Investigations into metabolism, transport and function of sulfonated steroids in the porcine testicular-epididymal compartment

Sulfonated steroids have been traditionally regarded as inactive metabolites destined for excretion, as they are incapable of binding to classical nuclear steroid receptors. However, by the enzyme steroid sulfatase (STS) they may be converted into free steroids, which may be biologically active directly or after a few additional enzymatic reactions. Thus, as sulfonated steroids commonly circulate at relatively high concentrations, they may form an important pool of precursors for the local (intra-tissue) production of active free steroids. This so-called sulfatase pathway has received increased attention over recent years especially with respect to estrogen metabolism in human hormone-dependent breast cancer, where the intratumoral estrogen production from sulfonated precursors obviously has a much higher capacity in comparison to the de novo synthesis via free steroids. This study is composed of two parts of which the first one addresses the secretory patterns of free and sulfonated steroids in vivo, whereas in the second part the expression of STS and of the steroid sulfotransferases SULT1E1 (estrogen specific) and SULT2B1 (specific for beta-hydroxysteroids) was characterized in the testis and in different segments of the epididymis. Other subjects of the second part of this study were hydrolysis of steroid sulfates and the sulfonation of estrone (E1), dehydroepiandrosterone (DHEA) and pregnenolone (P5) in the tissues investigated. Concentrations of androstenedione, testosterone, pregnenolone sulfate (P5S), dehydroepiandrosterone sulfate (DHEAS), estrone-3-sulfate (E1S)and 17beta-estradiol-3-sulfate were performed in the Steroid Research & Mass Spectrometry Unit, Division of Pediatric Endocrinology & Diabetology, Center of Child and Adolescent Medicine, Justus-Liebig-University, Giessen (head: Prof. Dr. S. Wudy) applying liquid chromatography tandem mass spectrometry (LC-MS-MS). Moreover, 17beta-estradiol (E2) and E1 were measured by inhouse radioimmunoassays to cope with the low concentrations of free estrogens in boars. In order to get new information on the sulfonation of free steroids and the hydrolysis of steroid sulfates in the porcine testicular-epididymal compartment, subcellular fractions were prepared from tissue samples collected from the testis and from defined sites of the epididymis (EH1, EH2: proximal/distal part of epididymal head; EB1-4: epididymal body, from proximal to distal; ET1, ET2: proximal/distal part of epididymal tail) using differential centrifugation. STS and steroid sulfotransferase activities were measured based on the differential distribution of free and sulfonated steroids between an aqueous phase and an organic solvent, tert butyl-methylether. The immunostaining results were shown that SULTs 1E1 and 2B1, immunostaining was especially prominent in superficial epithelial protrusions. Sporadic staining of weaker intensity was also found in the muscular layer and in the vascular endothelium. With WB, a specific band of the expected molecular size (approx. 61 kDa) was found in the testis and all segments of the epididymis. These results show that STS is widely expressed in the porcine testicular-epididymal compartment, indicating a high potential for sulfatase pathways especially in Leydig cells and the epithelial cells of the rete testis and epididymis. The co-expression of STS with SULTs 1E1 and 2B1 in the epididymal epithelium and especially their colocalization in superficial protrusions are very intriguing. In the epididymal duct, apocrine secretion has been described to give rise to the formation of epididymosomes, small vesicles which are considered as vehicles for the transfer of certain molecules to the maturing sperm cells. Other intriguing findings are the virtual absence of a sulfonation of E1, DHEA and P5 in testicular cytosols as well as the absent or questionable detection of SULTs 1E1 and 2B1 in light of the high efflux of various steroid sulfates from the testis. A plausible explanation could be a significant use of sulfonated steroids as precursors/intermediates in porcine testicular steroidogenesis starting from cholesterol sulfate. The concept of a “sulfate pathway” of steroidogenesis would not only provide an explanation for the production of high amounts of steroid sulfates in the virtual absence of relevant steroid sulfotransferase activities but also for the high STS expression in Leydig cells. According to this concept, STS could play a crucial role in the control of the substrate flow through the steroidogenic enzyme cascade by mediating the transition of sulfonated precursors into the pool of free steroids, with the exact subcellular localization being of importance for the step of the enzyme cascade at which this transition(s) may occur. Thus, in order to corroborate this concept investigations into the utilization of sulfonated substrates by steroidogenic enzymes and on the subcellular localization of STS are necessary

Expanding the chemical scope of RNA:methyltransferases to site-specific alkynylation of RNA for click labeling

This work identifies the combination of enzymatic transfer and click labeling as an efficient method for the site-specific tagging of RNA molecules for biophysical studies. A double-activated analog of the ubiquitous co-substrate S-adenosyl-l-methionine was employed to enzymatically transfer a five carbon chain containing a terminal alkynyl moiety onto RNA. The tRNA:methyltransferase Trm1 transferred the extended alkynyl moiety to its natural target, the N2 of guanosine 26 in tRNAPhe. LC/MS and LC/MS/MS techniques were used to detect and characterize the modified nucleoside as well as its cycloaddition product with a fluorescent azide. The latter resulted from a labeling reaction via Cu(I)-catalyzed azide-alkyne 1,3-cycloaddition click chemistry, producing site-specifically labeled RNA whose suitability for single molecule fluorescence experiments was verified in fluorescence correlation spectroscopy experiments

Characterizing Structural Transitions Using Localized Free Energy Landscape Analysis

Structural changes in molecules are frequently observed during biological processes like replication, transcription and translation. These structural changes can usually be traced to specific distortions in the backbones of the macromolecules involved. Quantitative energetic characterization of such distortions can greatly advance the atomic-level understanding of the dynamic character of these biological processes.Molecular dynamics simulations combined with a variation of the Weighted Histogram Analysis Method for potential of mean force determination are applied to characterize localized structural changes for the test case of cytosine (underlined) base flipping in a GTCAGCGCATGG DNA duplex. Free energy landscapes for backbone torsion and sugar pucker degrees of freedom in the DNA are used to understand their behavior in response to the base flipping perturbation. By simplifying the base flipping structural change into a two-state model, a free energy difference of upto 14 kcal/mol can be attributed to the flipped state relative to the stacked Watson-Crick base paired state. This two-state classification allows precise evaluation of the effect of base flipping on local backbone degrees of freedom.The calculated free energy landscapes of individual backbone and sugar degrees of freedom expectedly show the greatest change in the vicinity of the flipping base itself, but specific delocalized effects can be discerned upto four nucleotide positions away in both 5' and 3' directions. Free energy landscape analysis thus provides a quantitative method to pinpoint the determinants of structural change on the atomic scale and also delineate the extent of propagation of the perturbation along the molecule. In addition to nucleic acids, this methodology is anticipated to be useful for studying conformational changes in all macromolecules, including carbohydrates, lipids, and proteins

Structure Analysis of Entamoeba histolytica DNMT2 (EhMeth)

In eukaryotes, DNA methylation is an important epigenetic modification that is generally involved in gene regulation. Methyltransferases (MTases) of the DNMT2 family have been shown to have a dual substrate specificity acting on DNA as well as on three specific tRNAs (tRNAAsp, tRNAVal, tRNAGly). Entamoeba histolytica is a major human pathogen, and expresses a single DNA MTase (EhMeth) that belongs to the DNMT2 family and shows high homology to the human enzyme as well as to the bacterial DNA MTase M.HhaI. The molecular basis for the recognition of the substrate tRNAs and discrimination of non-cognate tRNAs is unknown. Here we present the crystal structure of the cytosine-5-methyltransferase EhMeth at a resolution of 2.15 Å, in complex with its reaction product S-adenosyl-L-homocysteine, revealing all parts of a DNMT2 MTase, including the active site loop. Mobility shift assays show that in vitro the full length tRNA is required for stable complex formation with EhMeth

DNA methylation in the promoter region of the p16 (CDKN2/MTS-1/INK4A) gene in human breast tumours

The p16 (CDKN2/MTS-1/INK4A) gene is one of several tumour-suppressor genes that have been shown to be inactivated by DNA methylation in various human cancers including breast tumours. We have used bisulphite genomic sequencing to examine the detailed sequence specificity of DNA methylation in the CpG island promoter/exon 1 region in the p16 gene in DNA from a series of human breast cancer specimens and normal human breast tissue (from reductive mammaplasty). The p16 region examined was unmethylated in the four normal human breast specimens and in four out of nine breast tumours. In the other five independent breast tumour specimens, a uniform pattern of DNA methylation was observed. Of the nine major sites of DNA methylation in the amplified region from these tumour DNAs, four were in non-CG sequences. This unusual concentration of non-CG methylation sites was not a general phenomenon present throughout the genome of these tumour cells because the methylated CpG island regions of interspersed L1 repeats had a pattern of (almost exclusively) CG methylation similar to that found in normal breast tissue DNA and in DNA from tumours with unmethylated p16 genes. These data suggest that DNA methylation of the p16 gene in some breast tumours could be the result of an active process that generates a discrete methylation pattern and, hence, could ultimately be amenable to theraputic manipulation. © 1999 Cancer Research Campaig

Epigenetic polypharmacology: from combination therapy to multitargeted drugs

The modern drug discovery process has largely focused its attention in the so-called magic bullets, single chemical entities that exhibit high selectivity and potency for a particular target. This approach was based on the assumption that the deregulation of a protein was causally linked to a disease state, and the pharmacological intervention through inhibition of the deregulated target was able to restore normal cell function. However, the use of cocktails or multicomponent drugs to address several targets simultaneously is also popular to treat multifactorial diseases such as cancer and neurological disorders. We review the state of the art with such combinations that have an epigenetic target as one of their mechanisms of action. Epigenetic drug discovery is a rapidly advancing field, and drugs targeting epigenetic enzymes are in the clinic for the treatment of hematological cancers. Approved and experimental epigenetic drugs are undergoing clinical trials in combination with other therapeutic agents via fused or linked pharmacophores in order to benefit from synergistic effects of polypharmacology. In addition, ligands are being discovered which, as single chemical entities, are able to modulate multiple epigenetic targets simultaneously (multitarget epigenetic drugs). These multiple ligands should in principle have a lower risk of drug-drug interactions and drug resistance compared to cocktails or multicomponent drugs. This new generation may rival the so-called magic bullets in the treatment of diseases that arise as a consequence of the deregulation of multiple signaling pathways provided the challenge of optimization of the activities shown by the pharmacophores with the different targets is addressed