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Updated: Jun 7, 2025
In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans
Published on: April 1, 2020
Identification of DAF-16/FOXO Binding Partners in Caenorhabditis elegans by Immunoprecipitation and Mass Spectrometry
Patryk Marcinkowski1, Christian G Riedel2,3
1Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden.
Abstract:
The conserved transcription factor DAF-16/FOXO is a central hub in the regulation of stress responses and aging. It was first discovered as a protein activated by reduced insulin/IGF-like signaling (IIS) that would drive aging-preventive transcriptional outcomes. However, research from the last two decades has shown that its functions extend much further, with it responding to a broad spectrum of stress and deprivation-related stimuli and relaying them into optimal transcriptional outcomes that promote stress resistance, slow aging, and ultimately help the organism to survive each given threat. Evidence is mounting that DAF-16/FOXO is not self-sufficient in this important role but relies on numerous binding partners that help it achieve appropriate activity and target gene selectivity, with prominent examples being the chromatin remodeling complex SWI/SNF, the DAF-16-inhibitory protein HCF-1, or the transcription factor HLH-30/TFEB-just to mention a few. Here we present a protocol for the identification of such DAF-16/FOXO binding partners in Caenorhabditis elegans, comprising the large-scale growth and harvest of such animals, their lysis, and the eventual purification of DAF-16/FOXO, to yield samples that can be analyzed for co-purifying proteins und thus potential binding partners by tandem mass spectrometry.
Insights
This study details a method to identify DAF-16/FOXO binding partners in C. elegans. This research helps understand how DAF-16/FOXO regulates stress resistance and aging through protein interactions.
Area of Science:
- Molecular Biology
- Genetics
- Aging Research
Background:
- DAF-16/FOXO is a key transcription factor regulating stress responses and aging.
- It is activated by reduced insulin/IGF-like signaling (IIS) and promotes longevity.
- DAF-16/FOXO's functions extend beyond IIS, responding to various stress stimuli.
Purpose of the Study:
- To present a protocol for identifying DAF-16/FOXO binding partners in Caenorhabditis elegans.
- To facilitate the understanding of DAF-16/FOXO's regulatory mechanisms.
- To uncover novel proteins that modulate DAF-16/FOXO activity and target gene selectivity.
Main Methods:
- Large-scale cultivation and harvesting of Caenorhabditis elegans.
- Lysis of nematodes and purification of DAF-16/FOXO protein.
- Tandem mass spectrometry analysis of co-purifying proteins to identify binding partners.
Main Results:
- A detailed protocol for DAF-16/FOXO binding partner identification is established.
- The method allows for the analysis of proteins that interact with DAF-16/FOXO.
- This facilitates the discovery of novel regulators of stress response and aging.
Conclusions:
- The presented protocol is crucial for dissecting the molecular network of DAF-16/FOXO.
- Understanding these interactions will elucidate DAF-16/FOXO's role in stress resistance and aging.
- This work provides a foundation for future studies on DAF-16/FOXO-mediated pathways.
