High-throughput tandem-microwell assay identifies inhibitors of the hydrogen sulfide signaling pathway.

We report a high-throughput assay for H2S-producing enzymes, which is based on a newly designed tandem-well plate. Screening of 21,599 agents identified several potent inhibitors of cystathionine ß-synthase and cystathionine γ-lyase, the two key enzymes generating H2S in mammals, with IC50 values in the low two-digit micromolar range.

Structure and mechanism of a cysteine sulfinate desulfinase engineered on the aspartate aminotransferase scaffold.

The joint substitution of three active-site residues in Escherichia coli (L)-aspartate aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 10(5)-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, k(cat)' for desulfination of l-cysteine sulfinate increased to 0.5s(-1) (from 0.05s(-1) in wild-type enzyme), whereas k(cat)' for transamination of the same substrate was reduced from 510s(-1) to 0.05s(-1). Similarly, k(cat)' for ß-decarboxylation of l-aspartate increased from<0.0001s(-1) to 0.07s(-1), whereas k(cat)' for transamination was reduced from 530s(-1) to 0.13s(-1). l-Aspartate aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-aspartate ß-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate form or liganded with a covalent coenzyme-substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme-substrate intermediates by bulk water.

Tyrosine Phosphorylation in the C-Terminal Nuclear Localization and Retention Signal (C-NLS) of the EWS Protein.

Ewing sarcoma (EWS) proto-oncoprotein, an RNA-binding protein, is involved in DNA recombination and repair, gene expression, RNA processing and transport, as well as cell signalling. Chimeric EWS oncoproteins generated by chromosomal translocations between EWSR1 and the genes of transcription factors cause malignant tumors. To understand the loss of function by these translocations, the role of the intact EWS protein has to be investigated. The predominantly nuclear localization of the EWS protein via a transportin-1-mediated mechanism is dependent on the recently identified C-NLS (also known as PY-NLS). Among other residues in the C-NLS, Y656 interacts with transportin-1 and is essential for its nuclear localization. Here, we show that Y656 is phosphorylated, which seems to be a critical factor for transportin-1-mediated nuclear import. If Y656 was mutated cytosolic aggregates of the EWS protein, colocalized with transportin-1, were observed, similar to those described with mutants of the closely related FUS/TLS protein that had amino acid substitutions in the PY-NLS causing familial amyothrophic lateral sclerosis.

A novel approach to inhibit intracellular vitamin B6-dependent enzymes: proof of principle with human and plasmodium ornithine decarboxylase and human histidine decarboxylase.

Pyridoxal-5'-phosphate (vitamin B(6))-dependent enzymes play central roles in the metabolism of amino acids. Moreover, the synthesis of polyamines, which are essential for cell growth, and of biogenic amines, such as histamine and other signal transmitters, relies on these enzymes. Certain B(6) enzymes thus are prime targets for pharmacotherapeutic intervention. We have devised a novel, in principle generally applicable strategy for obtaining small-molecule cell-permeant inhibitors of specific B(6) enzymes. The imine adduct of pyridoxal-5'-phosphate and the specific amino acid substrate, the first intermediate in all pyridoxal-5'-phosphate-dependent reactions of amino acids, was reduced to a stable secondary amine. This coenzyme-substrate-conjugate was modified further to make it membrane-permeant and, guided by structure-based modeling, to boost its affinity to the apoform of the target enzyme. Inhibitors of this type effectively decreased the respective intracellular enzymatic activity (IC(50) in low micromolar range), providing lead compounds for inhibitors of human ornithine decarboxylase (hODC), plasmodium ornithine decarboxylase, and human histidine decarboxylase. The inhibitors of hODC interfere with the metabolism of polyamines and efficiently prevent the proliferation of tumor cell lines (IC(50)∼ 25 µM). This approach to specific inhibition of intracellular B(6) enzymes might be applied in a straightforward manner to other B(6) enzymes of emerging medicinal interest.

Analysis of Ewing sarcoma (EWS)-binding proteins: interaction with hnRNP M, U, and RNA-helicases p68/72 within protein-RNA complexes.

The human Ewing Sarcoma (EWS) protein belongs to the TET family of RNA-binding proteins and consists of an N-terminal transcriptional activation domain (EAD) and a C-terminal RNA-binding domain (RBD), which is extensively methylated at arginine residues. This multifunctional protein acts in transcriptional co-activation, DNA-recombination, -pairing and -repair, in splicing, and mRNA transport. The role of arginine methylation in these processes as well as the time and place of methylation within cells is still unclear. In this study, we show that methylation of recombinant EWS protein in HEK cells occurs immediately after or even during translation. Pull-down experiments with recombinant EWS protein as bait, followed by mass spectrometric analysis identified more than 30 interacting proteins independent of whether the EWS protein was methylated or not. The EWS protein interacts via its RBD with RNase-sensitive protein complexes consisting of mainly heterogeneous nuclear ribonucleoproteins (hnRNPs) and RNA helicases. HnRNP M and U, the RNA-helicases p68 and p72, but also actin and tubulin were found to interact directly with the EWS protein. Co-precipitation experiments with recombinant proteins confirmed the interaction of the EWS protein with p68 via its RBD. Colocalization of the EWS protein and the RNA-helicases in the nucleus of HEK cells was visualized by expressing labeled EWS protein and p68 or p72. When co-expressed, the labeled proteins relocated from the nucleoplasm to nucleolar capping structures. As arginine methylation within the RBD of the EWS protein are neither needed for its subcellular localization nor for its protein-protein interaction, a role of EWS protein methylation in RNA-binding and affecting the activation/repression activity or even in the stabilization of the EWS protein seems very likely.

Poisoning pyridoxal 5-phosphate-dependent enzymes: a new strategy to target the malaria parasite Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum is able to synthesize de novo pyridoxal 5-phosphate (PLP), a crucial cofactor, during erythrocytic schizogony. However, the parasite possesses additionally a pyridoxine/pyridoxal kinase (PdxK) to activate B6 vitamers salvaged from the host. We describe a strategy whereby synthetic pyridoxyl-amino acid adducts are channelled into the parasite. Trapped upon phosphorylation by the plasmodial PdxK, these compounds block PLP-dependent enzymes and thus impair the growth of P. falciparum. The novel compound PT3, a cyclic pyridoxyl-tryptophan methyl ester, inhibited the proliferation of Plasmodium very efficiently (IC(50)-value of 14 microM) without harming human cells. The non-cyclic pyridoxyl-tryptophan methyl ester PT5 and the pyridoxyl-histidine methyl ester PHME were at least one order of magnitude less effective or completely ineffective in the case of the latter. Modeling in silico indicates that the phosphorylated forms of PT3 and PT5 fit well into the PLP-binding site of plasmodial ornithine decarboxylase (PfODC), the key enzyme of polyamine synthesis, consistent with the ability to abolish ODC activity in vitro. Furthermore, the antiplasmodial effect of PT3 is directly linked to the capability of Plasmodium to trap this pyridoxyl analog, as shown by an increased sensitivity of parasites overexpressing PfPdxK in their cytosol, as visualized by GFP fluorescence.

Dynamic subcellular localization of the Ewing sarcoma proto-oncoprotein and its association with and stabilization of microtubules.

The Ewing sarcoma (EWS) protein is a member of a large family of RNA-binding proteins. Chimeric EWS oncoproteins generated by chromosomal translocations between the EWS protein and several transcription factors cause various malignant tumors. Due to its multifunctional properties, the EWS protein is involved in such processes as meiotic DNA pairing/recombination, cellular senescence, gene expression, RNA processing and transport, and cell signaling. The EWS protein is predominantly located in the nucleus. It was found in the cytoplasm and associated with the cell membrane. In this study, analysis of the localization of endogenous and fluorescently labeled recombinant EWS protein in different phases of the cell cycle in different cell lines revealed a very dynamic subcellular distribution of the EWS protein. In Cos7 and HeLa cells, an association of the EWS protein with the centrosomal compartments was shown. Furthermore, in HEK (human embryonic kidney)-293 (T) cells, an interaction of the overexpressed recombinant EWS-yellow fluorescent protein fusion protein with microtubules, leading to their stabilization and cell cycle arrest, was demonstrated. As an outlook, the present findings provide an important insight into temporally and spatially regulated functions of the EWS protein and, particularly, into its role in the regulation of the cell cycle and possibly cell differentiation.

Structural requirements for novel coenzyme-substrate derivatives to inhibit intracellular ornithine decarboxylase and cell proliferation.

Creating transition-state mimics has proven to be a powerful strategy in developing inhibitors to treat malignant diseases in several cases. In the present study, structurally diverse coenzyme-substrate derivatives mimicking this type for pyridoxal 5'-phosphate-dependent human ornithine decarboxylase (hODC), a potential anticancer target, were designed, synthesized, and tested to elucidate the structural requirements for optimal inhibition of intracellular ODC as well as of tumor cell proliferation. Of 23 conjugates, phosphopyridoxyl- and pyridoxyl-L-tryptophan methyl ester (pPTME, PTME) proved significantly more potent in suppression proliferation (IC(50) up to 25 microM) of glioma cells (LN229) than alpha-DL-difluoromethylornithine (DFMO), a medically used irreversible inhibitor of ODC. In agreement with molecular modeling predictions, the inhibitory action of pPTME and PTME toward intracellular ODC of LN229 cells exceeded that of the previous designed lead compound POB. The inhibitory active compounds feature hydrophobic side chain fragments and a kind of polyamine motif (-NH-(CH(X))(4)-NH-). In addition, they induce, as polyamine analogs often do, the activity of the polyamine catabolic enzymes polyamine oxidase and spermine/spermidine N(1)-acetyltransferase up to 250 and 780%, respectively. The dual-action mode of these compounds in LN229 cells affects the intracellular polyamine metabolism and might underlie the more favorable cell proliferation inhibition in comparison with DFMO.

Identification of proteins interacting with protein arginine methyltransferase 8: the Ewing sarcoma (EWS) protein binds independent of its methylation state.

Protein arginine methylation is a eukaryotic posttranslational modification that plays a role in transcription, mRNA splicing and transport, in protein-protein interaction, and cell signaling. The type I protein arginine methyltransferase (PRMT) 8 is the only member of the human PRMT family that is localized at the cell membrane and its endogenous substrates have remained unknown as yet. Although PRMT8 was supposed to be expressed only in brain tissue, its presence in HEK 293 (T) cells could be demonstrated. We identified more than 20 PRMT8-binding partners in pull-down experiments using recombinant PRMT8 as bait followed by mass spectrometric identification of the bound proteins. Among the extracted proteins were several heterogeneous nuclear ribonucleoproteins (hnRNP), RNA-helicases (DEAD box proteins), the TET-family proteins TLS, Ewing's sarcoma (EWS), and TAF(II)68, and caprin, which all contain RGG methylation motifs and are potential substrates of PRMT8. Additionally, actin, tubulin, and heat shock proteins belong to the identified proteins. The interaction between PRMT8 and the EWS protein was characterized in more detail. Although binding of endogenous and recombinant EWS protein to PRMT8 as well as colocalization in HEK cells was observed, in vitro methylation assays revealed a rather poor methyltransferase activity of PRMT8 towards the EWS protein and a synthetic RGG-rich reference peptide (K(m), 1.3 microM; k(cat)/K(m), 2.8 x 10(-4) microM(-1) s(-1)) in comparison to PRMT1 (K(m), 0.8 microM; k(cat)/K(m), 8.1 x 10(-3) microM(-1) s(-1)). In contrast, substrate proteins within a cell extract could be methylated by PRMT8 as efficient as by PRMT1. The main interaction site of the EWS protein with PRMT8 was determined to be the C-terminal RGG box 3. Remarkably, complete methylation of the EWS protein did not abrogate the binding to PRMT8, pointing to an adapter role of PRMT8 for nuclear proteins at the cell membrane in addition to its methyltransferase activity.

Inhibitory and structural studies of novel coenzyme-substrate analogs of human histidine decarboxylase.

Histamine, a biogenic amine with important biological functions, is produced from histidine by histidine decarboxylase (HDC), a pyridoxal 5'-phosphate-dependent enzyme. HDC is thus a potential target to attenuate histamine production in certain pathological states. Targeting mammalian HDC with novel inhibitors and elucidating the structural basis of their specificity for HDC are challenging tasks, because the three-dimensional structure of mammalian HDC is still unknown. In the present study, we designed, synthesized, and tested potentially membrane-permeable pyridoxyl-substrate conjugates as inhibitors for human (h) HDC and modeled an active site of hHDC, which is compatible with the experimental data. The most potent inhibitory compound among nine tested structural variants was the pyridoxyl-histidine methyl ester conjugate (PHME), indicating that the binding site of hHDC does not tolerate groups other than the imidazole side chain of histidine. PHME inhibited 60% of the fraction of 12-O-tetradecanoylphorbol-13-acetate-induced newly synthesized HDC in human HMC-1 cells at 200 microM and was also inhibitory in cell extracts. The proposed model of hHDC, containing phosphopyridoxyl-histidine in the active site, revealed the binding specificity of HDC toward its substrate and the structure-activity relationship of the designed and investigated compounds.

New transition state-based inhibitor for human ornithine decarboxylase inhibits growth of tumor cells.

Pyridoxal 5'-phosphate (PLP)-dependent ornithine decarboxylase (ODC) is the key enzyme in polyamine synthesis. ODC is overexpressed in many tumor cells and thus a potential drug target. Here we show the design and synthesis of a coenzyme-substrate analogue as a novel precursor inhibitor of ODC. Structural analysis of the crystal structure of human ODC disclosed an additional hydrophobic pocket surrounding the epsilon-amino group of its substrate ornithine. Molecular modeling methods showed favorable interactions of the BOC-protected pyridoxyl-ornithine conjugate, termed POB, in the active site of human ODC. The synthesized and purified POB completely inhibited the activity of newly induced ODC activity at 100 micromol/L in glioma LN229 and COS7 cells. In correlation with the inhibition of ODC activity, a time-dependent inhibition of cell growth was observed in myeloma, glioma LN18 and LN229, Jurkat, COS7, and SW2 small-cell lung cancer cells if DNA synthesis and cell number were measured, but not in the nontumorigenic human aortic smooth muscle cells. POB strongly inhibited cell proliferation not only of low-grade glioma LN229 cells in a dose-dependent manner (IC(50) approximately 50 micromol/L) but also of high-grade glioblastoma multiforme cells. POB is much more efficient in inhibiting proliferation of several types of tumor cells than alpha-DL-difluoromethylornithine, the best known irreversible inhibitor of ODC.

Protein arginine methylation: Cellular functions and methods of analysis.

During the last few years, new members of the growing family of protein arginine methyltransferases (PRMTs) have been identified and the role of arginine methylation in manifold cellular processes like signaling, RNA processing, transcription, and subcellular transport has been extensively investigated. In this review, we describe recent methods and findings that have yielded new insights into the cellular functions of arginine-methylated proteins, and we evaluate the currently used procedures for the detection and analysis of arginine methylation.

Identification and characterization of the nuclear localization/retention signal in the EWS proto-oncoprotein.

Ewing sarcoma (EWS) protein, a member of a large family of RNA-binding proteins, contains an N-terminal transcriptional activation domain (EAD) and a C-terminal RNA-binding domain (RBD). Due to its multifunctional properties EWS protein is involved in processes such as gene expression, RNA processing and transport, and cell signaling. Chimeric EWS proteins generated by chromosomal translocations cause malignant tumors. EWS protein is located predominantly in the nucleus, but was found also in the cytosol and associated with the cell membrane. The determinants responsible for the nuclear localization of the protein were as yet unknown. We identified the nuclear localization signal of EWS protein at its C terminus (C-NLS), which is required for the nuclear import and retention of the protein. The C-NLS sequence is conserved in related proto-oncoproteins suggesting an NLS function also in these proteins. Two arginine residues, due to their positive charge, a proline residue and a tyrosine residue are essential for C-NLS function. The nuclear localization of EWS protein is independent of the regions in RBD containing numerous arginine methylation sites, RNA-recognition and zinc finger motifs. Regions in EAD guide the subnuclear partition of EWS protein and contain another but different NLS that allows nucleocytoplasmic shuttling of the N-terminal domain.

Different methylation characteristics of protein arginine methyltransferase 1 and 3 toward the Ewing Sarcoma protein and a peptide.

The multifunctional Ewing Sarcoma (EWS) protein, a member of a large family of RNA-binding proteins, is extensively asymmetrically dimethylated at arginine residues within RGG consensus sequences. Using recombinant proteins we examined whether type I protein arginine methyltransferase (PRMT)1 or 3 is responsible for asymmetric dimethylations of the EWS protein. After in vitro methylation of the EWS protein by GST-PRMT1, we identified 27 dimethylated arginine residues out of 30 potential methylation sites by mass spectrometry-based techniques (MALDI-TOF MS and MS/MS). Thus, PRMT1 recognizes most if not all methylation sites of the EWS protein. With GST-PRMT3, however, only nine dimethylated arginines, located mainly in the C-terminal region of EWS protein, could be assigned, indicating that structural determinants prevent complete methylation. In contrary to previous reports this study also revealed that trypsin is able to cleave after methylated arginines. Pull-down experiments showed that endogenous EWS protein binds efficiently to GST-PRMT1 but less to GST-PRMT3, which is in accordance to the in vitro methylation results. Furthermore, methylation of a peptide containing different methylation sites revealed differences in the site selectivity as well as in the kinetic properties of GST-PRMT1 and GST-PRMT3. Kinetic differences due to an inhibition effect of the methylation inhibitor S-adenosyl-L-homocysteine could be excluded by determining the corresponding K(i) values of the two enzymes and the K(d) values for the methyl donor S-adenosyl-L-methionine. The study demonstrates the strength of MS-based methods for a qualitative and quantitative analysis of enzymic arginine methylation, a posttranslational modification that becomes more and more the object of investigations.

Structural studies of the catalytic reaction pathway of a hyperthermophilic histidinol-phosphate aminotransferase.

In histidine biosynthesis, histidinol-phosphate aminotransferase catalyzes the transfer of the amino group from glutamate to imidazole acetol-phosphate producing 2-oxoglutarate and histidinol phosphate. In some organisms such as the hyperthermophile Thermotoga maritima, specific tyrosine and aromatic amino acid transaminases have not been identified to date, suggesting an additional role for histidinol-phosphate aminotransferase in other transamination reactions generating aromatic amino acids. To gain insight into the specific function of this transaminase, we have determined its crystal structure in the absence of any ligand except phosphate, in the presence of covalently bound pyridoxal 5'-phosphate, of the coenzyme histidinol phosphate adduct, and of pyridoxamine 5'-phosphate. The enzyme accepts histidinol phosphate, tyrosine, tryptophan, and phenylalanine, but not histidine, as substrates. The structures provide a model of how these different substrates could be accommodated by histidinol-phosphate aminotransferase. Some of the structural features of the enzyme are more preserved between the T. maritima enzyme and a related threonine-phosphate decarboxylase from S. typhimurium than with histidinol-phosphate aminotransferases from different organisms.

Human connective tissue growth factor expressed in Escherichia coli is a non-mitogenic inhibitor of apoptosis.

After cloning and sequencing chicken connective tissue growth factor (cCTGF) we showed that in serum-deprived chicken embryo fibroblasts (CEF) the initial decline of cCTGF mRNA was followed by an increase. Human CTGF that was produced as a soluble protein in Escherichia coli by thioredoxin co-expression inhibited serum-deprivation induced apoptosis of CEF at I(50) of approximately 8pM, demonstrating a function of CTGF at concentrations observed in supernatants of fibroblasts. CTGF was neither mitogenic for CEF nor for other cell types. In conclusion CTGF supports very efficiently survival of certain cells without stimulating their cell growth.

Expression and subcellular localization of Ewing sarcoma (EWS) protein is affected by the methylation process.

Ewing sarcoma (EWS) protein contains an N-terminal transcriptional activation domain (EAD) and a C-terminal RNA-binding domain (RBD). Recently, we had shown that EWS protein is not only localized in the nucleus and cytosol, but also on the surface of T cells and that its RBD is extensively asymmetrically dimethylated on arginine residues. Here we show that stimulation of T cells with phytohemagglutinin (PHA) caused a time-dependent 10-fold increase in expression of methylated EWS protein on the cell surface and a sixfold increase in the nuclei of peripheral blood mononuclear cells (PBMC). Mitogenic stimulation of malignant T cell lines, however, did not increase their inherently high expression of EWS protein. This expression seemed to correlate with methionine adenosyltransferase activity and S-adenosyl-L-methionine (AdoMet) utilization in PBMC and tumor cells and thus indicates dependence on the methylation process. Inhibition of methylation in normal and malignant cells with the methylation inhibitor adenosine dialdehyde (AdOx) resulted in a three to fivefold decreased expression of EWS protein not only in the nucleus but also on the cell surface. The inhibitory effect of AdOx was compensated and negligible in PBMC, but not in tumor cells if they were treated simultaneously with mitogenic PHA concentrations. The present findings indicate that expression of EWS protein in the various subcellular compartments is affected by the methylation process, in particular by the availability of intracellular AdoMet.