A RIESKE STEROL C7-DESATURASE, HIGHLY CONSERVED IN ANIMALS, IS PRESENT IN BOTH STEROL-AUXOTROPH AND PROTOTROPH CILIATE SPECIES.

SEBASTIÁN R. NAJLE; NUSBLAT ALEJANDRO; CLARA B. NUDEL; ANTONIO D. UTTARO

Mar del Plata

Congreso; IX Congreso Argentino de Protozoología y Enfermedades Parasitarias; 2011

Sociedad Argentina de Protozoología

A RIESKE STEROL C7-DESATURASE, HIGHLY CONSERVED IN ANIMALS, IS PRESENT IN BOTH STEROL-AUXOTROPH ANDPROTOTROPH CILIATE SPECIES.Sebastián R. Najle1, Alejandro D. Nusblat2, Clara B. Nudel2, Antonio D. Uttaro11Parasitología Molecular. Instituto de Biología Molecular y Celular de Rosario, 2Cátedra de Microbiología Industrial y Biotecnología, FFyB, UBACholesterol and other sterols serve, besides its structural role in eukaryotic membranes, as indispensable precursors for the biosynthesis of steroid hormones.The first step in the biosynthesis of ecdysteroids in protostome animals is the conversion of cholesterol into 7-dehydrocholesterol by a Rieske[2Fe-2S]-monooxygenase (named Neverland in insects and DAF-36 in nematodes). Several orthologous proteins from different animal phyla, includingvertebrates, were shown to have the same activity highlighting the relevance of its conservation. The metabolism of exogenous sterols in ciliates hasbeen studied since the 60’s. Tetrahymena thermophila does not require sterols for living, but when sterols are present in the media they are incorporatedby the cells, inhibiting the synthesis of the sterol surrogate tetrahymanol, and modified by desaturation in positions C5(6), C7(8) and C22(23). Inthe case of phytosterols the ethyl (but not methyl) group in C24 is removed. Paramecium tetraurelia is a sterol auxotroph and its growth is supportedonly by 29 carbon phytosterols. It is known that the incorporated sterols in P. tetraurelia cells are modified by desaturations in the same positions abovementioned for T. thermophila. Some enzymes responsible for sterol modifications in Tetrahymena were characterized by our group during the past fewyears. DES5A was the first sterol C5-desaturase identified in a ciliate and DES24 the first sterol deethylase enzyme identified in nature. This workreports the identification of Neverland/DAF-36 homologs in both ciliate species. T. thermophila possesses one protein (TTHERM_00310640) with29% identity and 44% similarity with Drosophila Neverland. The sterol C7-desaturase activity of this protein was demonstrated by RNA interferenceby feeding bacteria overexpressing dsRNA corresponding to a portion of the targeted gene. GC/MS analysis of lipid extracts from interfered culturessupplemented with cholestanol exhibited a significant decrease on C7-desaturase activity, as compared with controls. A bioinformatic analysis on theParamecium tetraurelia genome database revealed the presence of 6 paralogs, indicating that this gene was present before the three successive wholegenome duplications occurred through the life history of this ciliate species. This data raise the hypothesis that the Rieske sterol C7-desaturase is anancient character, present in ciliates before the divergence of the clade Oligohymenophorea.