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Identification de nouvelles protéines de l'épithélium pigmentaire de la rétine transitant par la voie sécrétoire intra-cellulaire par la méthode du Peptide Signal Yeast Trap

Using the peptide signal Yeast Trap method and a library of RPE cDNAs, our goal was to identify novel heterologous proteins using the intracellular secretory pathway such as secreted proteins, transmembrane receptors and transmembrane channels. Using this method, we previously identified the prosaposine, known nowadays to be involved in several neurodegenerative diseases and patented as a neuroprotective agent.

We used a special Yeast strain growing only in a sucrose medium. The invertase is normally secreted by this train of yeast in the sucrose milieu for cleaving the sucrose into two smaller sugars which are swallowed by the this yeast strain in order to survive and to proliferate. We deleted completely from the whole genome of this special yeast strain its two copies of the invertase gene. We introduced  a partially deleted yeast invertase cDNA  sequence (devoid of  its peptide signal cDNA sequence and its ATG initiation codon) in a particular plasmid. Using this particular plasmid, we produced a plasmid cDNA library where human RPE cDNAs  were introduced in the 5' position of the altered yeast invertase cDNA sequence.  Then, we transfected the plasmidic library into the special yeast strain homozygously completely deleted for its invertase genes. Only the fused plasmidic cDNAs bringing an initiation ATG codon an a peptide signal sequence could allow the secretion of the invertase and the survival of select yeast colonies; Then, we retrieved the plasmids from each yeast colony and sequenced the unknown human cDNA sequenced introduced. In one step, we were able to identify hundreds of unexpected human sequences. 

We report  the identification by this method of three novel cDNAs expressed in the human and rat RPE : 1) a novel cDNA encoding a special protocadherin which inhibits the canonical Wnt pathway; 2) A novel cDNA sequence encoding a very special protein of the Serinc proteins family, the mutation of which is the cause of a novel syndromic retinal degeneration, 3) The PAX6 cDNA sequence. This last unexpected sequence contributes to further validate the discoveries made by Professor Alain Proschiantz and collaborators.

Although several sets of SAGE data and numerous sets of RNA seq data are available for the RPE and the neural retina, they frequently miss very important cDNA sequences for each cell type analyzed. Additionally, The RNA-Seq Technology requires repeated deep sequencing, a strong normalization of the procedures used and cutting edge computational methods. This Peptide Signal peptide Yeast Trap Method allows in one step to identify ptoteins utilizing the secretory pathway and constitutes a low cost method for identifying novel therapeutic targets and novel molecular links between unexpected develpmental pathways

This work brings additional proof of the efficiency of the peptide signal Yeast Trap for discovering novel proteins produced through the intracellular secretory pathway. These proteins are of utmost importance both for the discovery of genes causing inherited diseases, for unravelling novel pathophysiological mechanisms and for providing a source of novel drugs for retinal dystrophies and neurodegenerative diseases.