Ligand-induced changes in estrogen receptor conformation as measured by site-directed spin labeling

Site-directed spin labeling (SDSL), the site-specific incorporation of nitroxide spin-labels into a protein, has allowed us to investigate ligand-induced conformational changes in the ligand-binding domain of human estrogen receptor alpha (hERalpha-LBD). EPR (electron paramagnetic resonance) spectroscopy of the nitroxide probe attached to ER produces different spectra depending upon the identity of the bound ligand; these differences are indicative of changes in the type and degree of motional character of the spin-label induced by different ligand-induced conformations of labeled ER. Visual inspection of EPR spectra, construction of B versus C cross-correlation plots, and cross-comparison of spectral pairs using a relative squared difference (RSD) calculation allowed receptor-ligand complexes to be profiled according to their conformational character. Plotting B and C parameters allowed us to evaluate the liganded receptor according to the motional characteristics of the attached spin-label, and they were particularly illustrative for the receptor labeled at position 530, which had motion between the fast and intermediate regimes. RSD analysis allowed us to directly compare the similarity or difference between two different spectra, and these comparisons produced groupings that paralleled those seen in B versus C cross-correlation plots, again relating meaningfully with the pharmacological nature of the bound ligand. RSD analysis was also particularly useful for qualifying differences seen with the receptor labeled at position 417, which had motion between the intermediate and slow motional regimes. This work demonstrates that B and C formulas from EPR line shape theory are useful for qualitative analysis of spectra with differences subtler than those that are often analyzed by EPR spectroscopists. This work also provides evidence that the ER can exist in a range of conformations, with specific conformations resulting from preferential stabilization of ER by the bound ligand. Furthermore, it documents the complexity and uniqueness of the ligand-receptor structure, and highlights the fact that structural differences exist between the receptor bound with ligands of different pharmacological character that, nevertheless, produce similar crystal structures.     

Bovine Brain Myelin Glycerophosphocholine Choline Phosphodiesterase is an Alkaline Lysosphingomyelinase of the eNPP-Family, Regulated by Lysosomal Sorting

Glycerophosphocholine choline phosphodiesterase (GPC-Cpde) is a glycosylphosphatidylinositol (GPI)-anchored alkaline hydrolase that is expressed in the brain and kidney. In brain the hydrolase is synthesized by the oligodendrocytes and expressed on the myelin membrane. There are two forms of brain GPC-Cpde, a membrane-linked (mGPC-Cpde) and a soluble (sGPC-Cpde). Here we report the characterisation sGPC-Cpde from bovine brain. The amino acid sequence was identical to ectonucleotide pyrophosphatase/phosphodiesterase 6 (eNPP6) precursor, lacking the N-terminal signal peptide region and a C-terminal stretch, suggesting that the hydrolase was solubilised by C-terminal proteolysis, releasing the GPI-anchor. sGPC-Cpde existed as two isoforms, a homodimer joined by a disulfide bridge linking C414 from each monomer, and a monomer resulting from proteolysis N-terminally to this disulfide bond. The only internal disulfide bridge, linking C142 and C154, stabilises the choline-binding pocket. sGPC-Cpde was specific for lysosphingomyelin, displaying 1 to 2 orders of magnitude higher catalytic activity than towards GPC and lysophosphatidylcholine, suggesting that GPC-Cpde may function in the sphingomyelin signaling, rather than in the homeostasis of acylglycerophosphocholine metabolites. The truncated high mannose and bisected hybrid type glycans linked to N118 and N341 of sGPC-Cpde is a hallmark of glycans in lysosomal glycoproteins, subjected to GlcNAc-1-phosphorylation en route through Golgi. Thus, sGPC-Cpde may originate from the lysosomes, suggesting that lysosomal sorting contributes to the level of mGPC-Cpde on the myelin membrane.     

Trypanothione Reductase: A Target Protein for a Combined In Vitro and In Silico Screening Approach

With the goal to identify novel trypanothione reductase (TR) inhibitors, we performed a combination of in vitro and in silico screening approaches. Starting from a highly diverse compound set of 2,816 compounds, 21 novel TR inhibiting compounds could be identified in the initial in vitro screening campaign against T. cruzi TR. All 21 in vitro hits were used in a subsequent similarity search-based in silico screening on a database containing 200,000 physically available compounds. The similarity search resulted in a data set containing 1,204 potential TR inhibitors, which was subjected to a second in vitro screening campaign leading to 61 additional active compounds. This corresponds to an approximately 10-fold enrichment compared to the initial pure in vitro screening. In total, 82 novel TR inhibitors with activities down to the nM range could be identified proving the validity of our combined in vitro/in silico approach. Moreover, the four most active compounds, showing IC₅₀ values of <1 μM, were selected for determining the inhibitor constant. In first on parasites assays, three compounds inhibited the proliferation of bloodstream T. brucei cell line 449 with EC₅₀ values down to 2 μM.     

Endogenous and environmental factors influence 35S promoter methylation of a maize A1 gene construct in transgenic petunia and its colour phenotype

Summary 30000 transgenic petunia plants carrying a single copy of the maize A1 gene, encoding a dihydroflavonol reductase, which confers a salmon red flower colour phenotype on the petunia plant, were grown in a field test. During the growing season plants with flowers deviating from this salmon red colour, such as those showing white or variegated phenotypes and plants with flowers exhibiting only weak pigmentation were observed with varying frequencies. While four white flowering plants were shown at the molecular level to be mutants in which part of the A1 gene had been deleted, other white flowering plants, as well as 13 representative plants tested out of a total of 57 variegated individuals were not mutants but rather showed hypermethylation of the 35S promoter directing A1 gene expression. This was in contrast to the homogeneous fully red flowering plants in which no methylation of the 35S promoter was observed. While blossoms on plants flowering early in the season were predominantly red, later flowers on the same plants showed weaker coloration. Once again the reduction of the A1-specific phenotype correlated with the methylation of the 35S promoter. This variation in coloration seems to be dependent not only on exogenous but also on endogenous factors such as the age of the parental plant from which the seed was derived or the time at which crosses were made.     

De novo and maintenance DNA methylation by a mouse plasmacytoma cell DNA methyltransferase

A DNA methyltransferase of Mr = 140,000 that is active on both unmethylated and hemimethylated DNA substrates has been purified from the murine plasma-cytoma cell line MPC 11. The maximal rate of methylation was obtained with maintenance methylation of hemimethylated Micrococcus luteus or M13 DNAs. At low enzyme concentrations, the highest rate of de novo methylation occurred with single-stranded DNA or relatively short duplex DNA containing single-stranded regions. Strong substrate inhibition was observed with hemimethylated but not unmethylated DNA substrates. Fully methylated single-stranded M13 phage DNA inhibited neither the de novo nor the maintenance reactions, but unmethylated single-stranded M13 DNA strongly inhibited the maintenance reaction. The kinetics observed with hemimethylated and single-stranded substrates could be explained if the enzyme were to bind irreversibly to a DNA molecule and to aggregate if present in molar excess. Such aggregates would be required for activity upon hemimethylated but not single-stranded DNA. For de novo methylation of duplex DNA, single-stranded regions or large amounts of methyltransferase appear to be required. The relative substrate preference for the enzyme is hemimethylated DNA greater than fully or partially single-stranded DNA greater than fully duplex DNA.