The redox reactions at position C17 of the steroid molecule are catalyzed by a number of different 17␤-hydroxysteroid dehydrogenases (17␤-HSD) 1 (1-3). Until now, four human 17␤-HSDs were characterized. The soluble 17␤-HSD type I consisting of 327 amino acids (aa) was cloned from human placenta and performs the oxidation of estradiol at the same efficiency as the reduction of estrone (4 -6). The 17␤-HSD type II is a microsomal enzyme of 387 aa that slightly prefers the oxidation over the reduction of estrogens and androgens and is expressed at high levels in the human placenta (7,8). The testes predominantly express the microsomal 17␤-HSD type III consisting of 310 aa and is responsible for the reduction of estrogens and androgens (9). The porcine 17␤-HSD type IV inactivates hormones very efficiently because of its 360-fold preference for steroid oxidation (10,11) and is the first steroid metabolizing enzyme localized in peroxisomes (12). The enzyme is primarily translated as an 80-kDa protein from a 2.9-kilobase message (13). The post-translational modifications include an N-terminal cleavage leading to a 32-kDa peptide (10). A fraction of the 32-kDa peptide is covalently linked to actin through an ⑀(␥-glutamyl)-lysine bond (14). Recently, cloning of the human and mouse 80-kDa 17␤-HSD type IV showed a close relationship revealing 85% amino acid similarity, the same multidomain structure, and identical kinetic parameters of the 17␤-HSD IV (15, 16). In contrast, the overall similarity between sequences of four human 17␤-HSD type I-IV is less than 25%.The amino acid sequence comparison with the Swissprot and EMBL data bases (17) revealed several interesting features of the type IV enzyme (Fig. 1). The N-terminal part shows homologies to the family of short chain alcohol dehydrogenases (18 -20), especially to the two short chain alcohol dehydrogenases domains of the multifunctional (hydratase-dehydrogenase) enzymes of peroxisomal ␤-oxidation of fatty acids in Saccharomyces cerevisae (21) and Candida tropicalis (22). The central domain of the 17␤-HSD type IV is 40 and 38% identical ( Fig. 1) with the C-terminal parts of the S. cerevisae and C. tropicalis multidomain proteins, respectively. The C-terminal extension of the 80-kDa protein shows an intriguing similarity to the sterol carrier protein 2 (SCP2) which is assumed to participate in the intracellular transport of sterols and lipids (23)(24)(25)(26)(27). Although the SCP2 was first identified as a 13-kDa protein it is, however, as well part of a 60-kDa fusion protein between SCP2 and a peroxisomal 3-oxoacyl-CoA thiolase named . Recently, it was demonstrated that SCP2 and SCPx are expressed from a single gene via alternative transcription initiation from two distinct promoters (31, 32).The 80-kDa protein reveals a complex structure which was unknown among other 17␤-hydroxysteroid dehydrogenases and enzymes of peroxisomal ␤-oxidation of fatty acids. To evaluate the activities suggested by the amino acid similarities, the functionalities of the purified porcine 17...

Success of human gene therapy depends upon the development of delivery vehicles or vectors, which can selectively deliver therapeutic genes to target cells with efficiency and safety. Previous studies have shown an efficient, systemic trans-gene expression in many cell lines (in vitro) by using an anionic liposomal vector, based on the composition of retroviral envelopes (artificial viral envelopes, AVEs). The AVE-liposomes and their complexes with plasmid (DNA) were characterized according to zeta potential measurements and transmission electron microscopy (TEM). We successfully demonstrated that AVE liposomes, dispersed in 10% serum-containing growth medium, efficiently delivered plasmid DNA to HuH-7 (human hepatoma cell line) cells. We assessed the utility of liver-targeted vesicles as a drug/gene delivery system for the treatment of liver diseases. We found that small unilamellar AVE vesicles containing 15 mol% digalactosyl diglyceride (DGDG) are efficiently targeted to the liver via the hepatic asialoglycoprotein receptor.

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The 17beta-hydroxysteroid dehydrogenase (17betaHSD) enzyme system governs important redox reactions at the C17 position of steroid hormones. Different 17betaHSD types (no. 1-4) have been identified to date in peripheral human tissues, such as placenta, testis, and breast. However, there is little information on their expression and activity in either normal or malignant prostate. In the present work, we have inspected pathways of 17beta-oxidation of either androgen or estrogen in human prostate cancer cells (LNCaP, DU145, and PC3) in relation to the expression of messenger RNAs (mRNAs) for 17betaHSD types 1-4. These cell systems feature distinct steroid receptor status and response to hormones. We report here that high expression levels of 17betaHSD4 were consistently observed in all three cell lines, whereas even greater amounts of 17betaHSD2 mRNA were detected solely in PC3 cells. Neither 17betaHSD1 nor 17betaHSD3 mRNAs could be detected in any cell line. From a metabolic standpoint, intact cell analysis showed a much lower extent of 17beta-oxidation of both androgen [testosterone (T)] and estrogen [estradiol (E2)] in LNCaP and DU145 cells compared to PC3 cells, where a greater precursor degradation and higher formation rates of oxidized derivatives (respectively, androstenedione and estrone) were observed. Using subcellular fractionation, we have been able to differentiate among 17betaHSD types 1-4 on the basis of their distinct substrate specificities and subcellular localization. This latter approach gave rise to equivalent results. PC3 cells, in fact, displayed a high level of microsomal activity with a low E2/T activity ratio and approximately equal apparent Km values for E2 and T, suggesting the presence of 17betaHSD2. Dehydrogenase specific activity with both E2 and T was also detected, although at lower levels, in LNCaP and DU145 cells. No evidence for reductase activity could be obtained in either the soluble or microsomal fraction of any cell line. As comparable expression levels of 17betaHSD4 were seen in the three cell lines, 17betaHSD2 is a likely candidate to account for the predominant oxidative activity in PC3 cells, whereas 17betaHSD4 may account for the lower extent of E2 oxidation seen in both LNCaP and DU145 cells. This is the first report on the expression of four different 17betaHSD types in human prostate cancer cells. It ought to be emphasized that for the first time, analysis of different 17betaHSD activities in either intact or fractionated cells harmonizes with the expression of relevant mRNAs species.

The product of the porcine HSD17B4 gene is a peroxisomal 80 kDa polypeptide containing three functionally distinct domains. The N-terminal part reveals activities of 17beta-estradiol dehydrogenase type IV and D-specific 3-hydroxyacyl CoA dehydrogenase, the central part shows D-specific hydratase activity with straight and 2-methyl-branched 2-enoyl-CoAs. The C-terminal part is similar to sterol carrier protein 2. The 80 kDa polypeptide chain ends with the tripeptide AKI, which resembles the motif SKL, the first identified peroxisome targeting signal PTS1. So far AKI, although being similar to the consensus sequence PTS1, has neither been reported to be present in mammalian peroxisomal proteins, nor has it been shown to be functional. We investigated whether the HSD17B4 gene product is targeted to peroxisomes by this C-terminal motif. Recombinant human PTS1 binding protein Pex5p interacted with the bacterially expressed C-terminal domain of the HSD17B4 gene product. Binding was competitively blocked by a SKL-containing peptide. Recombinant deletion mutants of the C-terminal domain lacking 3, 6, and 14 amino acids and presenting KDY, MIL, and IML, respectively, at their C-termini did not interact with Pex5p. The wild-type protein and mutants were also transiently expressed in the HEK 293 cells. Immunofluorescence analysis with polyclonal antibodies against the C-terminal domain showed a typical punctate peroxisomal staining pattern upon wild-type transfection, whereas all mutant proteins localized in the cytoplasm. Therefore, AKI is a functional PTS1 signal in mammals and the peroxisome targeting of the HSD17B4 gene product is mediated by Pex5p.