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Papers In Press, published online ahead of print May 19, 2004
J. Biol. Chem, 10.1074/jbc.M313666200
Submitted on December 15, 2003
Revised on April 28, 2004
Accepted on May 19, 2004

Appropriate function of 11beta -hydroxysteroid dehydrogenase type 1 in the endoplasmic reticulum lumen is dependent on its N-terminal region sharing similar topological determinants with 50-kDa esterase

Christoph Frick, Atanas G. Atanasov, Peter Arnold, Juris Ozols, and Alex Odermatt

Department of Clinical Research, University of Berne, Childrens Hospital G4-847, Berne 3010

Corresponding Author: alex.odermatt{at}dkf2.unibe.ch

By interconverting glucocorticoids, 11beta -hydroxysteroid dehydrogenase type 1 (11beta -HSD1) exerts an important pre-receptor function and is currently considered a promising therapeutic target. In addition, 11beta -HSD1 plays a potential role in 7-ketocholesterol (7KC) metabolism. Here, we investigated the role of the N-terminal region on enzymatic activity and addressed the relevance of 11beta -HSD1 orientation into the endoplasmic reticulum (ER) lumen. Previous studies revealed that the lumenal orientation of 11beta -HSD1 and 50 kDa-esterase/arylacetamide deacetylase (E3) is determined by their highly similar N-terminal transmembrane domains. Substitution of Lys5 by Ser in 11beta -HSD1, but not of the analogous Lys4 by Ile in E3, led to an inverted topology in the ER-membrane, indicating the existence of a second topological determinant. Here, we identified Glu25/Glu26 in 11beta -HSD1 and Asp25 in E3 as the second determinant for lumenal orientation. Our results suggest that the exact location of specific residues rather than net charge distribution on either side of the helix are critical for membrane topology. Analysis of charged residues in the N-terminal domain revealed an essential role of Lys35/Lys36 and Glu25/Glu26 on enzymatic activity, suggesting that these residues are responsible for the observed stabilizing effect of the N-terminal membrane anchor on the catalytic domain of 11beta -HSD1. Moreover, activity measurements in intact cells expressing wild-type 11beta -HSD1, facing the ER-lumen, or mutant K5S/K6S, facing the cytoplasm, revealed that the lumenal orientation is essential for efficient oxidation of cortisol. Furthermore, we demonstrate that 11beta -HSD1, but not mutant K5S/K6S with cytoplasmic orientation, catalyzes the oxoreduction of 7KC. 11beta -HSD1 and E3 constructs with cytosolic orientation of their catalytic moiety should prove useful in future studies addressing the physiological function of these proteins.


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