Heinz Gehring

Isolation, crystallization and preliminary crystallographic data of aspartate aminotransferase from chicken heart mitochondria☆

Abstract The mitochondrial isoenzyme of aspartate aminotransferase (E.C. 2.6.1.1) has been isolated from chicken heart in an electrophoretically and immunologically homogeneous form. Large, well-diffracting single crystals of this enzyme, a dimeric molecule with a molecular weight of 90,000, have been grown by vapour phase diffusion against polyethylene glycol solutions. The crystals belong to space group P1. The unit cell, with the dimensions a = 55.6 A , 6 = 58.7 A , c = 76.0 A , α = 85.3 °, β = 109.2 °, γ = 115.6 °, contains a single dimer. The diffraction pattern extends to at least 2.1 A resolution.

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 (IC50-value of 14 µM) 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.

Peptide mapping of protein bands from polyacrylamide gel electrophoresis by chemical cleavage in gel pleces and re-electrophoresis☆

Abstract A simple method has been developed for peptide mapping of protein bands obtained by polyacrylamide gel electrophoresis. The procedure is based on selective acid hydrolysis of aspartyl-prolyl bonds which occur in proteins with an average frequency of 1 per 400 amino acid residues. A gel piece containing the protein to be analyzed is soaked with 75% formie acid. For the subsequent incubation at 37°C for 24 h the gel piece is immersed in liquid paraffin. After removal of formic acid by lyophilization the gel piece is rehydrated in buffer and placed into the sample well of a second polyacrylamide gel on which the generated peptides are electrophoretically separated.

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, Ewings sarcoma (EWS), and TAFII68, 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 (Km, 1.3 μM; kcat/Km, 2.8 × 10−4 μM−1 s−1) in comparison to PRMT1 (Km, 0.8 μM; kcat/Km, 8.1 × 10−3 μM−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. Proteins 2008.

Maturation-induced Conformational Changes of HIV-1 Capsid Protein and Identification of Two High Affinity Sites for Cyclophilins in the C-terminal Domain

Viral incorporation of cyclophilin A (CyPA) during the assembly of human immunodeficiency virus type-1 (HIV-1) is crucial for efficient viral replication. CyPA binds to the previously identified Gly-Pro90 site of the capsid protein p24, but its role remained unclear. Here we report two new interaction sites between cyclophilins and p24. Both are located in the C-terminal domain of p24 around Gly-Pro157 and Gly-Pro224. Peptides corresponding to these regions showed higher affinities (K d ∼ 0.3 μm) for both CyPA and cyclophilin B than the best peptide derived from the Gly-Pro90 site (∼8 μm) and thus revealed new sequence motifs flanking Gly-Pro that are important for tight interaction of peptide ligands with cyclophilins. Between CyPA and an immature (unprocessed) form of p24, a K d of ∼8 μm was measured, which corresponded with theK d of the best of the Gly-Pro90peptides, indicating an association via this site. Processing of immature p24 by the viral protease, yielding mature p24, elicited a conformational change in its C-terminal domain that was signaled by the covalently attached fluorescence label acrylodan. Consequently, CyPA and cyclophilin B bound with much higher affinities (∼0.6 and 0.25 μm) to the new, i.e. maturation-generated sites. Since this domain is essential for p24 oligomerization and capsid cone formation, CyPA bound to the new sites might impair the regularity of the capsid cone and thus facilitate in vivocore disassembly after host infection.

Conversion of aspartate aminotransferase into an L-aspartate beta-decarboxylase by a triple active-site mutation.

The conjoint substitution of three active-site residues in aspartate aminotransferase (AspAT) of Escherichia coli (Y225R/R292K/R386A) increases the ratio ofl-aspartate β-decarboxylase activity to transaminase activity >25 million-fold. This result was achieved by combining an arginine shift mutation (Y225R/R386A) with a conservative substitution of a substrate-binding residue (R292K). In the wild-type enzyme, Arg386 interacts with the α-carboxylate group of the substrate and is one of the four residues that are invariant in all aminotransferases; Tyr225 is in its vicinity, forming a hydrogen bond with O-3′ of the cofactor; and Arg292interacts with the distal carboxylate group of the substrate. In the triple-mutant enzyme, k cat′ for β-decarboxylation of l-aspartate was 0.08 s−1, whereas k cat′ for transamination was decreased to 0.01 s−1. AspAT was thus converted into an l-aspartate β-decarboxylase that catalyzes transamination as a side reaction. The major pathway of β-decarboxylation directly produces l-alanine without intermediary formation of pyruvate. The various single- or double-mutant AspATs corresponding to the triple-mutant enzyme showed, with the exception of AspAT Y225R/R386A, no measurable or only very low β-decarboxylase activity. The arginine shift mutation Y225R/R386A elicits β-decarboxylase activity, whereas the R292K substitution suppresses transaminase activity. The reaction specificity of the triple-mutant enzyme is thus achieved in the same way as that of wild-type pyridoxal 5′-phosphate-dependent enzymes in general and possibly of many other enzymes, i.e. by accelerating the specific reaction and suppressing potential side reactions.

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 Ki values of the two enzymes and the Kd 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. Proteins 2005.

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.

MAGE-C1/CT7 is the dominant cancer-testis antigen targeted by humoral immune responses in patients with multiple myeloma

MAGE-C1/CT7 is the dominant cancer-testis antigen targeted by humoral immune responses in patients with multiple myeloma

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.