Biomedical subjects

Rudolf Volkmer-Engert

Publications and source records attributed to Rudolf Volkmer-Engert.

At least 37 records · Page 2Linked to original sources

PubMed2004

Quantification of PDZ domain specificity, prediction of ligand affinity and rational design of super-binding peptides.

Transient macromolecular complexes are often formed by protein-protein interaction domains (e.g. PDZ, SH2, SH3, WW) which recognize linear sequence motifs with in vitro affinities typically in the micromolar range. The analysis of the resulting interaction networks requires a quantification of domain specificity and selectivity towards all possible ligands with physiologically relevant affinity. As representative examples, we determined specificity as a function of ligand sequence-dependent affinity contributions by statistical analysis of peptide library screens for the AF6, ERBIN and SNA1 (alpha-1-syntrophin) PDZ domains. For this purpose, the three PDZ domains were first screened for binding with a peptide library comprising 6223 human C termini created by SPOT synthesis. Based on the detected ligand preferences, we designed focused peptide libraries (profile libraries). These libraries were used to quantify the affinity contributions of the four C-terminal ligand residues by means of ANOVA models (analysis of variance) relating the C-terminal ligand sequences to the corresponding dissociation constants. Our models agreed well with experimentally determined dissociation constants and allowed us to design super binding peptides. The latter were shown experimentally to bind to their cognate PDZ domains with the highest affinity. In addition, we determined structure-activity relationships and thereby rationalized the position-specific affinity contributions. Furthermore, we used the statistical models to predict the dissociation constants for the complete ligand sequence space and thus determined the specificity overlap for the three investigated PDZ domains (). Altogether, we present an efficient method for profiling protein-protein interaction domains that provides a biophysical picture of specificity and selectivity. This approach allows the rational design of functional experiments and provides a basis for simulating interaction networks in the field of systems biology.

Amino Acid Sequence↗

PubMed2004

Peroxisomal membrane proteins contain common Pex19p-binding sites that are an integral part of their targeting signals.

Targeting of peroxisomal membrane proteins (PMPs) is a multistep process that requires not only recognition of PMPs in the cytosol but also their insertion into the peroxisomal membrane. As a consequence, targeting signals of PMPs (mPTS) are rather complex. A candidate protein for the PMP recognition event is Pex19p, which interacts with most PMPs. However, the respective Pex19p-binding sites are ill-defined and it is currently disputed whether these sites are contained within mPTS. By using synthetic peptide scans and yeast two-hybrid analyses, we determined and characterized Pex19p-binding sites in Pex11p and Pex13p, two PMPs from Saccharomyces cerevisiae. The sites turned out to be composed of a short helical motif with a minimal length of 11 amino acids. With the acquired data, it proved possible to predict and experimentally verify Pex19p-binding sites in several other PMPs by applying a pattern search and a prediction matrix. A peroxisomally targeted Pex13p fragment became mislocalized to the endoplasmic reticulum in the absence of its Pex19p-binding site. By adding the heterologous binding site of Pex11p, peroxisomal targeting of the Pex13p fragment was restored. We conclude that Pex19p-binding sites are well-defined entities that represent an essential part of the mPTS.

Amino Acid Sequence↗

PubMed2004

Protein interaction networks by proteome peptide scanning.

A substantial proportion of protein interactions relies on small domains binding to short peptides in the partner proteins. Many of these interactions are relatively low affinity and transient, and they impact on signal transduction. However, neither the number of potential interactions mediated by each domain nor the degree of promiscuity at a whole proteome level has been investigated. We have used a combination of phage display and SPOT synthesis to discover all the peptides in the yeast proteome that have the potential to bind to eight SH3 domains. We first identified the peptides that match a relaxed consensus, as deduced from peptides selected by phage display experiments. Next, we synthesized all the matching peptides at high density on a cellulose membrane, and we probed them directly with the SH3 domains. The domains that we have studied were grouped by this approach into five classes with partially overlapping specificity. Within the classes, however, the domains display a high promiscuity and bind to a large number of common targets with comparable affinity. We estimate that the yeast proteome contains as few as six peptides that bind to the Abp1 SH3 domain with a dissociation constant lower than 100 microM, while it contains as many as 50-80 peptides with corresponding affinity for the SH3 domain of Yfr024c. All the targets of the Abp1 SH3 domain, identified by this approach, bind to the native protein in vivo, as shown by coimmunoprecipitation experiments. Finally, we demonstrate that this strategy can be extended to the analysis of the entire human proteome. We have developed an approach, named WISE (whole interactome scanning experiment), that permits rapid and reliable identification of the partners of any peptide recognition module by peptide scanning of a proteome. Since the SPOT synthesis approach is semiquantitative and provides an approximation of the dissociation constants of the several thousands of interactions that are simultaneously analyzed in an array format, the likelihood of each interaction occurring in any given physiological settings can be evaluated. WISE can be easily extended to a variety of protein interaction domains, including those binding to modified peptides, thereby offering a powerful proteomic tool to help completing a full description of the cell interactome.

Animals↗

PubMed2004

An improved method for the synthesis of cellulose membrane-bound peptides with free C termini is useful for PDZ domain binding studies.

SPOT synthesis permits parallel synthesis and screening of thousands of cellulose membrane-bound peptides to study protein-protein interactions in a proteomic context. Recognition of C-terminal residues is one of the most common binding features of PDZ domains. Unfortunately, most solid support-bound peptide libraries lack a free C terminus due to C-terminal fixation on the solid support. To overcome this restriction, we developed a robust methodology based on our previous strategy for generating peptides with authentic C termini. To validate this improved method, we screened a human peptide library of 6223 C termini with the syntrophin PDZ domain. Furthermore, using the same library, new peptide ligands derived from membrane proteins and receptors were found for the ERBIN PDZ domain. Finally, we identified the protein kinase breakpoint cluster region, which is known as a negative regulator of cell proliferation and oncogenic transformation, as an ERBIN ligand.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Analysis of antigen-specific T-cell responses with synthetic peptides--what kind of peptide for which purpose?

The analysis of T-cell responses to peptides has recently become a busy area of immunologic research. Peptides may be used as single stimulants, pools or libraries, or as part of peptide/major histocompatibility complexes (MHC) for direct T-cell receptor staining. For stimulating T cells, peptides must be bound to MHC molecules. In this study we have used 9- or 10-amino acid peptides, 15-amino acid peptides containing stimulating shorter sequences, and peptides with modified C-terminal function. On average 67% of the T cells from healthy cytomegalovirus-positive donors that bound a frequently used cytomegalovirus pp65/HLA-A*0201 tetramer were able to produce interferon-gamma on stimulation with the respective 9-amino acid peptide. Peptides of 15 amino acids length used at the same concentration (in microg/ml) stimulated CD8 T cells somewhat less efficiently (on average 77% of the frequencies induced with the respective shorter peptides). Modifications of 9-amino acid peptides such as addition of amino acids or functional groups often resulted in a decreased ability to stimulate. However, based on our own results, published data, and theoretic considerations, we conclude that sets of peptides of 15 amino acids length with 11 amino acids overlap represent a good compromise for stimulating both CD8 and CD4 T cells in a number of applications. These parameters may be modified subject to the purpose of a study.

Amides↗

PubMed2004

Cross-reactivity of T lymphocytes in infection and autoimmunity.

T helper (Th) lymphocytes mediate critical effector and regulatory functions in infectious, allergic, or autoimmune diseases. Th cells possess clonal receptors that recognize antigenic peptides that are complexed with self-molecules of the major histocompatibility complex (MHC) on the surface of antigen presenting cells. An organism's repertoire of T cell receptors must be broad enough to recognize any possible microbial antigen. At the same time, tissue destruction resulting from the attack of autoreactive T lymphocytes that recognize self-peptides must be avoided. It was therefore believed that the immune system could distinguish between self and non-self antigens. This hypothesis was supported by several lines of evidence, including the seemingly exquisite specificity of immune responses. What, then, triggers autoaggressive attacks by the immune system? Clinical and epidemiological observations strongly suggest a link between infection and autoimmunity. A popular hypothesis considers autoimmunity as a side effect of antimicrobial immune responses. Cross-reactive T cells, capable of recognizing both microbial and self-peptides, have been prime suspects as instigators of autoimmunity ever since computerized data base searches revealed astonishing sequence homologies between microbial and self-peptides. Here we review recent data that show a previously unexpected degeneracy of antigen recognition by T cells. It has become clear that each individual T cell receptor can recognize a large number of different ligands. Furthermore, structural criteria rather than sequence homology dictate the antigen recognition process. Thus, the idea that cross-reactivity per se would cause autoimmune disease is most likely too simple. Instead, a variety of different molecular mechanisms dictate the immunological outcome of ligand recognition by T cells.

Amino Acid Sequence↗

PubMed2004

Crystallization and preliminary x-ray analysis of complexes of porcine pancreatic elastase with two natural inhibitors.

Two different natural protease inhibitors, the squash inhibitor MCEI III and the third domain of turkey ovomucoid inhibitor OMTKY3, were crystallized in complexes with porcine pancreatic elastase (PPE). About 700 conditions were screened altogether. Crystals of the complex between MCEI III and PPE were grown in citrate buffer with and without ammonium acetate. X-ray diffraction data were collected to 1.9 angstroms resolution at room temperature using synchrotron radiation. The crystals belong to space group P2(1), with unit-cell parameters a = 49.17, b = 44.59, c = 67.08 angstroms, beta = 110.97 degrees. Crystals of the OMTKY3/PPE complex were obtained in the presence of ammonium sulfate, MES buffer and polyethylene glycol monomethylether (PEG). These crystals of this complex diffracted to 2.1 angstroms resolution and belongs to space group I222, with unit-cell parameters a = 84.58, b = 84.61, c = 89.92 angstroms and diffracted to 2.2 angstroms resolution. The diffraction data were collected using a conventional rotating anode X-ray generator at room temperature. In both cases the presence of inhibitor in the crystals was confirmed by crystallography.

Amino Acid Sequence↗

PubMed2003

Interaction of the Bacillus subtilis chaperone CsaA with the secretory protein YvaY.

Bacillus subtilis CsaA was previously characterised as a molecular chaperone with export-related activities. In order to elucidate the functionality of CsaA further, interaction with its postulated substrate YvaY was investigated. Similar binding to carrier immobilised mature and preYvaY revealed that the interaction was not mediated via the signal peptide of preYvaY. Higher affinity to denatured peptides compared to native peptides indicated preferred binding to unfolded proteins. To characterise affinity of CsaA more detailed, binding to preYvaY derived peptides was analysed. CsaA showed affinity to multiple peptides in the scan, mainly correlated to a positive net charge. Affinity of export-specific Escherichia coli chaperone SecB to the carrier immobilised peptides indicated partially overlapping binding characteristics of SecB and CsaA.

Bacillus subtilis↗

PubMed2003

Sequence-specific binding of prePhoD to soluble TatAd indicates protein-mediated targeting of the Tat export in Bacillus subtilis.

The Tat (twin-arginine protein translocation) system initially discovered in the thylakoid membrane of chloroplasts has been described recently for a variety of eubacterial organisms. Although in Escherichia coli four Tat proteins with calculated membrane spanning domains have been demonstrated to mediate Tat-dependent transport, a specific transport system for twin-arginine signal peptide containing phosphodiesterase PhoD of Bacillus subtilis consists of one TatA/TatC (TatAd/TatCd) pair of proteins. Here, we show that TatAd was found beside its membrane-integrated localization in the cytosol were it interacted with prePhoD. prePhoD was efficiently co-immunoprecipitated by TatAd. Inefficient co-immunoprecipitation of mature PhoD and missing interaction to Sec-dependent and cytosolic peptides by TatAd demonstrated a particular role of the twin-arginine signal peptide for this interaction. Affinity of prePhoD to TatAd was interfered by peptides containing the twin-arginine motif but remained active when the arginine residues were substituted. The selective binding of TatAd to peptides derived from the signal peptide of PhoD elucidated the function of the twin-arginine motif as a target site for pre-protein TatAd interaction. Substitution of the binding motif demonstrated the pivotal role of basic amino acid residues for TatA binding. These features suggest that TatA interacts prior to membrane integration with its pre-protein substrate and could therefore assist targeting of twin-arginine pre-proteins.

Amino Acid Motifs↗

PubMed2003

Design of N-substituted peptomer ligands for EVH1 domains.

Ena/VASP proteins are implicated in cytoskeletal reorganization during actin-dependent motility processes. Recruitment to subcellular sites of actin polymerization is mediated by the highly conserved N-terminal EVH1 domain, which interacts with target proteins containing proline-rich motifs. The VASP EVH1 domain specifically binds peptides with the consensus motif FPPPP present in all its binding partners, including the Listerial ActA protein. Previous studies have shown that the Phe and first and final Pro residues are highly conserved and cannot be substituted with any other natural amino acid without significant loss of binding affinity. We have incorporated peptoid building blocks (sarcosine derived, non-natural amino acids) into the peptide SFEFPPPPTEDEL from the Listerial ActA protein and were able to substitute the most highly conserved residues of this motif while maintaining binding to the VASP EVH1 domain with affinities in the range of 45-180 microm. We then used NMR chemical shift perturbations to locate specific domain residues involved in particular interactions. These studies may open up the way for designing selective modulators of VASP function for biological studies and for the development of novel therapeutics for diseases involving pathologically altered cell adhesion or cell motility.

Actins↗

PubMed2003

The ScPex13p SH3 domain exposes two distinct binding sites for Pex5p and Pex14p.

Pex13p is an essential component of the peroxisomal protein import machinery and interacts via its C-terminal SH3 domain with the type II SH3-ligand Pex14p and the non-PXXP protein Pex5p. We report the solution structure of the SH3 domain of Pex13p from Saccharomyces cerevisiae and the identification of a novel-binding pocket, which binds a non-PXXP-peptide representing the binding site of Pex5p. Chemical shift assays revealed the binding sites for Pex5p and Pex14p ligand peptides to be distinct and spatially separated. Competition assays demonstrated that the two ligand peptides can bind simultaneously to the SH3 domain.

Amino Acid Motifs↗

PubMed2003

Sensitive visualization of peptide presentation in vitro and ex vivo.

BACKGROUND: The presentation of antigenic peptides to specific T cells is one of the key events for the induction of a T-cell-dependent immune response. The nature of the antigen-presenting cells which present distinct peptides has been difficult to analyze so far due to the low number of peptides presented in vivo by a single antigen-presenting cell. METHODS: We have used magnetofluorescent liposomes to identify and characterize antigen-presenting cells according to presentation of a hapten-labeled antigenic peptide in vitro and ex vivo. RESULTS: Magnetofluorescent liposomes allowed the identification and isolation of antigen-presenting cells according to the presentation of less than 100 peptides per cell, the physiological threshold for activation of specific T cells. Ex vivo, we could demonstrate peptide presentation by B lymphocytes and dendritic cells already 1 h after intravenous peptide injection; this rapidly declined to background level after 12-24 h. CONCLUSIONS: The sensitive visualization of peptide presentation allows the phenotypical and functional characterization of those antigen-presenting cells which present specific peptides at physiological relevant quantities. This technology will help to characterize the antigen-presenting cells (APC) which are responsible for the induction of distinct immune reactions in vivo, e.g., the generation of tolerance or immunity.

Animals↗

PubMed2003

Volatile polydimethylcyclosiloxanes in the ambient laboratory air identified as source of extreme background signals in nanoelectrospray mass spectrometry.

Polydimethylcyclosiloxanes, an almost ubiquitous air contaminant, can interfere with nanoelectrospray analysis. The sensitivity of nanoelectrospray to these volatile air contaminants was demonstrated in this study. The intensity of the interfering ion signals caused by these compounds can be decreased by changing the position of the nanoESI needle and almost completely suppressed by applying a flow of pure nitrogen around the needle and the sample cone. The nitrogen flow causes a slight shift in charge distribution, but does not influence the sensitivity for peptide detection.

Air↗

PubMed2003

WW domain sequence activity relationships identified using ligand recognition propensities of 42 WW domains.

WW domains mediate protein-protein interactions in a number of different cellular functions by recognizing proline-containing peptide sequences. We determined peptide recognition propensities for 42 WW domains using NMR spectroscopy and peptide library screens. As potential ligands, we studied both model peptides and peptides based on naturally occurring sequences, including phosphorylated residues. Thirty-two WW domains were classified into six groups according to detected ligand recognition preferences for binding the motifs PPx(Y/poY), (p/phi)P(p,g)PPpR, (p/phi)PPRgpPp, PPLPp, (p/xi)PPPPP, and (poS/poT)P (motifs according to modified Seefeld Convention 2001). In addition to these distinct binding motifs, group-specific WW domain consensus sequences were identified. For PPxY-recognizing domains, phospho-tyrosine binding was also observed. Based on the sequences of the PPx(Y/poY)-specific group, a profile hidden Markov model was calculated and used to predict PPx(Y/poY)-recognition activity for WW domains, which were not assayed. PPx(Y/poY)-binding was found to be a common property of NEDD4-like ubiquitin ligases.

Amino Acid Motifs↗

PubMed2003

Potent inhibition of angiogenesis by D,L-peptides derived from vascular endothelial growth factor receptor 2.

Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells and plays a central role in angiogenesis and vasculogenesis. Therefore, VEGF and its receptors VEGFR-1 and VEGFR-2 are prime targets for anti-angiogenic intervention which is thought to be one of the most promising approaches in cancer therapy. Recently, we have discovered a VEGFR-2-derived peptide ((247)RTELNVGIDFNWEYP(261)) representing a potential binding site to VEGF. Using the spot synthesis technique, systematic D-amino acid substitutional analyses of this peptide were conducted and the resulting D,L-peptides inhibit VEGF binding to VEGFR-2 at half maximal concentration of 30 nM. The serum-stable D,L-peptides further inhibited autophosphorylation of the VEGFR-2 at nanomolar concentrations. Testing of the peptides in a spheroid-based angiogenesis assay demonstrated a potent anti-angiogenic effect in vitro. The rational design of potent and stable anti-angiogenic peptide inhibitors from their parent receptors provides a feasible route to develop novel leads for anti-angiogenic medicines.

Amino Acid Sequence↗

PubMed2002

Identification of distinct antibody epitopes and mimotopes from a peptide array of 5520 randomly generated sequences.

We used a relatively small library of 5520 randomly generated single 15-mer peptides prepared by SPOT synthesis as an array of 28.5x19.0 cm to identify epitopes for three distinct monoclonal antibodies, namely anti-p24 (human immunodeficiency virus (HIV)-1) monoclonal anibody (mab) CB4-1, anti-interleukin-10 (IL-10) mab CB/RS/13, and anti-transforming growth factor alpha (TGFalpha) mab Tab2. Initially identified peptide ligands mostly had very low affinities for the antibodies with dissociation constants around 10(-4) M. Subsequent identification of residues critical for the antibody interactions involved complete L-amino acid substitutional analyses. Several substitutions resulted in analogs with dissociation constants in the low micromolar and high nanomolar range. Specifically binding peptides with key residue patterns matching the wild-type epitopes were identified for all three antibodies. In addition, for antibody CB4-1 mimotopes that showed no homology to the known epitope were selected. Our results suggest that a very limited library diversity, although far from covering the entire sequence repertoire, can suffice to rapidly and economically select peptidic antibody epitopes and mimotopes.

Combinatorial Chemistry Techniques↗

PubMed2002

Cytomegalovirus (CMV) phosphoprotein 65 makes a large contribution to shaping the T cell repertoire in CMV-exposed individuals.

Antigen-specific, cytokine flow cytometry was used to analyze the prevalence and frequency of CD4 and CD8 memory T cells specific for the abundantly expressed cytomegalovirus (CMV) phosphoprotein 65 (pp65) in healthy CMV IgG-seropositive individuals. Stimulation of peripheral blood mononuclear cells with peptide pools and individual peptides derived from the pp65 amino acid sequence in 40 donors revealed that 63% of donors had a detectable CD4 T cell response and that 83% of donors had a detectable CD8 T cell response against this protein. The overall frequencies of T cells directed against pp65 were analyzed for 20 donors by stimulation with peptide pools covering the complete pp65 protein and were as high as 2 in 1000 and 9 in 1000 (median) peripheral blood CD4 and CD8 T cells, respectively. In addition, a comparison between CD4 responses to a CMV lysate containing various CMV proteins and pp65-specific responses in 9 donors indicated that pp65 was a dominant target of the CMV-specific CD4 T cell response in some, but not all, donors. Several new T cell epitopes were identified.

Amino Acid Sequence↗

PubMed2002

Isoaspartyl dipeptidase activity of plant-type asparaginases.

Recombinant plant-type asparaginases from the cyanobacteria Synechocystis sp. PCC (Pasteur culture collection) 6803 and Anabaena sp. PCC 7120, from Escherichia coli and from the plant Arabidopsis thaliana were expressed in E. coli with either an N-terminal or a C-terminal His tag, and purified. Although each of the four enzymes is encoded by a single gene, their mature forms consist of two protein subunits that are generated by autoproteolytic cleavage of the primary translation products at the Gly-Thr bond within the sequence GTI/VG. The enzymes not only deamidated asparagine but also hydrolysed a range of isoaspartyl dipeptides. As various isoaspartyl peptides are known to arise from proteolytic degradation of post-translationally altered proteins containing isoaspartyl residues, and from depolymerization of the cyanobacterial reserve polymer multi-L-arginyl-poly-L-aspartic acid (cyanophycin), plant-type asparaginases may not only function in asparagine catabolism but also in the final steps of protein and cyanophycin degradation. The properties of these enzymes are compared with those of the sequence-related glycosylasparaginases.

Amidohydrolases↗