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Structural investigation of proteins containing large stretches of sequences without predicted secondary structure is the focus of much increased attention. Here, we have produced an unglycosylated 30 kDa peptide from the chondroitin sulphate (CS)-attachment region of human aggrecan (CS-peptide), which was predicted to be intrinsically disordered and compared its structure with the adjacent aggrecan G3 domain. Biophysical analyses, including analytical ultracentrifugation, light scattering, and circular dichroism showed that the CS-peptide had an elongated and stiffened conformation in contrast to the globular G3 domain. The results suggested that it contained significant secondary structure, which was sensitive to urea, and we propose that the CS-peptide forms an elongated wormlike molecule based on a dynamic range of energetically equivalent secondary structures stabilized by hydrogen bonds. The dimensions of the structure predicted from small-angle X-ray scattering analysis were compatible with EM images of fully glycosylated aggrecan and a partly glycosylated aggrecan CS2-G3 construct. The semiordered structure identified in CS-peptide was not predicted by common structural algorithms and identified a potentially distinct class of semiordered structure within sequences currently identified as disordered. Sequence comparisons suggested some evidence for comparable structures in proteins encoded by other genes (PRG4, MUC5B, and CBP). The function of these semiordered sequences may serve to spatially position attached folded modules and/or to present polypeptides for modification, such as glycosylation, and to provide templates for the multiple pleiotropic interactions proposed for disordered proteins. Proteins 2010. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Steroid sulfatase (STS) regulates the hydrolysis of steroid sulfates to their unconjugated forms. Estrone sulfate and dehydroepiandrosterone sulfate can be hydrolyzed by STS to estrone and dehydroepiandrosterone, respectively, with these steroids being the precursors for the synthesis of more biologically active estrogens or androgens. A number of potent STS inhibitors have now been developed including STX64, which entered a phase I trial for the treatment of postmenopausal women with advanced metastatic hormone-dependent breast cancer. The results from this phase I trial were encouraging, suggesting that STS inhibitors may also have a role in the treatment of other hormone-dependent cancers including those of the endometrium, ovary, and prostate. In this paper the potential use of STS inhibitors to treat these hormone-dependent cancers is reviewed. In addition, results from in vitro studies show that Ishikawa endometrial cancer cells, OVCAR-3 ovarian cancer cells, and LNCaP prostate cancer cells all possess significant STS activity. Furthermore, STS activity in these cells can be almost completely inhibited by STX64 or the second-generation STS inhibitor, STX213. Results from these investigations therefore suggest that STS inhibitors could have therapeutic potential for the treatment of a range of hormone-dependent cancers. [ABSTRACT FROM AUTHOR]

Oestradiol (E2) stimulates the growth of hormone-dependent breast cancer. 17β-hydroxysteroid dehydrogenases (17β-HSDs) catalyse the pre-receptor activation/inactivation of hormones and other substrates. 17β-HSD1 converts oestrone (E1) to active E2, but it has recently been suggested that another 17β-HSD, 17β-HSD12, may be the major enzyme that catalyses this reaction in women. Here we demonstrate that it is 17β-HSD1 which is important for E2 production and report the inhibition of E1-stimulated breast tumor growth by STX1040, a non-oestrogenic selective inhibitor of 17β-HSD1, using a novel murine model. 17β-HSD1 and 17β-HSD12 mRNA and protein expression, and E2 production, were assayed in wild type breast cancer cell lines and in cells after siRNA and cDNA transfection. Although 17β-HSD12 was highly expressed in breast cancer cell lines, only 17β-HSD1 efficiently catalysed E2 formation. The effect of STX1040 on the proliferation of E1-stimulated T47D breast cancer cells was determined in vitro and in vivo. Cells inoculated into ovariectomised nude mice were stimulated using 0.05 or 0.1 μg E1 (s.c.) daily, and on day 35 the mice were dosed additionally with 20 mg/kg STX1040 s.c. daily for 28 days. STX1040 inhibited E1-stimulated proliferation of T47D cells in vitro and significantly decreased tumor volumes and plasma E2 levels in vivo. In conclusion, a model was developed to study the inhibition of the major oestrogenic 17β-HSD, 17β-HSD1, in breast cancer. Both E2 production and tumor growth were inhibited by STX1040, suggesting that 17β-HSD1 inhibitors such as STX1040 may provide a novel treatment for hormone-dependent breast cancer. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The mechanism, by which transhydrogenase couples transfer of H equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane, has been investigated in the solubilised, purified enzyme from Escherichia coil using analogues of the nucleotide substrates. The key observation was that, at low pH and ionic strength, solubilised transhydrogenase catalysed the very rapid reduction of acetylpyridine adenine dinucleotide (an analogue of NAD+) by NADH, but only in the presence of either NADP[SUP+] or NADPH. This indicates that the rates of release of NADP+ and NADPH from their binary complexes with the enzyme are slow. The dependences on pH and salt concentration suggest that (a) release of both NADP+ and NADPH are accompanied by the release of H[SUP+] from the enzyme and (b) increased ionic strength decreases the value of the pK[SUBa] of the group responsible for H[SUP+] release. Modification of the enzyme with N,N[SUP1]dicyclohexylcarbodiimide led to inhibition of the rate of release of NADP+ and NADPH from the enzyme, but had a much smaller effect on the binding and release of NAD[SUP+], NADH and their analogues and on the interconversion of the ternary complexes of the enzyme with its substrates. It is considered that the binding and release of H[SUP+], which accompany the binding and release of NADP[SUP+]/NADPH, might be central to the mechanism of proton translocation by the enzyme in its membrane-bound state. [ABSTRACT FROM AUTHOR]