Dissection of the CMV specific T-cell response is required for optimized cardiac transplant monitoring.

Despite the success of antivirals in preventing clinically overt CMV disease in cardiac allograft recipients, sub-clinical active CMV infection remains a major concern because of its association with allograft rejection and vasculopathy. The measurement of CMV specific T-cell responses is a promising approach to assessing this situation. For simplicity, class-I MHC/peptide-multimers staining CD8 T-cells directly are often used but this ignores a much wider range of responses including the whole CD4 T-cell compartment.

Conservation of PEX19-binding motifs required for protein targeting to mammalian peroxisomal and trypanosome glycosomal membranes.

Glycosomes are divergent peroxisomes found in trypanosomatid protozoa, including those that cause severe human diseases throughout much of the world. While peroxisomes are dispensable for both yeast (Saccharomyces cerevisiae and others) and mammalian cells in vitro, glycosomes are essential for trypanosomes and hence are viewed as a potential drug target. The import of proteins into the matrix of peroxisomes utilizes multiple peroxisomal membrane proteins which require the peroxin PEX19 for insertion into the peroxisomal membrane.

Characterization of GD2 peptide mimotope DNA vaccines effective against spontaneous neuroblastoma metastases.

Disialoganglioside GD2 is an established target for immunotherapy in neuroblastoma. We tested the hypothesis that active immunization against the glycolipid GD2 using DNA vaccines encoding for cyclic GD2-mimicking decapeptides (i.e.

Sequence requirements and an optimization strategy for short antimicrobial peptides.

Short antimicrobial host-defense peptides represent a possible alternative as lead structures to fight antibiotic resistant bacterial infections. Bac2A is a 12-mer linear variant of the naturally occurring bovine host defense peptide, bactenecin, and demonstrates moderate, broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria as well as against the yeast Candida albicans. With the assistance of a method involving peptide synthesis on a cellulose support, the primary sequence requirements for antimicrobial activity against the human pathogen Pseudomonas aeruginosa of 277 Bac2A variants were investigated by using a luciferase-based assay.

Targeting of the tail-anchored peroxisomal membrane proteins PEX26 and PEX15 occurs through C-terminal PEX19-binding sites.

Tail-anchored proteins contain a single transmembrane domain (TMD) followed by a short C-terminal domain extending into the organellar lumen. Tail-anchored proteins are thought to target to the correct subcellular compartment by virtue of general physicochemical properties of their C-termini; however, the machineries that enable correct sorting remain largely elusive. Here we analyzed targeting of the human peroxisomal tail-anchored protein PEX26.

Probing the ligand-binding specificity and analyzing the folding state of SPOT-synthesized FBP28 WW domain variants.

The WW domains are known as the smallest naturally occurring, monomeric, triple-stranded, antiparallel beta-sheet domains. Hence, we chose the FBP28 WW domain as a model to investigate the stability of the beta-sheet structure at the amino acid level in the context of its function (ligand binding). The structure-function relationship was investigated through a complete substitution analysis of the FBP28 WW domain, with variants synthesized as a cellulose-bound peptide array.

Topography of the surface of the signal-transducing protein EIIA(Glc) that interacts with the MalK subunits of the maltose ATP-binding cassette transporter (MalFGK2) of Salmonella typhimurium.

The signal-transducing protein EIIA(Glc), a component of the phosphoenolpyruvate-glucose phosphotransferase system, plays a key role in carbon regulation in enteric bacteria, such as Escherichia coli and Salmonella typhimurium. The phosphorylation state of EIIA(Glc) governs transport and metabolism of a number of carbohydrates. When glucose as preferred carbon source is transported, EIIA(Glc) becomes predominantly unphosphorylated and allosterically inhibits several permeases, including the maltose ATP-binding cassette transport system (MalFGK2) in a process termed "inducer exclusion.

Oncostatin M receptor-mediated signal transduction is negatively regulated by SOCS3 through a receptor tyrosine-independent mechanism.

Down-regulation of interleukin (IL)-6-type cytokine signaling has been shown to occur, among other mechanisms, via induction of the feedback inhibitor SOCS3 (suppressor of cytokine signaling 3). Binding of SOCS3 to the phosphorylated Tyr(759) in the cytoplasmic region of gp130, the common signal transducing receptor chain of all IL-6-type cytokines, is necessary for inhibition of Janus kinase-mediated signaling. In the present study, we analyzed the effect of SOCS3 on signal transduction by the proinflammatory cytokine oncostatin M (OSM), which signals through a receptor complex of gp130 and the OSM receptor (OSMR).

Identification of VCP/p97, carboxyl terminus of Hsp70-interacting protein (CHIP), and amphiphysin II interaction partners using membrane-based human proteome arrays.

Proteins mediate their biological function through interactions with other proteins. Therefore, the systematic identification and characterization of protein-protein interactions have become a powerful proteomic strategy to understand protein function and comprehensive cellular regulatory networks. For the screening of valosin-containing protein, carboxyl terminus of Hsp70-interacting protein (CHIP), and amphiphysin II interaction partners, we utilized a membrane-based array technology that allows the identification of human protein-protein interactions with crude bacterial cell extracts.

Complete substitutional analysis of a sunflower trypsin inhibitor with different serine proteases.

Here we present a method to simultaneously characterize and/or optimize both the binding loop towards the protease and a cysteine-stabilized scaffold. The small peptidic sunflower trypsin inhibitor (SFTI-1) was chosen as a model system for these experiments. The inhibitor was investigated for positional specificity against trypsin, elastase and proteinase K using complete substitutional analyses based on cellulose-bound peptide spot synthesis.

Influence of antigen on the development of MALT lymphoma.

Mucosa-associated lymphoid tissue (MALT) B-cell lymphomas develop in the context of autoimmune or chronic inflammations like Helicobacter pylori-induced gastritis. Remission of most gastric MALT lymphomas after eradication of H pylori links tumor cell proliferation to antigen-induced inflammation and the need for antigenic contact. Furthermore, the tumor cells correspond to antigen-activated memory B cells.

High-throughput generation of small antibacterial peptides with improved activity.

Cationic antimicrobial peptides are able to kill a broad variety of Gram-negative and Gram positive bacteria and thus are good candidates for a new generation of antibiotics to treat multidrug-resistant bacteria. Here we describe a high-throughput method to screen large numbers of peptides for improved antimicrobial activity. The method relies on peptide synthesis on a cellulose support and a Pseudomonas aeruginosa strain that constitutively expresses bacterial luciferase.

Lipopolysaccharide injection induces relapses of experimental autoimmune encephalomyelitis in nontransgenic mice via bystander activation of autoreactive CD4+ cells.

Infections sometimes associate with exacerbations of autoimmune diseases through pathways that are poorly understood. Ag-specific mechanisms such as cross-reactivity between a microbial Ag and a self-Ag have received no direct support. In this study, we show that injection of LPS induces experimental autoimmune encephalomyelitis in TCR-transgenic mice and relapse of encephalomyelitis in normal mice.

SPOT synthesis: reliability of array-based measurement of peptide binding affinity.

Peptide arrays prepared by the SPOT synthesis technology have emerged as a proteomic tool to study molecular recognition and identify biologically active peptides. However, it was previously not clear how accurately signal intensities obtained by probing peptide arrays for protein binding really reflect the dissociation constants of the protein-peptide complexes. Using the monoclonal antibody CB4-1 as a model system, we systematically compared dissociation constants of antibody-peptide complexes with signal intensities obtained using the SPOT technology.

Determinants governing the potency of STAT3 activation via the individual STAT3-recruiting motifs of gp130.

In recent years, the elucidation of the structures of many signalling molecules has allowed new insights into the molecular mechanisms that govern signal transduction events. In the field of cytokine signalling, the solved structures of cytokine/receptor complexes and of key components involved in signal transduction such as STAT factors or the tyrosine phosphatase SHP2 have broadened our understanding of the molecular basis of the signalling events and provided key information for the rational design of therapeutic approaches to modulate or block cytokine signal transduction. Unfortunately, no structural data on the intracellular parts of cytokine receptors are available.

The periplasmic chaperone SurA exploits two features characteristic of integral outer membrane proteins for selective substrate recognition.

The Escherichia coli periplasmic chaperone and peptidyl-prolyl isomerase (PPIase) SurA facilitates the maturation of outer membrane porins. Although the PPIase activity exhibited by one of its two parvulin-like domains is dispensable for this function, the chaperone activity residing in the non-PPIase regions of SurA, a sizable N-terminal domain and a short C-terminal tail, is essential. Unlike most cytoplasmic chaperones SurA is selective for particular substrates and recognizes outer membrane porins synthesized in vitro much more efficiently than other proteins.

Identification of a novel, intraperoxisomal pex14-binding site in pex13: association of pex13 with the docking complex is essential for peroxisomal matrix protein import.

The peroxisomal docking complex is a key component of the import machinery for matrix proteins. The core protein of this complex, Pex14, is thought to represent the initial docking site for the import receptors Pex5 and Pex7. Associated with this complex is a fraction of Pex13, another essential component of the import machinery.

Function of the PEX19-binding site of human adrenoleukodystrophy protein as targeting motif in man and yeast. PMP targeting is evolutionarily conserved.

We predicted in human peroxisomal membrane proteins (PMPs) the binding sites for PEX19, a key player in the topogenesis of PMPs, by virtue of an algorithm developed for yeast PMPs. The best scoring PEX19-binding site was found in the adrenoleukodystrophy protein (ALDP). The identified site was indeed bound by human PEX19 and was also recognized by the orthologous yeast PEX19 protein.

The tyrosine 974 within the LIF-R-chain of the gp130/LIF-R heteromeric receptor complex mediates negative regulation of LIF signalling.

Signalling of interleukin (IL)-6 and interleukin-11 through gp130 homodimeric receptor complexes has been analysed with respect to initiation and termination of signalling in great detail. Gp130 contains a crucial motif around tyrosine Y759, which mediates negative regulation through the feedback inhibitor SOCS3 and the protein tyrosine phosphatase SHP2. Signalling of leukaemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), CT-1-like factor (CLC) or oncostatin M (OSM) through gp130/LIF-R is believed to be similar due to the presence of the common signal transducer gp130 within the receptor complexes utilized, but the difference in the composition of gp130/gp130-homodimers and gp130/LIF-R-heterodimers is likely to be reflected in different signalling.

Bioluminescence resonance energy transfer reveals ligand-induced conformational changes in CXCR4 homo- and heterodimers.

Homo- and heterodimerization have emerged as prominent features of G-protein-coupled receptors with possible impact on the regulation of their activity. Using a sensitive bioluminescence resonance energy transfer system, we investigated the formation of CXCR4 and CCR2 chemokine receptor dimers. We found that both receptors exist as constitutive homo- and heterodimers and that ligands induce conformational changes within the pre-formed dimers without promoting receptor dimer formation or disassembly.

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.

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.

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.