense rhythmically), but none have analyzed how these patterns change with sleep deprivation.

      Thanks!

      1. Thanks, Koral!

        Maybe next time you’re at OSU, you can bring your proteins and do some peptide-sequencing. 🙂

        Have a great summer, and best of luck at medical school! I have a feeling you’ll be very successful.

        1. Thanks Lexie! I hope life as a post-doc is everything you hoped it would be! Hopefully we run into each other again.

  7. Hi Lori,

    This pathway is extremely conserved across organisms. This is why fruit flies are such a great model organism. Targeted ubiquitylation is the most prominent way for intracellular proteins to be degraded. Throughout each day, many proteins are made and degraded so this pathway is especially important to understand.

    Thanks for the question.

  8. Great job Koral! You’ve really completed some nice work here (and also gotten some really good questions as well)!

  9. Hello Koral. I was delighted to be able to follow along with your entire presentation. It’s well outside my field, but you did an excellent job at explaining things. Thank you! And good luck in med school. I hope you continue with your chosen specialization :-). I know it will be intense and busy, but I hope you take time to have some fun too! Congratulations, and take care.

  10. Very interesting! Is it known what proteins are involved in the ubiquitylation process? And if so is there possibly a way to study/perform an antibody test on those proteins to test your new hypothesis in the changes of ubiquitylation?

    1. Hi Kevin,

      Great thought. I actually dive heavily into that idea in the discussion of my IS. The process of ubiquitylation requires many ligases and hand-off reactions to covalently bind ubiquitin to the target protein. I think it is possible that the activity or expression of these proteins may account for the differences in ubiquitylation seen here. I would perform Western blots with antibodies to these proteins and then an activity assay to further characterize the ligases.

      Thanks

  11. Great job. I feel like one of the sleep derived flies during my home lock down. Good Luck!!

  12. Go Koral! You’re one step closer to changing the world. Congrats and best of luck!

  13. Hi Koral,

    I’m just curious – when you made your lysates for your Westerns, did you use the entire fly or are you able to make lysates from only the brain/fly neurons. I’m just thinking about how many flies it would take to get enough protein to run on a blot! 🙂

    1. Hi Kristy,

      We used whole head lysates. I removed the heads on dry ice and then essentially crushed the heads in RIPA buffer. Each sample contained between 5-15 heads. From there, I quantified the protein concentrations to eventually do the Westerns.

      1. Very cool. Interesting project and I wish you the best in medical school! 🙂

  14. Koral! What a fantastic project! I’m so intrigued by the idea that you effectively produced sleep deprivation using a genetic manipulation and produced more than one kind of sleep deprivation. What motivated a genetics-based approach rather than an environmental approach? Do flies sleep through bright lights or loud noises, or was there some other factor that prevented you from trying other approaches?

  15. Hi Koral,

    As an AP Biology teacher, your research and presentation are very interesting to me. Aside from the fact that we delve a bit into ubiquitins and protein degradation, I am encouraged by your mode of presentation because I have my students do something very similar towards the end of the course. From this I feel that I am preparing them well for future coursework! The changes that occur biologically due to sleep loss are also important for high school students to understand. Would you mind if I incorporate your presentation into my curriculum?

    Good luck in med school!

    Theresa Szczepanik

  16. Koral,
    The making of the fly food, hatching flies, keeping them alive, using their brains, making more food, being in the lab when they hatched…all of that has paid off big in your research. There was so much more behind the scenes that had to happen before the research could even begin. I am so proud of you and all that you have accomplished with Dr. Kelly. All your research experience as a sophomore led you to this point. What is really impressive is how you were able to relate this research to each and every one of us. We all need sleep! Well done. You are headed toward greatness.

  17. Nice job, Koral. Very interesting how sleep affects proteins. Any idea what proteins are affected from your research or the literature? Thank you for a job well done!

  18. Great work, Koral!
    It was great to work with you in the NT course, and I wish you the best as you are beginning the next chapter of your life journey!

  19. Koral,

    How timely this research topic is! Many of us are struggling with sleep due to Covid 19 stress. I’m glad to know that people like you are out there figuring out what is going on biochemically with those of us struggling to get sleep. Looks like much work still needs to be done.

  20. Great work, Koral! This is such a relatable and much-needed study. Thanks for sharing and congratulations!

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