In Saccharomyces cerevisiae and Candida albicans, the steroid-3-ketoreductase (Erg27p) exerts a stabilizing action on oxidosqualene cyclase (Erg7p, OSC), with an effect so pronounced that in these yeast strains lacking the reductase gene, cyclase is completely inactive. In this study, we want to establish if Erg7p and Erg27p interact also in Schizosaccharomyces pombe and mammals. S. pombe ERG7 was overexpressed in an erg7/erg27 S. cerevisiae strain. OSC functionality of recombinant strain was tested by cell culture incubation with [14C]acetate and incubation of homogenates with [14C]oxidosqualene. The accumulation of 3-ketosteroids and the full enzymatic activity of OSC indicate that in S. pombe Erg27p doesn’t protect Erg7p. As a model to study the interaction in mammals, we used a cholesterol requiring murine myeloma cell line (NS0), deficient in the 17β-hydroxysteroid dehydrogenase type 7 (Hsd17b7), the ortologue of the yeast steroid-3-ketoreductase. Tracer experiments with intact NS0 cells followed by lipid analysis showed that OSC is fully active in these mammalian cells, suggesting that in mammals the ketosteroid reductase is not required for OSC activity. To determine if the mammalian 3-ketoreductase could complement both the catalytic and the protective function of Erg27p, mouse and human Hsd17b7 were overexpressed in an erg27 S. cerevisiae strain. Recombinant strains were tested for the ability to grow on different media, the 3-ketosteroid reductase activity, and the OSC activity. The full enzymatic functionality of mammalian Hsd17b7 expressed in S. cerevisiae along with an only negligible OSC activity point to the inability of the mammalian 3-ketoreductase to retain secondary function of assisting OSC activity in S. cerevisiae. Results demonstrate that in S. pombe and mammals, unlike in S. cerevisiae and C. albicans, OSC and steroid-3-ketoreductase are non-interacting proteins.

Different interaction between oxidosqualene cyclase and steroid-3-ketoreductase from yeasts to mammals

TARAMINO, Silvia;OLIARO BOSSO, Simonetta;VIOLA, Franca Cecilia;BALLIANO, Gianni
2011-01-01

Abstract

In Saccharomyces cerevisiae and Candida albicans, the steroid-3-ketoreductase (Erg27p) exerts a stabilizing action on oxidosqualene cyclase (Erg7p, OSC), with an effect so pronounced that in these yeast strains lacking the reductase gene, cyclase is completely inactive. In this study, we want to establish if Erg7p and Erg27p interact also in Schizosaccharomyces pombe and mammals. S. pombe ERG7 was overexpressed in an erg7/erg27 S. cerevisiae strain. OSC functionality of recombinant strain was tested by cell culture incubation with [14C]acetate and incubation of homogenates with [14C]oxidosqualene. The accumulation of 3-ketosteroids and the full enzymatic activity of OSC indicate that in S. pombe Erg27p doesn’t protect Erg7p. As a model to study the interaction in mammals, we used a cholesterol requiring murine myeloma cell line (NS0), deficient in the 17β-hydroxysteroid dehydrogenase type 7 (Hsd17b7), the ortologue of the yeast steroid-3-ketoreductase. Tracer experiments with intact NS0 cells followed by lipid analysis showed that OSC is fully active in these mammalian cells, suggesting that in mammals the ketosteroid reductase is not required for OSC activity. To determine if the mammalian 3-ketoreductase could complement both the catalytic and the protective function of Erg27p, mouse and human Hsd17b7 were overexpressed in an erg27 S. cerevisiae strain. Recombinant strains were tested for the ability to grow on different media, the 3-ketosteroid reductase activity, and the OSC activity. The full enzymatic functionality of mammalian Hsd17b7 expressed in S. cerevisiae along with an only negligible OSC activity point to the inability of the mammalian 3-ketoreductase to retain secondary function of assisting OSC activity in S. cerevisiae. Results demonstrate that in S. pombe and mammals, unlike in S. cerevisiae and C. albicans, OSC and steroid-3-ketoreductase are non-interacting proteins.
36th FEBS Congress, Biochemistry for Tomorrow's Medicine
Torino
25-30 giugno 2011
36th FEBS Congress, Biochemistry for Tomorrow's Medicine, Lingotto Conference Center, Torino, Italy, June 25-30, 2011
Richard Perham
278
189
189
S. Taramino; B. Teske; S. Oliaro-Bosso; F. Viola; M. Bard; G. Balliano
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2318/95887
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