The protein encoded by the HSD17B7 gene was originally described as a prolactin receptor-associated protein and as 17beta-hydroxysteroid dehydrogenase (HSD) type 7. Its ability to synthesize 17beta-estradiol in vitro has been reported previously. However, we demonstrate that HSD17B7 is the ortholog of the yeast 3-ketosteroid reductase Erg27p and converts zymosterone to zymosterol in vitro, using reduced nicotinamide adenine dinucleotide phosphate as cofactor. Expression of human and murine HSD17B7 in an Erg27p-deficient yeast strain complements the 3-ketosteroid reductase deficiency of the cells and restores growth on sterol-deficient medium. A fusion of HSD17B7 with green fluorescent protein is located in the endoplasmic reticulum, the site of postsqualene cholesterogenesis. Further critical evidence for a role of HSD17B7 in cholesterol metabolism is provided by the observation that its murine ortholog is a member of the same highly distinct embryonic synexpression group as hydroxymethyl-glutaryl-coenzyme A reductase, the rate-limiting enzyme of sterol biogenesis, and is specifically expressed in tissues that are involved in the pathogenesis of congenital cholesterol-deficiency disorders. We conclude that HSD17B7 participates in postsqualene cholesterol biosynthesis, thus completing the molecular cloning of all genes of this central metabolic pathway. In its function as the 3-ketosteroid reductase of cholesterol biosynthesis, HSD17B7 is a novel candidate for inborn errors of cholesterol metabolism.

Cultivation of retrovirus packaging cells at 32˚C represents a common procedure to achieve high titres in mouse retrovirus production. Gene expression profiling of mouse NIH 3T3 cells producing amphotropic mouse leukaemia virus 4070A revealed that 10 % of the 1176 cellular genes investigated were regulated by temperature shift (37/32˚C), while 5 % were affected by retrovirus infection. Strikingly, retrovirus production at 32˚C activated the cholesterol biosynthesis/transport pathway and caused an increase in plasma membrane cholesterol levels. Furthermore, these conditions resulted in transcriptional activation of smoothened (smo), patched (ptc) and gli-1; Smo, Ptc and Gli-1, as well as cholesterol, are components of the Sonic hedgehog (Shh) signalling pathway, which directs pattern formation, diversification and tumourigenesis in mammalian cells. These findings suggest a link between cultivation at 32˚C, production of MLV-A and the Shh signalling pathway.

Short-chain alcohol dehydrogenases (SCAD) constitute a large and diverse family of ancient origin. Several of its members play an important role in human physiology and disease, especially in the metabolism of steroid substrates (e.g., prostaglandins, estrogens, androgens, and corticosteroids). Their involvement in common human disorders such as endocrine-related cancer, osteoporosis, and Alzheimer disease makes them an important candidate for drug targets. Recent phylogenetic analysis of SCAD is incomplete and does not allow any conclusions on very ancient divergences or on a functional characterization of novel proteins within this complex family. We have developed a 3D structure-based approach to establish the deep-branching pattern within the SCAD family. In this approach, pairwise superpositions of X-ray structures were used to calculate similarity scores as an input for a tree-building algorithm. The resulting phylogeny was validated by comparison with the results of sequence-based algorithms and biochemical data. It was possible to use the 3D data as a template for the reliable determination of the phylogenetic position of novel proteins as a first step toward functional predictions. We were able to discern new patterns in the phylogenetic relationships of the SCAD family, including a basal dichotomy of the 17beta-hydroxysteroid dehydrogenases (17beta-HSDs). These data provide an important contribution toward the development of type-specific inhibitors for 17beta-HSDs for the treatment and prevention of disease. Our structure-based phylogenetic approach can also be applied to increase the reliability of evolutionary reconstructions in other large protein families.

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Isopentenyl diphosphate isomerase (IDI) activates isopentenyl diphosphate (IPP) for polymerization by converting it to its highly nucleophilic isomer dimethylallyl diphosphate (DMAPP). In plants, this central reaction of isoprenoid biosynthesis is catalyzed by various highly conserved isozymes that differ in expression pattern and subcellular localization. Here we report the identification of an IDI duplication in mammals. In contrast to the situation in plants, only one of the two isoforms (IDI1) is highly conserved, ubiquitously expressed and most likely responsible for housekeeping isomerase activity. The second isoform (IDI2) is much more divergent. We demonstrate that after the initial duplication IDI2 underwent a short phase of apparently random change, during which its active center became modified. Afterwards, IDI2 was exapted for a novel function and since then has been under strong purifying selection for at least 70 million years. Molecular modeling shows that the modified IDI2 is still likely to catalyze the isomerization of IPP to DMAPP. In humans, IDI2 is expressed at high levels only in skeletal muscle, where it may be involved in the specialized production of isoprenyl diphosphates for the posttranslational modification of proteins. The significant positive fitness effect of IDI2, revealed by the pattern of sequence conservation, as well as its specific expression pattern underscores the importance of the IDI gene duplication in mammals.