Type of Submission

Poster

Keywords

Tetrahymena, netrin, signaling

Proposal

Netrins are pleiotropic guidance proteins that are involved in developmental signaling of branched structures within vertebrates. However, like many developmental pathways, dysregulation of the netrin pathway has been implicated in cancer progression and metastasis. Since Tetrahymena respond to guidance proteins, showing chemoattractant and chemorepellent behavior, we hypothesized that we could use these organisms as a model system for cancer signaling. We have previously found that netrin-1-peptided, netrin-3-peptide, and recombinant netrin-4 are all chemorepellents in this organism. Since netrin-1-peptide signals through a tyrosine kinase in Tetrahymena, we hypothesized that Tetrahymena might possess tyrosine kinases as well as a receptor homologous to UNC-5, a netrin receptor which relays signals via tyrosine kinases in vertebrates. Using immunoprecipitation with a polyclonal anti-UNC-5-B antibody, we purified a 250 kD protein from Tetrahymena whole cell extract. Similarly, we immunoprecipitated several proteins, including a 60 kD protein and a 75 kD protein using a polyclonal anti-src-antibody. Our purified samples were sent out for identification by mass spectroscopy. Mass spectroscopy indicated that we have purified a number of novel peptides not currently found in the Tetrahymena Genome Database. Our data indicate that the proteome database in this organism is incomplete, and that there are additional proteins waiting to be discovered in this organism.

Start Date

4-8-2020 1:00 PM

End Date

4-22-2020 6:00 PM

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

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Apr 8th, 1:00 PM Apr 22nd, 6:00 PM

Biochemical Evidence for Netrin-Signaling Homologues in Tetrahymena thermophila

Netrins are pleiotropic guidance proteins that are involved in developmental signaling of branched structures within vertebrates. However, like many developmental pathways, dysregulation of the netrin pathway has been implicated in cancer progression and metastasis. Since Tetrahymena respond to guidance proteins, showing chemoattractant and chemorepellent behavior, we hypothesized that we could use these organisms as a model system for cancer signaling. We have previously found that netrin-1-peptided, netrin-3-peptide, and recombinant netrin-4 are all chemorepellents in this organism. Since netrin-1-peptide signals through a tyrosine kinase in Tetrahymena, we hypothesized that Tetrahymena might possess tyrosine kinases as well as a receptor homologous to UNC-5, a netrin receptor which relays signals via tyrosine kinases in vertebrates. Using immunoprecipitation with a polyclonal anti-UNC-5-B antibody, we purified a 250 kD protein from Tetrahymena whole cell extract. Similarly, we immunoprecipitated several proteins, including a 60 kD protein and a 75 kD protein using a polyclonal anti-src-antibody. Our purified samples were sent out for identification by mass spectroscopy. Mass spectroscopy indicated that we have purified a number of novel peptides not currently found in the Tetrahymena Genome Database. Our data indicate that the proteome database in this organism is incomplete, and that there are additional proteins waiting to be discovered in this organism.

 

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