Αμιδοϋδρολάση των λιπαρών οξέων στην Tetrahymena: κινητικές παράμετροι, ενδογενή υποστρώματα και προσδιορισμός του αντίστοιχου γονιδίου

Abstract

The endocannabinoid system is a lipid signaling system in mammalian cells. The major components of the endocannabinoid system such as Fatty Acid Amide Hydrolase, Monoacylglycerol Lipase and NAPE-PLD were previously reported to be present in the protist Tetrahymena, with characteristics similar to those in mammals. Tetrahymena is a model organism for molecular and cellular biology studies as its genome sequence is available. The main aims of this study were the identification of the endoannabinoids of Tetrahymena and the search of the gene responsible for the FAAH expression. Using LC-MS/MS it has been clearly shown that N-acylethanolamines and their respective 2-acylglycerols are present in Tetrahymena thermophila and Tetrahymena pyriformis; the former consists a new lipid class for the protist. The identified molecules are, N-gamma-linolenoyl, N-eicosenoyl, N-linoleoyl, N-palmitoyl, N-stearoyl and N-oleoylethanolamines as well as the corresponding monoacylglycerols. Anandamide and 2-arachidonoyglycerol were also present but in trace amounts. The levels of acylglycerols were much higher than the corresponding N-acylethanolamines, as reported also for mammals. To our knowledge, N-β-linolenoylethanolamine was found for the first time in nature. Moreover the levels of those molecules were altered with the increase of the cultivation temperature from 27oC to 33oC. N-acylethanolamines were significantly increased while monoacylglycerols were decreased, suggesting a different role for the two lipid signaling classes. Using the online database of Tetrahymena‘s macronuclear genome and PCR method, it was found that a number of FAAH isomorphisms seem to exist in Tetrahymena. Three of those FAAH like proteins encoding genes were identified in both Tetrahymena pyriformis and Tetrahymena thermopila. One of them was cloned using TA cloning method for further study of the gene and the expressed protein. Nevertheless, the presence of major endocannabinopid systems components including endocannabinoids in a lower organism, indicates its importance and suggest that Tetrahymena could be used as a model organism for the pharmacological modulation of the ECS enzymes.