Historic nucleic acids isolated by Friedrich Miescher contain RNA besides DNA.

One hundred fifty years ago, Friedrich Miescher discovered DNA when he isolated "Nuclein"-as he named it-from nuclei of human pus cells. Miescher recognized his isolate as a new type of molecule equal in importance to proteins. He realised that it is an acid of large molecular weight and high phosphorus content. Subsequently, he discovered Nuclein also in the nuclei of other cell types, realised that it chemically defines the nucleus, and speculated on its role in proliferation, heredity and fertilisation. While now universally recognised as the discoverer of DNA, whether Miescher also discovered RNA has not yet been addressed. To determine whether his isolation also yielded RNA, we first reproduced his historic protocols. Our resulting modern Nuclein contained a significant percentage of RNA. Encouraged by this result, we then analysed a sample of Nuclein isolated by Miescher from salmon sperm. Assuming that the RNA present in this sample had degraded to nucleobases, we tested for the presence of uracil in the historic Nuclein. Detection of significant levels of uracil by LC-UV-MS demonstrates that Miescher isolated both forms of nucleic acid-DNA and RNA-and underlines the fundamental nature of his discovery for the field of molecular genetics.

High-throughput-screening-based identification and structure-activity relationship characterization defined (S)-2-(1-aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole as a highly antimycotic agent nontoxic to cell lines.

Novel nontoxic (S)-2-aminoalkylbenzimidazole derivatives were found to be effective against Candida spp. at low micromolar concentrations using high-throughput screening with infected HeLa cells. A collection of analogues defined the chemical groups relevant for activity. The most active compound was characterized by transcriptional analysis of the response of C. albicans Sc5314. (S)-2-(1-Aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole had a strong impact on membrane biosynthesis. Testing different clinically relevant pathogenic fungi showed the selectivity of the antimycotic activity against Candida species.

Characterization of structural features controlling the receptiveness of empty class II MHC molecules.

MHC class II molecules (MHC II) play a pivotal role in the cell-surface presentation of antigens for surveillance by T cells. Antigen loading takes place inside the cell in endosomal compartments and loss of the peptide ligand rapidly leads to the formation of a non-receptive state of the MHC molecule. Non-receptiveness hinders the efficient loading of new antigens onto the empty MHC II. However, the mechanisms driving the formation of the peptide inaccessible state are not well understood. Here, a combined approach of experimental site-directed mutagenesis and computational modeling is used to reveal structural features underlying "non-receptiveness." Molecular dynamics simulations of the human MHC II HLA-DR1 suggest a straightening of the α-helix of the ß1 domain during the transition from the open to the non-receptive state. The movement is mostly confined to a hinge region conserved in all known MHC molecules. This shift causes a narrowing of the two helices flanking the binding site and results in a closure, which is further stabilized by the formation of a critical hydrogen bond between residues αQ9 and ßN82. Mutagenesis experiments confirmed that replacement of either one of the two residues by alanine renders the protein highly susceptible. Notably, loading enhancement was also observed when the mutated MHC II molecules were expressed on the surface of fibroblast cells. Altogether, structural features underlying the non-receptive state of empty HLA-DR1 identified by theoretical means and experiments revealed highly conserved residues critically involved in the receptiveness of MHC II. The atomic details of rearrangements of the peptide-binding groove upon peptide loss provide insight into structure and dynamics of empty MHC II molecules and may foster rational approaches to interfere with non-receptiveness. Manipulation of peptide loading efficiency for improved peptide vaccination strategies could be one of the applications profiting from the structural knowledge provided by this study.

Bidirectional binding of invariant chain peptides to an MHC class II molecule.

T-cell recognition of peptides bound to MHC class II (MHCII) molecules is a central event in cell-mediated adaptive immunity. The current paradigm holds that prebound class II-associated invariant chain peptides (CLIP) and all subsequent antigens maintain a canonical orientation in the MHCII binding groove. Here we provide evidence for MHCII-bound CLIP inversion. NMR spectroscopy demonstrates that the interconversion from the canonical to the inverse alignment is a dynamic process, and X-ray crystallography shows that conserved MHC residues form a hydrogen bond network with the peptide backbone in both orientations. The natural catalyst HLA-DM accelerates peptide reorientation and the exchange of either canonically or inversely bound CLIP against antigenic peptide. Thus, noncanonical MHC-CLIP displays the hallmarks of a structurally and functionally intact antigen-presenting complex.

Identification of full length bovine TLR1 and functional characterization of lipopeptide recognition by bovine TLR2/1 heterodimer.

Toll-like receptors (TLR) are highly conserved pattern recognition receptors of the innate immune system. Toll-like receptor 2 (TLR2) recognizes bacterial lipopeptides in a heterodimeric complex with TLR6 or TLR1, thereby discriminating between di- or triacylated lipopeptides, respectively. Previously, we found that HEK293 cells transfected with bovine TLR2 (boTLR2) were able to respond to diacylated lipopeptides but did not recognize triacylated lipopeptides, even after cotransfection with the so far published sequence of boTLR1. In this study we now could show that primary bovine cells were in general able to detect triacylated lipopetides. A closer investigation of the boTLR1 gene locus revealed an additional ATG 195 base pairs upstream from the published start codon. Its transcription would result in an N-terminus with high identity to human and murine TLR1 (huTLR1, muTLR1). Cloning and cotransfection of this longer boTLR1 with boTLR2 now resulted in the recognition of triacylated lipopeptides by HEK293 cells, thereby resembling the ex vivo observation. Analysis of the structure-activity relationship showed that the ester-bound acid chains of these lipopeptides need to consist of at least 12 carbon atoms to activate the bovine heterodimer showing similarity to the recognition by huTLR2/huTLR1. In contrast, HEK293 cell cotransfected with muTLR2 and muTLR1 could already be activated by lipopeptides with shorter fatty acids of only 6 carbon atoms. Thus, our data indicate that the additional N-terminal nucleotides belong to the full length and functionally active boTLR1 (boTLR1-fl) which participates in a species-specific recognition of bacterial lipopeptides.

Regulators of G-protein signalling are modulated by bacterial lipopeptides and lipopolysaccharide.

Regulators of G-protein signalling accelerate the GTPase activity of G(alpha) subunits, driving G proteins in their inactive GDP-bound form. This property defines them as GTPase activating proteins. Here the effect of different Toll-like receptor agonists on RGS1 and RGS2 expression in murine bone marrow-derived macrophages and J774 cells was analysed. After stimulation with TLR2/1 or TLR2/6 lipopeptide ligands and the TLR4/MD2 ligand lipopolysaccharide, microarray analyses show only modulation of RGS1 and RGS2 among all the regulators of G-protein signalling tested. Real-time PCR confirmed modulation of RGS1 and RGS2. In contrast to RGS2, which was always downregulated, RGS1 mRNA was upregulated during the first 30 min after stimulation, followed by downregulation. Similar results were also found in the murine macrophage cell line J774. The ligand for intracellular TLR9 modulates RGS1 and RGS2 in a similar manner. However, the TLR3 ligand poly(I:C) permanently upregulates RGS1 and RGS2 expression indicating a different modulation by the MyD88- and TRIF-signalling pathway. This was confirmed using MyD88(-/-) and TRIF(-/-) bone marrow-derived macrophages. Modulation of RGS1 and RGS2 by Toll-like receptor ligands plays an important role during inflammatory and immunological reactions after bacterial and viral infection.

Complementary strategies to elucidate T helper cell epitopes in myasthenia gravis.

CD4(+) T cells specific for the acetylcholine receptor (AChR) are assumed to play an important role in pathogenesis of myasthenia gravis (MG). A large and diverse number of potential T cell epitopes have been reported for different experimental setups aiming at the identification of disease-relevant T cells in MG. Investigating the T cell response to the epsilon subunit of human AChR, we explore complementary in vitro and in vivo approaches (PBMC from MG patients and mice transgenic for HLA-DR3 and human CD4) to address the possibilities and limitations of different strategies for elucidating natural autoimmune T cell epitopes.

Anchor side chains of short peptide fragments trigger ligand-exchange of class II MHC molecules.

Class II MHC molecules display peptides on the cell surface for the surveillance by CD4+ T cells. To ensure that these ligands accurately reflect the content of the intracellular MHC loading compartment, a complex processing pathway has evolved that delivers only stable peptide/MHC complexes to the surface. As additional safeguard, MHC molecules quickly acquire a 'non-receptive' state once they have lost their ligand. Here we show now that amino acid side chains of short peptides can bypass these safety mechanisms by triggering the reversible ligand-exchange. The catalytic activity of dipeptides such as Tyr-Arg was stereo-specific and could be enhanced by modifications addressing the conserved H-bond network near the P1 pocket of the MHC molecule. It affected both antigen-loading and ligand-release and strictly correlated with reported anchor preferences of P1, the specific target site for the catalytic side chain of the dipeptide. The effect was evident also in CD4+ T cell assays, where the allele-selective influence of the dipeptides translated into increased sensitivities of the antigen-specific immune response. Molecular dynamic calculations support the hypothesis that occupation of P1 prevents the 'closure' of the empty peptide binding site into the non-receptive state. During antigen-processing and -presentation P1 may therefore function as important "sensor" for peptide-load. While it regulates maturation and trafficking of the complex, on the cell surface, short protein fragments present in blood or lymph could utilize this mechanism to alter the ligand composition on antigen presenting cells in a catalytic way.

Heterodimerization of TLR2 with TLR1 or TLR6 expands the ligand spectrum but does not lead to differential signaling.

TLR are primary triggers of the innate immune system by recognizing various microorganisms through conserved pathogen-associated molecular patterns. TLR2 is the receptor for a functional recognition of bacterial lipopeptides (LP) and is up-regulated during various disorders such as chronic obstructive pulmonary disease and sepsis. This receptor is unique in its ability to form heteromers with TLR1 or TLR6 to mediate intracellular signaling. According to the fatty acid pattern as well as the assembling of the polypeptide tail, LP can signal through TLR2 in a TLR1- or TLR6-dependent manner. There are also di- and triacylated LP, which stimulate TLR1-deficient cells and TLR6-deficient cells. In this study, we investigated whether heterodimerization evolutionarily developed to broaden the ligand spectrum or to induce different immune responses. We analyzed the signal transduction pathways activated through the different TLR2 dimers using the three LP, palmitic acid (Pam)octanoic acid (Oct)(2)C-(VPGVG)(4)VPGKG, fibroblast-stimulating LP-1, and Pam(2)C-SK(4). Dominant-negative forms of signaling molecules, immunoblotting of MAPK, as well as microarray analysis indicate that all dimers use the same signaling cascade, leading to an identical pattern of gene activation. We conclude that heterodimerization of TLR2 with TLR1 or TLR6 evolutionarily developed to expand the ligand spectrum to enable the innate immune system to recognize the numerous, different structures of LP present in various pathogens. Thus, although mycoplasma and Gram-positive and Gram-negative bacteria may activate different TLR2 dimers, the development of different signal pathways in response to different LP does not seem to be of vital significance for the innate defense system.

The activity of lipopeptide TLR2 agonists critically depends on the presence of solubilizers.

Lipoproteins activate cells of the innate immune system via heteromers of Toll-like receptor (TLR) 2 with either TLR1 or TLR6. In spite of progress in understanding TLR-dependent signal transduction and the pathophysiological relevance of TLR2, the molecular basis of ligand recognition by this receptor is poorly defined. Here, we show that the bioactivity of lipopeptides (LP) critically depends on the dilution protocol and especially the presence of proteins or detergents acting as solubilizers. Fluorescence correlation spectroscopy of fluorescently labeled analogs of synthetic LP revealed that the LP form aggregates in solution. Dilution into protein- and serum-free buffers led to a complete loss of activity due to formation of large and highly heterogeneous aggregates. When dimethylsulfoxide stock solutions were diluted into BSA or serum-containing buffers particles of strongly reduced size were obtained. For some LP, an intermediary dilution step either with tert.-butyl alcohol/H2O (4:1) or with octyl-beta-D-glucopyranoside further increased activity. For a panel of LP exhibiting very different activities when diluted directly into protein-containing solutions, introduction of this dilution step resulted in comparable bioactivities. These results demonstrate the significance of solubilizing agents for the bioactivity of LP and are highly relevant for analyzing structure-activity relationships of LP-dependent TLR2 activation.

Planktocyclin, a cyclooctapeptide protease inhibitor produced by the freshwater cyanobacterium Planktothrix rubescens.

The freshwater cyanobacterium Planktothrix rubescens produces the cyclooctapeptide cyclo(Pro-Gly-Leu-Val-Met-Phe-Gly-Val). The chemical structure is new. This homodetic cyclic octapeptide was named planktocyclin ( 1). It consists solely of proteinogenic l-amino acids and is a strong inhibitor of mammalian trypsin and alpha-chymotrypsin and a moderately active inhibitor of human recombinant caspase-8. Mass spectrometric and 2D-NMR spectroscopic data allowed the determination of its structure. Synthetic planktocyclin was identical to the natural product.

Insecticidal activity of 12-epi-hapalindole J isonitrile.

12-epi-Hapalindole J isonitrile (1) and three previously described hapalindoles, 12-epi-hapalindole C isonitrile (2), hapalindole L (3) and 12-epi-hapalindole E isonitrile (4) were isolated and identified as insecticidal alkaloids of the biofilm-forming freshwater cyanobacterium Fischerella ATCC 43239 (Stigonematales). The structures of the purified compounds were elucidated by ESI-FTICR-MS, GC-EI-MS and various 2D NMR experiments. At 26 microM hapalindole 1 killed 100% of the larvae of the dipteran Chironomus riparius within 48h. Insecticidal activities were also found at similar concentration for the hapalindoles 2-4. The bioactivity of hapalindoles demonstrates that cyanobacterial biofilms can be considered as promising sources of insecticidal metabolites which might be useful for the biocontrol of dipterans.

Physicochemical and biological analysis of synthetic bacterial lipopeptides: validity of the concept of endotoxic conformation.

The importance of the biological function and activity of lipoproteins from the outer or cytoplasmic membranes of Gram-positive and Gram-negative bacteria is being increasingly recognized. It is well established that they are like the endotoxins (lipopolysaccharide (LPS)), which are the main amphiphilic components of the outer membrane of Gram-negative bacteria, potent stimulants of the human innate immune system, and elicit a variety of proinflammatory immune responses. Investigations of synthetic lipopeptides corresponding to N-terminal partial structures of bacterial lipoproteins defined the chemical prerequisites for their biological activity and in particular the number and length of acyl chains and sequence of the peptide part. Here we present experimental data on the biophysical mechanisms underlying lipopeptide bioactivity. Investigation of selected synthetic diacylated and triacylated lipopeptides revealed that the geometry of these molecules (i.e. the molecular conformations and supramolecular aggregate structures) and the preference for membrane intercalation provide an explanation for the biological activities of the different lipopeptides. This refers in particular to the agonistic or antagonistic activity (i.e. their ability to induce cytokines in mononuclear cells or to block this activity, respectively). Biological activity of lipopeptides was hardly affected by the LPS-neutralizing antibiotic polymyxin B, and the biophysical interaction characteristics were found to be in sharp contrast to that of LPS with polymyxin B. The analytical data show that our concept of "endotoxic conformation," originally developed for LPS, can be applied also to the investigated lipopeptide and suggest that the molecular mechanisms of cell activation by amphiphilic molecules are governed by a general principle.

Ubiquitin binds to a short peptide segment of hydrolase UCH-L3: a study by FCS, RIfS, ITC and NMR.

Screening for small peptidic affinity tags for the detection of ubiquitin and ubiquitinated proteins yielded the dodecapeptide amide DPDELRFNAIAL-NH(2) as a specific ubiquitin-interacting ligand. A peptide collection--based on crystal structures with ubiquitin-interacting proteins--was designed and confirmed by sequence comparison of ubiquitin-interacting motifs. Four independent physical detection methods demonstrated that the peptide binds to monomeric ubiquitin with an affinity of about 10 muM and with fast on and off rates. Fluorescence correlation spectroscopy with fluorescent peptides showed specific interaction with ubiquitin. Reflectometric interference spectroscopy with surface-immobilized peptides and isothermal calorimetry measurements confirmed the specific binding of ubiquitin and fast rate constants. (1)H,(15)N heteronuclear NMR localised the interaction site across the beta sheet of ubiquitin. The peptide aligns well with the ubiquitin-interacting motif and represents a lead structure for the rational design of high-affinity tags for targeting ubiquitinated protein in vitro and in vivo.

A fluorescence-based synthetic LPS sensor.

For the detection of bioanalytes, there is an ongoing search for synthetic sensors to replace enzyme-based assays which are sensitive to contaminants or suboptimal storage conditions. Lipopolysaccharide (LPS), a bacteria-borne endotoxin that may lead to life-threatening conditions such as septic shock, is one such case. Fluorescently labeled analogues of two peptide variants derived from the putative ligand-binding domain of the LPS-binding protein CD14 were developed that detect and discriminate LPS and lipids down to the submicromolar concentration range. Peptides are terminally labeled with carboxyfluorescein and tetramethylrhodamine. For one given peptide, sensitivity and specificity for the detection of LPS and discrimination from other lipids are achieved by spectral signatures that combine changes in the fluorescence resonance energy transfer (FRET) between both dyes and the total emission of tetramethylrhodamine. Alternatively, specificity is obtained by combining the FRET efficiencies of both peptide variants. In comparison to published synthetic LPS sensors, the CD14-derived sensors yield an increase in sensitivity by about 3 orders of magnitude and exhibit specificity for analytes for which the design of synthetic recognition elements is a challenging task. Moreover, one of the sensors enabled the detection of LPS in the presence of up to 50% fetal calf serum, thereby demonstrating the feasibility of this peptide-based approach for clinically relevant samples.

Small organic compounds enhance antigen loading of class II major histocompatibility complex proteins by targeting the polymorphic P1 pocket.

Major histocompatibility complex (MHC) molecules are a key element of the cellular immune response. Encoded by the MHC they are a family of highly polymorphic peptide receptors presenting peptide antigens for the surveillance by T cells. We have shown that certain organic compounds can amplify immune responses by catalyzing the peptide loading of human class II MHC molecules HLA-DR. Here we show now that they achieve this by interacting with a defined binding site of the HLA-DR peptide receptor. Screening of a compound library revealed a set of adamantane derivatives that strongly accelerated the peptide loading rate. The effect was evident only for an allelic subset and strictly correlated with the presence of glycine at the dimorphic position beta86 of the HLA-DR molecule. The residue forms the floor of the conserved pocket P1, located in the peptide binding site of MHC molecule. Apparently, transient occupation of this pocket by the organic compound stabilizes the peptide-receptive conformation permitting rapid antigen loading. This interaction appeared restricted to the larger Gly(beta86) pocket and allowed striking enhancements of T cell responses for antigens presented by these "adamantyl-susceptible" MHC molecules. As catalysts of antigen loading, compounds targeting P1 may be useful molecular tools to amplify the immune response. The observation, however, that the ligand repertoire can be affected through polymorphic sites form the outside may also imply that environmental factors could induce allergic or autoimmune reactions in an allele-selective manner.

Immunocytochemical analysis of rat vagus nerve by antibodies against glycogen phosphorylase isozymes.

Glycogen is an endogenous store of glucose equivalents for energy metabolism in many tissues. The brain contains a significant amount of glycogen the role of which as an energy reserve is currently under debate. Apparently little is known concerning a possible role of glycogen in peripheral nerves. We have demonstrated immunocytochemically the presence of glycogen phosphorylase (GP), a key enzyme in glycogen metabolism, in large and small axons of the rat vagus nerve, but not in Schwann cells. Furthermore, the isozyme-specific antibodies applied detected only the presence of the brain isoform BB of GP, but not the muscle isoform MM. This is in agreement with the occurrence of solely the BB isoform in the few brain and spinal cord neurons that contain GP. In contrast, astroglial cells in brain and spinal cord have previously been shown to contain both isoforms. Since GP isozymes are regulated differentially, the expression of isoform BB may provide hints to possible functions of glycogen in the vagus nerve.

HLA-DQ2 and -DQ8 signatures of gluten T cell epitopes in celiac disease.

Celiac disease is associated with HLA-DQ2 and, to a lesser extent, HLA-DQ8. Type 1 diabetes is associated with the same DQ molecules in the opposite order and with possible involvement of trans-encoded DQ heterodimers. T cells that are reactive with gluten peptides deamidated by transglutaminase 2 and invariably restricted by DQ2 or DQ8 can be isolated from celiac lesions. We used intestinal T cells from celiac patients to map DQ2 and DQ8 epitopes within 2 representative gluten proteins, alpha-gliadin AJ133612 and gamma-gliadin M36999. For alpha-gliadin, DQ2- and DQ8-restricted T cells recognized deamidated peptides of 2 separate regions. For gamma-gliadin, DQ2- and DQ8-restricted T cells recognized deamidated peptides of the same region. Some gamma-gliadin peptides were recognized by T cells in the context of DQ2 or DQ8 when bound in exactly the same registers, but with different requirements for deamidation; deamidation at peptide position 4 (P4) was important for DQ2-restricted T cells, whereas deamidation at P1 and/or P9 was important for DQ8-restricted T cells. Peptides combining the DQ2 and DQ8 signatures could be presented by DQ2, DQ8, and trans-encoded DQ heterodimers. Our findings shed light on the basis for the HLA associations in celiac disease and type 1 diabetes.