Hans Voshol

Triggering the succinate receptor GPR91 on dendritic cells enhances immunity

Succinate acts as an extracellular mediator signaling through the G protein–coupled receptor GPR91. Here we show that dendritic cells had high expression of GPR91. In these cells, succinate triggered intracellular calcium mobilization, induced migratory responses and acted in synergy with Toll-like receptor ligands for the production of proinflammatory cytokines. Succinate also enhanced antigen-specific activation of human and mouse helper T cells. GPR91-deficient mice had less migration of Langerhans cells to draining lymph nodes and impaired tetanus toxoid–specific recall T cell responses. Furthermore, GPR91-deficient allografts elicited weaker transplant rejection than did the corresponding grafts from wild-type mice. Our results suggest that the succinate receptor GPR91 is involved in sensing immunological danger, which establishes a link between immunity and a metabolite of cellular respiration.

Preparative two‐dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients

A reproducible high‐resolution protein separation method is the basis for a successful differential proteome analysis. Of the techniques currently available, two‐dimensional gel electrophoresis is most widely used, because of its robustness under various experimental conditions. With the introduction of narrow range immobilized pH gradient (IPG) strips (also referred to as ultra‐zoom gels) in the first dimension, the depth of analysis, i.e. the number of proteins that can be resolved, has increased substantially. However, for poorly understood reasons isoelectric focusing on ultra‐zoom gels in the alkaline region above pH 7 has suffered from problems with resolution and reproducibility. To tackle these difficulties we have optimized the separation of semipreparative amounts of proteins on alkaline IPG strips by focusing on two important phenomena: counteracting water transport during isoelectric focusing and migration of dithiothreitol (DTT) in alkaline pH gradients. The first problem was alleviated by the addition of glycerol and isopropanol to the focusing medium, leading to a significant improvement in the resolution above pH 7. Even better results were obtained by the introduction of excess of the reducing agent DTT at the cathode. With these adaptations together with an optimized composition of the IPG strip, separation efficiency in the pH 6.2–8.2 range is now comparable to the widely used acidic ultra‐zoom gels. We further demonstrated the usefulness of these modifications up to pH 9.5, although further improvements are still needed in that range. Thus, by extending the range covered by conventional ultra‐zoom gels, the depth of analysis of two‐dimensional gel electrophoresis can be significantly increased, underlining the importance of this method in differential proteomics.

Neuroproteomics: expression profiling of the brain's proteomes in health and disease.

The term “proteome” describes the protein complement of a genome. Proteomes of cells are dynamic and are directly affected by environmental factors, such as stress and/or drug treatment, or as a result of aging and disease. One of the distinct advantages of proteomic analysis, not attainable with RNA expression data, is the ability to fractionate the cells proteins into various subpopulations. In neuroscience, “neuromics” (proteomics in the central nervous system) is in its infancy, with a paucity of studies in the context of the brain. One of the objectives of this review is to illustrate the potential of neuromics to profile differences in the distribution of thousands of proteins as a function of disease markers. We have previously used this approach to determine the effects of varied postmortem interval in examining human brain tissue and to identify biomarkers. Here we review proteomic studies of schizophrenia, Alzheimers disease, and Parkinsons disease. Experimental results regarding Parkinsons disease are presented to illustrate the potential of neuromics to identify pathways of pathogenesis and novel therapeutic targets.

Modulation of Activation-Loop Phosphorylation by JAK Inhibitors Is Binding Mode Dependent

Janus kinase (JAK) inhibitors are being developed for the treatment of rheumatoid arthritis, psoriasis, myeloproliferative neoplasms, and leukemias. Most of these drugs target the ATP-binding pocket and stabilize the active conformation of the JAK kinases. This type I binding mode can lead to an increase in JAK activation loop phosphorylation, despite blockade of kinase function. Here we report that stabilizing the inactive state via type II inhibition acts in the opposite manner, leading to a loss of activation loop phosphorylation. We used X-ray crystallography to corroborate the binding mode and report for the first time the crystal structure of the JAK2 kinase domain in an inactive conformation. Importantly, JAK inhibitor-induced activation loop phosphorylation requires receptor interaction, as well as intact kinase and pseudokinase domains. Hence, depending on the respective conformation stabilized by a JAK inhibitor, hyperphosphorylation of the activation loop may or may not be elicited.

Inhibition of Dengue Virus Polymerase by Blocking of the RNA Tunnel

ABSTRACT Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Neither vaccine nor antiviral therapy is currently available for DENV. We report here that N-sulfonylanthranilic acid derivatives are allosteric inhibitors of DENV RNA-dependent RNA polymerase (RdRp). The inhibitor was identified through high-throughput screening of one million compounds using a primer extension-based RdRp assay [substrate poly(C)/oligo(G)20]. Chemical modification of the initial “hit” improved the compound potency to an IC50 (that is, a concentration that inhibits 50% RdRp activity) of 0.7 μM. In addition to suppressing the primer extension-based RNA elongation, the compound also inhibited de novo RNA synthesis using a DENV subgenomic RNA, but at a lower potency (IC50 of 5 μM). Remarkably, the observed anti-polymerase activity is specific to DENV RdRp; the compound did not inhibit WNV RdRp and exhibited IC50s of >100 μM against hepatitis C virus RdRp and human DNA polymerase α and β. UV cross-linking and mass spectrometric analysis showed that a photoreactive inhibitor could be cross-linked to Met343 within the RdRp domain of DENV NS5. On the crystal structure of DENV RdRp, Met343 is located at the entrance of RNA template tunnel. Biochemical experiments showed that the order of addition of RNA template and inhibitor during the assembly of RdRp reaction affected compound potency. Collectively, the results indicate that the compound inhibits RdRp through blocking the RNA tunnel. This study has provided direct evidence to support the hypothesis that allosteric pockets from flavivirus RdRp could be targeted for antiviral development.

Antibody‐based proteomics

Protein kinases drive the cellular signal transduction networks that underlie the regulation of growth, survival and differentiation. To repair the deregulations of signaling cascades that are associated with numerous disease states, therapeutic strategies, based on controlling aberrant protein kinase activity, are emerging. To develop such therapies it is crucial to have knowledge of the full complexity of signaling networks at a molecular level in order to understand the information flow through signaling cascades and their cell and tissue specificity. Antibody‐based proteomic approaches (such as reverse‐phase protein microarrays) are a powerful tool for using to obtain those signaling maps, through the study of phosphorylation states of pathway components using antibodies that specifically recognize the phosphorylated form of kinase substrates.

Guidelines for reporting the use of gel electrophoresis in proteomics

Gibson, Frank Anderson, Leigh Babnigg, Gyorgy Baker, Mark Berth, Matthias Binz, Pierre-Alain Borthwick, Andy Cash, Phil Day, Billy W. Friedman, David B. Garland, Donita Gutstein, Howard B. Hoogland, Christine Jones, Neil A. Khan, Alamgir Klose, Joachim Lamond, Angus I. Lemkin, Peter F. Lilley, Kathryn S. Minden, Jonathan Morris, Nicholas J. Paton, Norman W. Pisano, Michael R. Prime, John E. Rabilloud, Thierry Stead, David A. Taylor, Chris F. Voshol, Hans Wipat, Anil Jones, Andrew R. 2 NATURE PUBLISHING GROUP NEW YORK 335WX

Phosphorylation disrupts the central helix in Op18/stathmin and suppresses binding to tubulin.

Protein phosphorylation represents a ubiquitous control mechanism in living cells. The structural prerequisites and consequences of this important post‐translational modification, however, are poorly understood. Oncoprotein 18/stathmin (Op18) is a globally disordered phosphoprotein that is involved in the regulation of the microtubule (MT) filament system. Here we document that phosphorylation of Ser63, which is located within a helix initiation site in Op18, disrupts the transiently formed amphipathic helix. The phosphoryl group reduces tubulin binding 10‐fold and suppresses the MT polymerization inhibition activity of Op18s C‐terminal domain. Op18 represents an example where phosphorylation occurs within a regular secondary structural element. Together, our findings have implications for the prediction of phosphorylation sites and give insights into the molecular behavior of a globally disordered protein.

Identification of a peptide mimic of the L2/HNK-1 carbohydrate epitope

The L2/HNK‐1 carbohydrate is carried by many neural recognition molecules and is involved in neural cell interactions during development, regeneration in the peripheral nervous system, synaptic plasticity, and autoimmune‐based neuropathies. Its key structure consists of a sulfated glucuronic acid linked to lactosaminyl residues. Because of its biological importance but limited availability, the phage display method was used to isolate a collection of peptide mimics that bind specifically to an L2/HNK‐1 antibody. The phages isolated from a 15‐mer peptide library by adsorption to this antibody share a consensus sequence of amino acids. The peptide mimicked several important functions of the L2/HNK‐1 carbohydrate, such as binding to motor neurons in vitro, and preferential promotion of in vitro neurite outgrowth from motor axons compared with sensory neurons. A scrambled version of the peptide had no activity. The combined observations indicate that we have isolated a mimic of the L2/HNK‐1 carbohydrate that is able to act as its functional substitute.

Tracing pathway activities with kinase inhibitors and reverse phase protein arrays.

Controlling aberrant protein kinase activity is a promising strategy for a variety of diseases, particularly cancer. Hence, the development of kinase inhibitors is currently a focal point for pharmaceutical research. In this study we utilize a chip‐based reverse phase protein array (RPA) platform for profiling of kinase inhibitors in cell‐based assays. In combination with the planar wave‐guide technology the assay system has an absolute LOD down to the low zeptomole range. A431 cell lysates were analyzed for the activation state of key effectors in the epidermal growth factor (EGF) and insulin signaling pathways to validate this model for compound screening. A microtiter‐plate format for growing, treating, and lysing cells was shown to be suitable for this approach, establishing the value of the technology as a screening tool for characterization of large numbers of kinase inhibitors against a wide variety of cellular signaling pathways. Moreover, the reverse array format allows rapid development of site‐specific phosphorylation assays, since in contrast to ELISA type systems only a single antigen‐specific antibody is required.