Investigating the effect of VEGF glycosylation on glycosaminoglycan binding and protein unfolding.

VEGF165 binding to endothelial cells is potentiated by glycosaminoglycans (GAGs). Here, we have investigated the impact of VEGF165 N-glycosylation on GAG binding. Although glycosylated VEGF165 bound to heparin with only slightly higher affinity than non-glycosylated VEGF165, the natural ligand heparan sulfate induced a conformational change only in the glycosylated protein. Unfolding studies of the VEGF proteins indicated a stabilising effect of heparin on the growth factor structure.

USF2 is connected to GAAAATATGATA element and associates with androgen receptor-dependent transcriptional regulation in prostate.

BACKGROUND: We have previously identified a GAAAATATGATA binding site (pros) of a transcription factor involved in prostatic and androgen-dependent gene regulation. We now purified the potential factors interacting with the pros and characterized their co-operation with the androgen receptor (AR). METHODS: Sequence-specific DNA affinity chromatography, mass-spectrometry, electromobility shift assays, supershifts, glutathione-S-transferase pull-downs, and transient transfections. RESULTS: Several proteins bound to the pros site, but only upstream stimulatory factor 2 (USF2) was confirmed to be part of the transcription factor complex. Weak interaction was detected between AR and the transcription factor complex. Physical proximity between the androgen response element (ARE) and the pros was shown to be important for their co-operation. In the presence of pros and androgen, AR achieves its maximal efficiency even at low concentrations. CONCLUSIONS: The protein complex binding to the GAAAATATGATA site does not have a significant independent function, but may interact with AR if GAAAATATGATA is physically close to the ARE and enhances the transactivation function of AR.

Production, purification, and functional analysis of recombinant human and mouse 17beta-hydroxysteroid dehydrogenase type 7.

17beta-Hydroxysteroid dehydrogenases (17HSDs) have a central role in the regulation of the biological activity of sex steroid hormones. There is increasing evidence that in addition to their importance in gonads, these hormones also have substantial metabolic roles in a variety of peripheral tissues. In the present study, the cDNA of human 17HSD type 7 was cloned. In silico, the gene corresponding to the cDNA was localized on chromosome 1q23, close to the locus of hereditary prostate cancer 1 (HPC1) (1q24-25) and primary open-angle glaucoma (GLC1A) (1q23-25). Further, a pseudogene was found on chromosome 1q44, close to the locus of predisposing for early-onset prostate cancer (PCaP) (1q42.2-43). Both human (h17HSD7) and mouse 17HSD type 7 (m17HSD7) were for the first time produced as recombinant proteins and purified for functional analyses. Further, kinetic parameters and specific activities were described. h17HSD7 converted estrone (E1) to a more potent estrogen, estradiol (E2), and dihydrotestosterone (DHT), a potent androgen, to an estrogenic metabolite 5alpha-androstane-3beta, 17beta-diol (3betaA-diol) equally, thereby catalyzing the reduction of the keto group in either 17- or 3-position of the substrate. Minor 3betaHSD-like activity towards progesterone (P) and 20-hydroxyprogesterone (20-OH-P), leading to the inactivation of P by h17HSD7, was also detected. m17HSD7 efficiently catalyzed the reaction from E1 to E2 and moderately converted DHT to an inactive metabolite 5alpha-androstane-3alpha,17beta-diol (3alphaA-diol) and to an even lesser degree 3betaA-diol. The mouse enzyme did not metabolize P or 20-OH-P. The expression of 17HSD type 7 was observed widely in human tissues, most distinctly in adrenal gland, liver, lung, and thymus. Based on the enzymatic characteristics and tissue distribution, we conclude that h17HSD7 might be an intracrine regulator of steroid metabolism, fortifying the estrogenic milieu in peripheral tissues.

Sex hormone metabolism in prostate cancer cells during transition to an androgen-independent state.

The progression of prostate cancer during androgen deprivation therapy is a serious clinical problem. Little is known, however, about the mechanisms behind the transition of the disease to an androgen-independent stage. In the present report, we provide evidence of substantial changes in both estrogen and androgen metabolism during the transition of cultured prostate cancer LNCaP (lymph node carcinoma of the prostate) cells. The results of enzyme activity measurements performed using HPLC suggest that, related to the transition, there exists a remarkable decrease in the oxidative 17 beta-hydroxysteroid dehydrogenase (17HSD) activity, whereas the reductive 17HSD activity seems to increase. Relative quantitative RT-PCR revealed that the decrease in oxidative activity largely coincided with the remarkable decrease in the expression of the HSD17B2 gene. Furthermore, the present data suggest that the observed increasing activity of 17HSD type 7 could lead to the increased intracellular production of 17 beta-estradiol during disease progression. This was supported by the cDNA microarray screening results, which showed a considerable overexpression of several estrogen up-regulated genes in the LNCaP cell line variant that represents progressive prostate cancer. Because 17HSDs critically contribute to the control of bioavailability of active sex steroid hormones locally in the prostate, the observed variation in intraprostatic 17HSD activity might be predicted to be crucially involved in the regulation of growth and function of the organ.