Publications (15)36.82 Total impact
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Article: New cardiolipin analogs synthesized by phospholipase d-catalyzed transphosphatidylation.
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ABSTRACT: Cardiolipin (CL) and related diphosphatidyl lipids are hardly accessible because of the complexity of their chemical synthesis. In the present paper, the transphosphatidylation reaction catalyzed by phospholipase D (PLD) from Streptomyces sp. has been proven as an alternative enzyme-assisted strategy for the synthesis of new CL analogs. The formation of this type of compounds from phosphatidylcholine was compared for a series of N- and C2-substituted ethanolamine derivatives as well as non-charged alcohols such as glycerol and ethylene glycol. The rapid exchange of the choline head group by ethanolamine derivatives having a low molecular volume (diethanolamine and serinol) gave rise to an efficient production of the corresponding CL analogs. In contrast, the yields were comparably low in the reaction with bulky nitrogenous acceptor alcohols (triethanolamine, tris(hydroxymethyl)aminomethane, tetrakis(hydroxyethyl)ammonium) or the non-charged alcohols. Therefore, a strong dependence of the conversion of the monophosphatidyl to the diphosphatidyl compound on steric parameters and the head group charge was concluded. The enzyme-assisted strategy was used for the preparation of purified diphosphatidyldiethanolamine and diphosphatidylserinol.Chemistry and physics of lipids 10/2012; · 2.15 Impact Factor -
Article: The folding pathway of onconase is directed by a conserved intermediate.
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ABSTRACT: A promising approach to unravel the relationship between sequence information, tertiary structure, and folding mechanism of proteins is the analysis of the folding behavior of proteins with low sequence identity but comparable tertiary structures. Ribonuclease A (RNase A) and its homologues, forming the RNase A superfamily, provide an excellent model system for respective studies. RNase A has been used extensively as a model protein for folding studies. However, little is known about the folding of homologous RNases. Here, we analyze the folding pathway of onconase, a homologous protein from the Northern leopard frog with great potential as a tumor therapeutic, by high-resolution techniques. Although onconase and RNase A significantly differ in the primary structure (28% sequence identity) and in thermodynamic stability (DeltaDeltaG = 20 kJ mol(-1)), both enzymes possess very similar tertiary structures. The present folding studies on onconase by rapid mixing techniques in combination with fluorescence and NMR spectroscopy allow the structural assignment of the three kinetic phases observed in stopped-flow fluorescence spectroscopy. After a slow peptidyl-prolyl cis-to-trans isomerization reaction in the unfolded state, ONC folds via an on-pathway intermediate to the native state. By quenched-flow hydrogen/deuterium exchange experiments coupled with 2D NMR spectroscopy, 31 amino acid residues were identified to be involved in the structure formation of the intermediate. Twelve of these residues are identical in the RNase A sequence, which is a significantly higher percentage (39%) than the overall 28% sequence identity. Moreover, the structure of this intermediate closely resembles two of the intermediates that occur early during the refolding of RNase A. Obviously, in spite of considerable differences in their amino acid sequence the initial folding events of both proteins are comparable, guided by a limited number of conserved residues.Biochemistry 08/2009; 48(35):8449-57. · 3.42 Impact Factor -
Article: Thermodynamic stability and native-state dynamics of porcine pancreatic phospholipase A2 studied by 1H NMR spectroscopic measurements.
Advances in experimental medicine and biology 02/2009; 611:67-8. · 1.09 Impact Factor -
Article: Phospholipase D-catalyzed synthesis of new phospholipids with polar head groups.
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ABSTRACT: A series of new phospholipids with polar head groups have been synthesized by enzymatic transphosphatidylation of 1,2-dioleoyl-sn-glycerophosphocholine and identified by 1H NMR and MALDI-TOF-MS. The acceptor alcohols were N- or C2-substituted derivatives of ethanolamine (diethanolamine, triethanolamine, serinol, Tris, BisTris). Phospholipases D from cabbage (PLDcab) and Streptomyces sp. (PLDStr) were compared with respect to product yield and purity as well as the initial rates in transphosphatidylation and competing hydrolysis. In all reactions, PLDStr showed a remarkably higher transphosphatidylation activity than PLDcab. However, higher yields of the phospholipids with diethanolamine, triethanolamine, and serinol were obtained by PLDcab because PLDStr resulted in the additional formation of diphosphatidyl derivatives. In the synthesis of the Tris and BisTris derivatives, PLD(Str) was much more appropriate because voluminous head group alcohols (>129A3) are poorly converted by PLDcab. With BisTris as acceptor alcohol two regioisomeric forms of phosphatidyl-BisTris were obtained.Chemistry and Physics of Lipids 05/2008; 152(2):71-7. · 2.57 Impact Factor -
Article: Amino acids allosterically regulate the thiamine diphosphate-dependent alpha-keto acid decarboxylase from Mycobacterium tuberculosis.
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ABSTRACT: The gene rv0853c from Mycobacterium tuberculosis strain H37Rv codes for a thiamine diphosphate-dependent alpha-keto acid decarboxylase (MtKDC), an enzyme involved in the amino acid degradation via the Ehrlich pathway. Steady state kinetic experiments were performed to determine the substrate specificity of MtKDC. The mycobacterial enzyme was found to convert a broad spectrum of branched-chain and aromatic alpha-keto acids. Stopped-flow kinetics showed that MtKDC is allosterically activated by alpha-keto acids. Even more, we demonstrate that also amino acids are potent activators of this thiamine diphosphate-dependent enzyme. Thus, metabolic flow through the Ehrlich pathway can be directly regulated at the decarboxylation step. The influence of amino acids on MtKDC catalysis was investigated, and implications for other thiamine diphosphate-dependent enzymes are discussed.Journal of Biological Chemistry 03/2008; 283(9):5344-54. · 4.77 Impact Factor -
Article: Non-substrate peptides influencing dipeptidyl peptidase IV/CD26 activity and immune cell function.
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ABSTRACT: Investigations using inhibitors of dipeptidyl peptidase IV (DP IV) activities and DP IV-/- mice indicated an immunoregulatory role of DP IV that could not be compensated by DP IV-like enzymes. The HIV-1 Tat protein was identified as the first natural inhibitor of DP IV and as immunosuppressor. This review summarizes our investigations on the identification of the amino acid motif of Tat responsible for DP IV inhibition and of endogenous DP IV-inhibitory ligands that suppress immune cell activation. Examinations on numerous peptides carrying the N-terminal Xaa-Xaa-Pro motif of Tat revealed that tryptophan at position two strongly enhanced DP IV inhibition and immunosuppression. Here, we present evidence that the thromboxane A2 receptor exposing N-terminal Met-Trp-Pro at the cell surface could be a potential endogenous, inhibitory DP IV ligand. Moreover, our data suggest that the major envelope proteins p37k of the orthopoxviruses variola virus and vaccinia virus, as well as the B2L antigen of the parapoxvirus orf, that also carry N-terminal Met-Trp-Pro, could mediate immunosuppressive effects. Further examinations are in progress to identify new physiologic, inhibitory DP IV ligands and to enlighten the mechanism underlying the DP IV-mediated effects.Frontiers in Bioscience 02/2008; 13:3194-201. · 3.52 Impact Factor -
Article: Synthesis and characterization of a new and radiolabeled high-affinity substrate for H+/peptide cotransporters.
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ABSTRACT: In this study we described the design, rational synthesis and functional characterization of a novel radiolabeled hydrolysis-resistant high-affinity substrate for H(+)/peptide cotransporters. L-4,4'-Biphenylalanyl-L-Proline (Bip-Pro) was synthesized according to standard procedures in peptide chemistry. The interaction of Bip-Pro with H(+)/peptide cotransporters was determined in intestinal Caco-2 cells constitutively expressing human H(+)/peptide cotransporter 1 (PEPT1) and in renal SKPT cells constitutively expressing rat H(+)/peptide cotransporter 2 (PEPT2). Bip-Pro inhibited the [(14)C]Gly-Sar uptake via PEPT1 and PEPT2 with exceptional high affinity (K(i) = 24 microm and 3.4 microm, respectively) in a competitive manner. By employing the two-electrode voltage clamp technique in Xenopus laevis oocytes expressing PEPT1 or PEPT2 it was found that Bip-Pro was transported by both peptide transporters although to a much lower extent than the reference substrate, Gly-Gln. Bip-Pro remained intact to > 98% for at least 8 h when incubated with intact cell monolayers. Bip-[(3)H]Pro uptake into SKPT cells was linear for up to 30 min and pH dependent with a maximum at extracellular pH 6.0. Uptake was strongly inhibited, not only by unlabeled Bip-Pro but also by known peptide transporter substrates such as dipeptides, cefadroxil, Ala-4-nitroanilide and delta-aminolevulinic acid, but not by glycine. Bip-Pro uptake in SKPT cells was saturable with a Michaelis-Menten constant (K(t)) of 7.6 microm and a maximal velocity (V(max)) of 1.1 nmol x 30 min(-1) x mg of protein(-1). Hence, the uptake of Bip-Pro by PEPT2 is a high-affinity, low-capacity process in comparison to the uptake of Gly-Sar. We conclude that Bip-[(3)H]Pro is a valuable substrate for both mechanistic and structural studies of H(+)/peptide transporter proteins.FEBS Journal 12/2007; 274(22):5905-14. · 3.79 Impact Factor -
Article: Improvement of lipophilicity and membrane transport of cefuroxime using in vitro models.
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ABSTRACT: Most beta-lactam antibiotics cannot be absorbed orally and, therefore, must be administered intravenously (i.v.) or intramuscularly (i.m.). Because of the obvious drawbacks of drug delivery by injection, the development of alternatives with enhanced oral bioavailability is receiving much attention in pharmaceutical research. Cefuroxime exhibiting significant advantages in the parental treatment of common infections, was used as model drug in the present study. The effect of the cationic absorption enhancers (four quaternary ammonium salts) on the lipophilicity of cefuroxime was investigated by means of the n-octanol/water system. The results on partitioning coefficients in the n-octanol/buffer system were confirmed using an in vitro transport model with artificial (dodecanol collodium membrane) and biological membranes (Charles-River guinea pig).European Journal of Pharmaceutics and Biopharmaceutics 12/2004; 58(3):653-7. · 4.27 Impact Factor -
Article: Different modes of dipeptidyl peptidase IV (CD26) inhibition by oligopeptides derived from the N-terminus of HIV-1 Tat indicate at least two inhibitor binding sites.
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ABSTRACT: Dipeptidyl peptidase IV (DP IV, CD26) plays an essential role in the activation and proliferation of lymphocytes, which is shown by the immunosuppressive effects of synthetic DP IV inhibitors. Similarly, both human immunodeficiency virus-1 (HIV-1) Tat protein and the N-terminal peptide Tat(1-9) inhibit DP IV activity and T cell proliferation. Therefore, the N-terminal amino acid sequence of HIV-1 Tat is important for the inhibition of DP IV. Recently, we characterized the thromboxane A2 receptor peptide TXA2-R(1-9), bearing the N-terminal MWP sequence motif, as a potent DP IV inhibitor possibly playing a functional role during antigen presentation by inhibiting T cell-expressed DP IV [Wrenger, S., Faust, J., Mrestani-Klaus, C., Fengler, A., Stöckel-Maschek, A., Lorey, S., Kähne, T., Brandt, W., Neubert, K., Ansorge, S. & Reinhold, D. (2000) J. Biol. Chem.275, 22180-22186]. Here, we demonstrate that amino acid substitutions at different positions of Tat(1-9) can result in a change of the inhibition type. Certain Tat(1-9)-related peptides are found to be competitive, and others linear mixed-type or parabolic mixed-type inhibitors indicating different inhibitor binding sites on DP IV, at the active site and out of the active site. The parabolic mixed-type mechanism, attributed to both non-mutually exclusive inhibitor binding sites of the enzyme, is described in detail. From the kinetic investigations and molecular modeling experiments, possible interactions of the oligopeptides with specified amino acids of DP IV are suggested. These findings give new insights for the development of more potent and specific peptide-based DP IV inhibitors. Such inhibitors could be useful for the treatment of autoimmune and inflammatory diseases.European Journal of Biochemistry 06/2003; 270(10):2147-56. · 3.58 Impact Factor -
Article: New results on the conformations of potent DP IV (CD26) inhibitors bearing the N-terminal MWP structural motif.
Advances in experimental medicine and biology 02/2003; 524:65-8. · 1.09 Impact Factor -
Article: Transcellular proteolysis demonstrated by novel cell surface-associated substrates of dipeptidyl peptidase IV (CD26).
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ABSTRACT: Proteolytic enzymes contribute to the regulation of cellular functions such as cell proliferation and death, cytokine production, and matrix remodeling. Dipeptidyl peptidase IV (DP IV) catalyzes the cleavage of several cytokines and thereby contributes to the regulation of cytokine production and the proliferation of immune cells. Here we show for the first time that cell surface-bound DP IV catalyzes the cleavage of specific substrates that are associated with the cellular surface of neighboring cells. Rhodamine 110 (R110), a highly fluorescent xanthene dye, was used to synthesize dipeptidyl peptidase IV (DP IV/CD26) substrates Gly(Ala)-Pro-R110-R, thus facilitating a stable binding of the fluorescent moiety on the cell surface. The fixation resulted from the interaction with the reactive anchor rhodamine and allowed the quantification of cellular DP IV activity on single cells. The reactivity, length, and hydrophobicity of rhodamine was characterized as the decisive factor that facilitated the determination of cellular DP IV activity. Using fluorescence microscopy, it was possible to differentiate between different DP IV activities. The hydrolysis of cell-bound substrates Xaa-Pro-R110-R by DP IV of neighboring cells and by soluble DP IV was shown using flow cytometry. These data demonstrate that ectopeptidases such as DP IV may be involved in communication between blood cells via proteolysis of cell-associated substrates.Journal of Biological Chemistry 10/2002; 277(36):33170-7. · 4.77 Impact Factor -
Chapter: N-Terminal HIV-1 Tat Nonapeptides as Inhibitors of Dipeptidyl Peptidase IV. Conformational Characterization
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ABSTRACT: Compared to the N-terminal nonapeptide of the HIV-1 Tat protein as inhibitor of activity of DP IV which is supposed to mediate the immunosuppressive effects of HIV-1 Tat, the Ile5 and Leu6 analogues showed strongly reduced inhibitory activity. Interestingly, replacement of Asp2 with Gly or Lys led to compounds with considerably enhanced inhibition. Therefore, we have applied 1H NMR spectroscopy and restrained molecular dynamics calculations to elucidate the molecular conformation of a series of Tat nonapeptides. Conformational backbone differences of these peptides as well as the nature and the arrangement of the side chains per se at significant positions preventing effective binding to DP IV might explain their different inhibitory activity on DP IV.12/2001: pages 125-129; -
Chapter: Effects of Nonapeptides Derived From the N-terminal Structure of Human Immunodeficiency Virus-1 (HIV-1) Tat on Suppression of CD26-Dependent T Cell Growth
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ABSTRACT: The human immunodeficiency virus-1 (HIV-1) transactivator Tat occurs extracellularly and exerts immunosuppressive effects. Interestingly, Tat inhibits dipeptidyl peptidase IV (DP IV) activity of the T cellactivation marker CD26. The short N-terminal nonapeptideTat(l-9), MDPVDPNIE, also inhibits DP IV activity and suppresses DNA synthesis of tetanus toxoid-stimulated peripheral blood mononuclear cells (PBMC). Here, we present the influence of amino acid exchanges in the first three positions of Tat(l-9). For instance, the replacement of D2 of Tat(l-9) by G or K generated peptides, which inhibit DP IV-catalyzed IL-2(1-12) cleavage nearly threefold stronger. Similar effects were observed on the suppression of DNA synthesis of Tetanus toxoid-stimulated PBMC. This correlation suggests that Tat(l-9)-deduced peptides mediate antiproliferative effects at least in part via specific DP IV interactions and supports the hypothesis that CD26 plays a key role in the regulation of lymphocyte growth.12/2001: pages 161-165; -
Article: Determination of the Solution Conformation of HIV-1 Tat(1–9) Peptides by Means of Molecular Dynamics Simulations Considering NMR Data and Docking Studies into an Active Site Model of DP IV
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ABSTRACT: The human immunodeficiency virus 1 Tat protein suppresses antigen-, anti-CD3-and mitogen-induced activation of human T cells when added to T cell cultures. This activity is important for the development of AIDS because lymphocytes from HIV-infected individuals exhibit a similar antigen-specific dysfunction. Moreover, Tat was found to interact with dipeptidyl peptidase IV (DP IV). To find out the amino acid sequence important for the inhibition of the DP IV enzymatic activity we investigated N-terminal Tat(1–9) peptide analogues with amino acid substitutions in different positions. Interestingly, the exchange of Pro6 with Leu and Asp5 with Ile strongly diminished the DP IV inhibition by Tat(1–9). Based on data derived from one-and two-dimensional 1H NMR investigations the solution conformations of the three nonapeptides in water were determined by means of molecular dynamics simulations. These conformations were used for studies of the docking behavior of the peptides into a model of the active site of DP IV. The results suggest that several attractive interactions between the native Tat(1–9) and DP IV lead to a stable complex and that the reduced affinity of both L6-Tat(1–9) and I5-Tat(1–9) derivatives might be caused by conformational alterations in comparison to the parent peptide.Journal of Molecular Modeling 01/1998; 4(6):200-210. · 1.80 Impact Factor -
Article: Thioxo amino acid pyrrolidides and thiazolidides: new inhibitors of proline specific peptidases
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ABSTRACT: Aminopeptidase P (APP), dipeptidyl peptidase II (DP II), dipeptidyl peptidase IV (DP IV) and prolyl oligopeptidase (POP) are proline specific peptidases. Hence, they are able to cleave peptide bonds containing the imino acid proline. Amino acid pyrrolidides (Pyrr) and thiazolidides (Thia) are well-known product analogue inhibitors of DP IV and POP. For the first time we describe the influence of a thioxo amide bond, incorporated into these compounds, on the inhibition of the proline specific peptidases. Taking into account the substrate specificity of these peptidases, we have synthesized Xaa-ψ[CS-N]-Pyrr and Xaa-ψ[CS-N]-Thia of the amino acids Ala, Phe, Val and Ile. The inhibition constants were determined for the above mentioned proline specific peptidases isolated from different sources. As a result, the serine proteases DP II, DP IV and POP were inhibited competitively, whereas metal-dependent APP displayed a linear mixed-type inhibition with inhibition constants up to 10−4 M. Thioxylation of Xaa-Pyrr and Xaa-Thia led to a slight decrease of inhibition of DP IV and POP compared to Xaa-Pyrr and Xaa-Thia, though the inhibition constants were still in the range up to 10−7 M. As Xaa-Thia exist as two isomers, we investigated isomer specific inhibition with regard to DP IV. Thus, our studies have revealed that DP IV was only inhibited by the Z isomer of the Xaa-ψ[CS-N]-Thia. For the first time, Xaa-Pyrr and Xaa-Thia were characterized as inhibitors of DP II with inhibition constants in the micromolar range. In contrast to DP IV inhibition, the Xaa-ψ[CS-N]-Pyrr and Xaa-ψ[CS-N]-Thia have proven to be more potent inhibitors of DP II than the corresponding Xaa-Pyrr and Xaa-Thia. Thus, these Xaa-ψ[CS-N]-Thia are new potent inhibitors especially suitable for DP II with Ki values ranging in the upper nanomolar concentration.Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology.
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1998–2003
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Martin-Luther-Universität Halle-Wittenberg
- Institut für Biochemie und Biotechnologie
Halle, Saxony-Anhalt, Germany
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