Jacob Piehler

Helical Structures of ESCRT-III Are Disassembled by VPS4

During intracellular membrane trafficking and remodeling, protein complexes known as the ESCRTs (endosomal sorting complexes required for transport) interact with membranes and are required for budding processes directed away from the cytosol, including the budding of intralumenal vesicles to form multivesicular bodies; for the budding of some enveloped viruses; and for daughter cell scission in cytokinesis. We found that the ESCRT-III proteins CHMP2A and CHMP3 (charged multivesicular body proteins 2A and 3) could assemble in vitro into helical tubular structures that expose their membrane interaction sites on the outside of the tubule, whereas the AAA-type adenosine triphosphatase VPS4 could bind on the inside of the tubule and disassemble the tubes upon adenosine triphosphate hydrolysis. CHMP2A and CHMP3 copolymerized in solution, and their membrane targeting was cooperatively enhanced on planar lipid bilayers. Such helical CHMP structures could thus assemble within the neck of an inwardly budding vesicle, catalyzing late steps in budding under the control of VPS4.

Integrated optical Mach-Zehnder biosensor

We present measurements on biomolecular surface multilayers using an integrated optical sensor based on the Mach-Zehnder interferometer (MZI). The sensor design is unique in that it incorporates a three-waveguide coupler structure at the interferometer output which gives advantages in terms of signal referencing and in establishing and maintaining a sensitive operating point. The sensor performance is characterized with respect to bulk superstrate index and by the formation of multiple protein adlayers using a biotin-avidin-based biochemical system. The detection limit for protein loading is estimated as 5 pg/mm/sup 2/.

The Receptor of the Type I Interferon Family

All type I IFNs act through a single cell surface receptor composed of the IFNAR1 and IFNAR2 subunits and two associated cytoplasmic tyrosine kinases of the Janus family, Tyk2 and Jak1. A central issue in type I IFN biology is to understand how a multitude of subtypes can generate similar signaling outputs but also govern specific cellular responses. This review summarizes results from the last decade that contributed to our current state of knowledge of IFN-receptor complex structure and assembly.

A high-density poly(ethylene glycol) polymer brush for immobilization on glass-type surfaces

Label-free heterogeneous phase detection critically depends on the properties of the interfacial layer. We have obtained high-density monomolecular poly(ethylene glycol) (PEG) layers by solvent-free coupling of homo-bifunctional PEGs (2,000 g/mol) at 75 degrees C to silica surfaces silanized with glycidyloxipropyltrimethoxysilane (GOPTS). Characterization by ellipsometry and contact angles revealed that PEG layers up to 3.4 ng/mm2 with low roughness and flexibility were obtained. Specific and non-specific binding at these PEG surfaces was monitored by reflectometric interference spectroscopy (RIfS). No significant non-specific adsorption upon incubation of 1 mg/ml ovalbumin was detectable (< 10 pg/mm2), and 150 pg/mm2 upon incubation of 10% calf serum, less than 10% of the amount adsorbed to the solely silanized surfaces. The terminal functional groups of the PEG layers were utilized to couple ligands and a protein. Specific protein interaction with these immobilized compounds was detected with saturation loadings in the range of protein monolayers (2-4 ng/mm2). The excellent functional properties, the high stability of the layers, the generic and practical coupling procedure and the versatility for immobilizing compounds of very different functionality make these PEG layers very attractive for application in label-free detection with silica or metal-oxide based transducers.

Inquiring into the Differential Action of Interferons (IFNs): an IFN-α2 Mutant with Enhanced Affinity to IFNAR1 Is Functionally Similar to IFN-β

ABSTRACT Alpha and beta interferons (IFN-α and IFN-β) are multifunctional cytokines that exhibit differential activities through a common receptor composed of the subunits IFNAR1 and IFNAR2. Here we combined biophysical and functional studies to explore the mechanism that allows the alpha and beta IFNs to act differentially. For this purpose, we have engineered an IFN-α2 triple mutant termed the HEQ mutant that mimics the biological properties of IFN-β. Compared to wild-type (wt) IFN-α2, the HEQ mutant confers a 30-fold higher binding affinity towards IFNAR1, comparable to that measured for IFN-β, resulting in a much higher stability of the ternary complex as measured on model membranes. The HEQ mutant, like IFN-β, promotes a differentially higher antiproliferative effect than antiviral activity. Both bring on a down-regulation of the IFNAR2 receptor upon induction, confirming an increased ternary complex stability of the plasma membrane. Oligonucleotide microarray experiments showed similar gene transcription profiles induced by the HEQ mutant and IFN-β and higher levels of gene induction or repression than those for wt IFN-α2. Thus, we show that the differential activities of IFN-β are directly related to the binding affinity for IFNAR1. Conservation of the residues mutated in the HEQ mutant within IFN-α subtypes suggests that IFN-α has evolved to bind IFNAR1 weakly, apparently to sustain differential levels of biological activities compared to those induced by IFN-β.

Surface modification for direct immunoprobes

The modification of glass-type surfaces by several hydrophilic polymers of different molecular masses and functional properties [chitosan, dextran, poly(oxyethylene), poly(ethyleneimine) and poply(acrylamide)] with respect to the application for direct immunoprobes was investigated. Activation of the surface was carried out by silanisation and the polymers were coupled to the surface via amide bonds. The carboxyl derivative of a hapten was attached to the functional groups of the polymers by carbodiimide-activated coupling. As a reference system, the ligand was directly coupled to the silanised surface. Non-specific protein adsorption, specific binding of antibodies and regeneration were monitored by evaluation of reflectance spectra obtained by white light interference at a thin silica layer (RifS). All polymer modified layers showed improved properties compared to those with direct attachment of the hapten. The non-specific adsorption was reduced to 5-50%. Binding of a specific antibody was significantly increased by the polymer modification: Mass transport limited binding of the specific antibody in low concentrations (30 nM) up to a surface coverage value of 2 ng/mm2 and a maximum surface coverage in the range of a monolayer of IgG (5-6 ng/mm2) was observed for most of the polymers. The surface coverage found for IgG bound specifically to the dextran-modified surface exceeded a protein monolayer.

USP18-Based Negative Feedback Control Is Induced by Type I and Type III Interferons and Specifically Inactivates Interferon α Response

Type I interferons (IFN) are cytokines that are rapidly secreted upon microbial infections and regulate all aspects of the immune response. In humans 15 type I IFN subtypes exist, of which IFN α2 and IFN β are used in the clinic for treatment of different pathologies. IFN α2 and IFN β are non redundant in their expression and in their potency to exert specific bioactivities. The more recently identified type III IFNs (3 IFN λ or IL-28/IL-29) bind an unrelated cell-type restricted receptor. Downstream of these two receptor complexes is a shared Jak/Stat pathway. Several mechanisms that contribute to the shut down of the IFN-induced signaling have been described at the molecular level. In particular, it has long been known that type I IFN induces the establishment of a desensitized state. In this work we asked how the IFN-induced desensitization integrates into the network built by the multiple type I IFN subtypes and type III IFNs. We show that priming of cells with either type I IFN or type III IFN interferes with the cells ability to further respond to all IFN α subtypes. Importantly, primed cells are differentially desensitized in that they retain sensitivity to IFN β. We show that USP18 is necessary and sufficient to induce differential desensitization, by impairing the formation of functional binding sites for IFN α2. Our data highlight a new type of differential between IFNs α and IFN β and underline a cross-talk between type I and type III IFN. This cross-talk could shed light on the reported genetic variation in the IFN λ loci, which has been associated with persistence of hepatitis C virus and patients response to IFN α2 therapy.

A direct optical immunosensor for atrazine detection

Abstract Immunoanalytical techniques represent one of the most important applications of biomolecules in analytical procedures. Direct monitoring of immunoreactions by an analytical device is a particularly attractive approach to environmental sensing as it offers speed, a simple test scheme and does not require labelled compounds. Target limits of detection for pesticides are imposed by the EU drinking water act (0.1 μg/l for a single pesticide). A competitive test format with surface immobilised antigen is common for pesticide detection with direct immunosensors. Free pesticide binds to antibody in solution decreasing the amount of antibody binding to the transducer. As the concentration of antibody in a competitive test must be comparable to the concentration of the analyte (0.1 ppb atrazine ≈ 0.5 nM), the transducer must be able to resolve changes in antibody concentration below 1 nM. A prototype atrazine sensor based on reflectometric interference spectroscopy (RIFS) was investigated. The slope of the binding curve was used as a measure for antibody concentration. Binding curves of monoclonal anti-atrazine to an atrazine-modified surface were recorded at different concentrations. A reproducibility of 10% was found for the determination of antibody concentrations (100 to 1000 ng/ml). In competitive binding experiments with free atrazine inhibition of 50% was observed at 0.5 ppb atrazine (500 ng/ml antibody) and at 1 ppb (1 μg/ml antibody). The results are discussed with respect to the theoretical performance of the device and practical requirements. A comparison to other optical transducers and a critical discussion about the feasibility of direct optical immunosensors in pesticide detection is given.

Determination of simazine in water samples by waveguide surface plasmon resonance

We assessed a new sensing device based on the monitoring of immunobinding reactions using waveguide surface plasmon resonance (WSPR) for the determination of simazine in water samples. Standard solutions between 0.1 and 1.0 μg l−1 analysed in triplicate showed a mean within-day variability of 5%. Calibration curves for the same standards conducted on five consecutive days showed a 14% mean day-to-day variability. The detection limit calculated as three standard deviations below the mean blank value was 0.2 μg l−1. The upper limit of the working range calculated as a 90% decrease in the blank signal was 2.4 μg l−1. The cross-reactivity of atrazine and terbuthylazine was 61 and 63%, respectively. The recovery from spiked natural ground- and surface-water samples ranged from 55 to 153% for spikes ranging from 0.1 to 1.0 μg l−1. For the 11 surface- and 8 ground-water samples tested, the correlation coefficient between WSPR and high pressure liquid chromatography/gas chromatography (HPLC/GC) values was significant (p<0.05) when the chromatography values were calculated as the weighted sum of simazine and atrazine, taking into account the predetermined cross-reactivity of the latter in the WSPR determination. The present system is therefore better suited for screening groups of pesticides than for the determination of a single molecule. An attempt at analysing a soil water sample proved unsuccessful due to interference probably resulting from strong non-selective polyanion-polycation binding to the transducer surface which includes a basic amino dextran. The total duration of one determination, 22 min, enables almost immediate measurements without any sample pretreatment other than 0.45 μm filtration. No significant alteration of the sensor was observed after 200 determinations.

Structural and dynamic determinants of type I interferon receptor assembly and their functional interpretation.

Type I interferons (IFNs) form a network of homologous cytokines that bind to a shared, heterodimeric cell surface receptor and engage signaling pathways that activate innate and adaptive immune responses. The ability of IFNs to mediate differential responses through the same cell surface receptor has been subject of a controversial debate and has important medical implications. During the past decade, a comprehensive insight into the structure, energetics, and dynamics of IFN recognition by its two‐receptor subunits, as well as detailed correlations with their functional properties on the level of signal activation, gene expression, and biological responses were obtained. All type I IFNs bind the two‐receptor subunits at the same sites and form structurally very similar ternary complexes. Differential IFN activities were found to be determined by different lifetimes and ligand affinities toward the receptor subunits, which dictate assembly and dynamics of the signaling complex in the plasma membrane. We present a simple model, which explains differential IFN activities based on rapid endocytosis of signaling complexes and negative feedback mechanisms interfering with ternary complex assembly. More insight into signaling pathways as well as endosomal signaling and trafficking will be required for a comprehensive understanding, which will eventually lead to therapeutic applications of IFNs with increased efficacy.