View: publication – Dualsystems Biotech AG

Identification of a receptor for house dust mite allergens

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Abstract

Development of therapies with the potential to change the allergic asthmatic disease course will require the discovery of targets that play a central role during the initiation of an allergic response, such as those involved in the process of allergen recognition. We use a receptor glycocapture technique to screen for house dust mite (HDM) receptors and identify LMAN1 as a candidate. We verify the ability of LMAN1 to directly bind HDM allergens and demonstrate that LMAN1 is expressed on the surface of dendritic cells (DCs) and airway epithelial cells (AECs) in vivo. Overexpression of LMAN1 downregulates NF-κB signaling in response to inflammatory cytokines or HDM. HDM promotes binding of LMAN1 to the FcRγ and recruitment of SHP1. Last, peripheral DCs of asthmatic individuals show a significant reduction in the expression of LMAN1 compared with healthy controls. These findings have potential implications for the development of therapeutic interventions for atopic disease.

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LRC-TriCEPS identified a completely new mechanism of monocyte activation that might be clinically relevant in Alzheimers’ and inflammation diseases. Feb 2023

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The emerging cytokine tissue inhibitor of metalloproteinases-1 (TIMP-1) correlates with the progression of inflammatory diseases, including cancer. However, the effects of TIMP-1 on immune cell activation and underlying molecular mechanisms are largely unknown. The LRC-TriCEPS platform revealed TIMP-1-interaction with Amyloid Precursor Protein (APP) family members. We found that TIMP-1 triggered glucose uptake and flammatory cytokine expression in human monocytes. In cancer patients, TIMP-1 expression positively correlated with flammatory cytokine expression and processes associated with monocyte activation. This novel monocyte activation pathway might be clinically relevant for inflammation diseases such as pancreatic cancer but might also be involved in Alzheimers’.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9837626/

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Sensei Biotherapeutics Presents Preclinical Data at the 37th Society for Immunotherapy of Cancer (SITC) Annual Meeting

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Identification of key extracellular binding proteins

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Identification of key extracellular binding proteins implicate role in inflammation and fibrosis for alanyl- and aspartyl-tRNA synthetases

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Akkermansia muciniphila secretes a gluc.-like peptide-1-inducing protein that improves glucose homeostasis and ameliorates metabolic disease in mice

Intro

Nature Microbiol (2021) https://doi.org/10.1038/s41564-021-00880-5

Abstract

Here, we report that A. muciniphila increases thermogenesis and gluc.-like peptide-1 (GLP-1) secretion in high-fat-diet (HFD)-induced C57BL/6J mice by induction of uncoupling protein 1 in brown adipose tissue and systemic GLP-1 secretion. We apply fast protein liquid chromatography and liquid chromatography coupled to mass spectrophotometry analysis to identify an 84 kDa protein, named P9, that is secreted by A. muciniphila. Using L cells and mice fed on an HFD, we show that purified P9 alone is sufficient to induce GLP-1 secretion and brown adipose tissue thermogenesis. Using ligand–receptor capture analysis, we find that P9 interacts with intercellular adhesion molecule 2 (ICAM-2). Interleukin-6 deficiency abrogates the effects of P9 in glucose homeostasis and downregulates ICAM-2 expression. Our results show that the interactions between P9 and ICAM-2 could be targeted by therapeutics for metabolic diseases.

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Neuroplastin Modulates Anti-inflammatory Effects of MANF

Intro

iScience Volume 23, ISSUE 12, 101810, December 18, 2020, DOI:https://doi.org/10.1016/j.isci.2020.101810

Abstract

Identification of Neuroplastin (NPTN) as receptor of MANF polypeptide by LRC-TriCEPS. Endoplasmic reticulum (ER) stress is known to induce pro-inflammatory response and ultimately leads to cell death. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an ER-localized protein whose expression and secretion is induced by ER stress and a crucial survival factor. However, the underlying mechanism of how MANF exerts its cytoprotective activity remains unclear due to the lack of knowledge of its receptor. Here we show that Neuroplastin (NPTN) is such a receptor for MANF. Biochemical analysis shows the physiological interaction between MANF and NPTN on the cell surface. Binding of MANF to NPTN mitigates the inflammatory response and apoptosis via suppression of NF-kβ signaling. Our results demonstrate that NPTN is a cell surface receptor for MANF, which modulates inflammatory responses and cell death, and that the MANF-NPTN survival signaling described here provides potential therapeutic targets for the treatment of ER stress-related disorders, including diabetes mellitus, neurodegeneration, retinal degeneration, and Wolfram syndrome.

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NKG2A/CD94 Is a New Immune Receptor for HLA-G and Distinguishes Amino Acid Differences in the HLA-G Heavy Chain

Intro

International Journal of Molecular Sciences. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352787/

Abstract

The immune checkpoint molecule human leukocyte antigen (HLA)-G is upregulated on malignant cells but not on healthy surrounding cells, the requirement of understanding the basis of receptor mediated events at the HLA-G/NK cell interface becomes obvious. The NK cell receptors ILT2 and KIR2DL4 have been described to bind to HLA-G; however, their differential function and expression levels on NK cell subsets suggest the existence of an unreported receptor. Here, we performed a ligand-based receptor capture on living cells utilizing sHLA-G*01:01 molecules coupled to TriCEPS and bound to NK cells followed by mass spectrometric analyses. We could define NKG2A/CD94 as a cognate receptor of HLA-G.

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Acidity changes immunology: a new VISTA pathway

Intro

Nature Immunology volume 21, pages13–16 (2020)

Abstract

Using the LRC-TriCEPS platform PSGL-1 was identified as the interaction partner of the co-inhibitor Vista on activated primary T-cells as at acidic pH mimicking the acidic tumour environment
Now, Vista tumour immunotherapy can rationally move forward
The results also suggest to use also acidity as therapeutic target for tumour therapy
Now also other interactions at acidic pH for drug candidates or co-inhibitor interactions should be tested on primary T-cells. It could explain why some drug candidates are not successful in later development.

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Nature Article VISTA interaction with PSGL-1 identified by LRC-TriCEPS

Intro

https://www.nature.com/articles/s41586-019-1674-5 Nature volume 574, pages565–570 (2019)

Abstract

Summary We then performed ligand-based receptor capture with VISTA–Fc chimeric protein and human CD4 + Tcells at acidic pH 19 . PSGL-1 was one of few proteins that were enriched relative to controls (Fig.3a). In addition to its well-characterized role facilitating adhesion interactions between leukocytes, platelets and endothelial cells 20,21 , PSGL-1 has been identified as a negative regulator of T cell responses in contexts of chronic viral infection, cancer, and some autoimmune diseases 22–28 .

Author

Robert J. Johnston, Linhui Julie Su, Jason Pinckney, David Critton, Eric Boyer, Arathi Krishnakumar, Martin Corbett, Andrew L. Rankin, Rose Dibella, Lynne Campbell, Gaelle H. Martin, Hadia Lemar, Thomas Cayton, Richard Y.-C. Huang, Xiaodi Deng, Akbar Nayeem, Haibin Chen, Burce Ergel, Joseph M. Rizzo, Aaron P. Yamniuk, Sanjib Dutta, Justine Ngo, Andrea Olga Shorts, Radha Ramakrishnan, Alexander Kozhich, Jim Holloway, Hua Fang, Ying-Kai Wang, Zheng Yang, Kader Thiam, Ginger Rakestraw, Arvind Rajpal, Paul Sheppard, Michael Quigley, Keith S. Bahjat & Alan J. Korman

Anti-VISTA antibody that inhibits Vista function and blocks interaction with PSGL-1 and VSIG3 proteins slows tumor growth

Intro

https://www.nature.com/articles/s41598-020-71519-4 Mehta, N., Maddineni, S., Kelly, R.L. et al. An engineered antibody binds a distinct epitope and is a potent inhibitor of murine and human VISTA. Sci Rep 10, 15171 (2020). https://doi.org/10.1038/s41598-020-71519-4 PDF

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Phage resistance at the cost of virulence

Intro

Phage resistance at the cost of virulence: Listeria monocytogenes serovar 4b requires galactosylated teichoic acids for InlBmediated invasion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1008032 October 7, 2019
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Abstract

L. monocytogenes is a Gram-positive, food-borne, intracellular pathogen that causes severe infection in susceptible individuals. Interestingly, almost all infections are caused by a subset of strains belonging to certain serovars featuring a complex glycosylation pattern on their cell surface. Using an engineered bacteriophage that specifically recognizes these modifications we selected for mutants that lost these sugars. We found that the resulting strains are severely deficient in invading host cells as we observed that a major virulence factor mediating host cell entry requires galactose decoration of the cell surface for its function. Without this galactose decoration, the strain represents a serovar not associated with disease. Altogether, we show a complex interplay between bacteriophages, bacteria, and the host, demonstrating that cellular invasiveness is dependent upon a serovar-defining structure, which also serves as a phage receptor.

Author

TFF3 interacts with LINGO2 to regulate EGFR activation for protection against colitis and gastrointestinal helminths

Intro

Nature Communications volume 10, Article number: 4408 (2019) https://doi.org/10.1038/s41467-019-12315-1

Abstract

Intestinal epithelial cells (IEC) have important functions in nutrient absorption, barrier integrity, regeneration, pathogen-sensing, and mucus secretion. Goblet cells are a specialized cell type of IEC that secrete Trefoil factor 3 (TFF3) to regulate mucus viscosity and wound healing, but whether TFF3-responsiveness requires a receptor is unclear. We postulated that TFF3 interacted with its receptor through low-affinity interactions reliant upon carbohydrates because TFF3 glycosylation has been shown critical for biological activity34,35, therefore U937 cells were subjected to the TRICEPSTM protocol as a biochemical screening strategy to identify glycosylated transmembrane protein(s) on the cell surface32,36. As bait, rhTFF3 was covalently linked to the TRICEPS probe and incubated with PMA-treated U937 that had been treated with sodium periodate. Cell pellets were subjected to glycosidase digestion and peptides generated using mass spectrometry. As a positive control to account for enrichment efficiency, recombinant human>INS was used as bait for the>INS receptor. Whereas the TFF3-probe induced an 8-fold enrichment of LINGO2 (LIGO2) peptide, the>INS-probe induced a 7.76-fold-enrichment in INSR peptide (Fig. 1b). No other enriched peptides in this screen met this level of enrichment or were derived from proteins that satisfied our selection criterion (e.g., extracellular domain, transmembrane region, cytoplasmic tail).Here, we show that leucine rich repeat receptor and nogo-interacting protein 2 (LINGO2) is essential for TFF3-mediated functions. LINGO2 immunoprecipitates with TFF3, co-localizes with TFF3 on the cell membrane of IEC, and allows TFF3 to block apoptosis. We further show that TFF3-LINGO2 interactions disrupt EGFR-LINGO2 complexes resulting in enhanced EGFR signaling. Excessive basal EGFR activation in Lingo2 deficient mice increases disease severity during colitis and augments immunity against helminth infection. Conversely, TFF3 deficiency reduces helminth immunity. Thus, TFF3-LINGO2 interactions de-repress inhibitory LINGO2-EGFR complexes, allowing TFF3 to drive wound healing and immunity.

Author

Nicole Maloney Belle 1, Yingbiao Ji 1,5, Karl Herbine 1,5, Yun Wei 2, 3,5, JoonHyung Park 1,5, Kelly Zullo1, Li-Yin Hung 1,2, Sriram Srivatsa 1, Tanner Young 1, Taylor Oniskey 2, Christopher Pastore 1, Wildaliz Nieves 4, Ma Somsouk 4 & De’Broski R. Herbert 1,2 1 Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19140, USA. 2 Division of Experimental Medicine, University of California, San Francisco, San Francisco, CA 94110, USA. 3 Department of Inflammation and Oncology, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA. 4 Division of Gastroenterology at ZSFG, University of California, San Francisco, San Francisco, CA 94110, USA. 5These authors contributed equally: Yingbiao Ji, Karl Herbine, Yun Wei, JoonHyung Park. Email: debroski@vet.upenn.edu

White Paper Ligand-Receptor Identification Methodologies Details Matter

Reference

European Biopharmaceutical Review April 2019, pages 16-20 / Maria P. Pavlou and Paul Helbling / White -Paper PDF

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Validation of extracellular ligand–receptor interactions by Flow‑TriCEPS

Reference

BMC Research Notes 2018 11:863 https://doi.org/10.1186/s13104-018-3974-5 Laura A. Lopez‑Garcia, Levent Demiray, Sandra Ruch‑Marder, Ann‑Katrin Hopp, Michael O. Hottiger, Paul M. Helbling and Maria P. Pavlou Received: 28 October 2018 – Accepted: 30 November 2018- Published: 5 December 2018 PDF

Abstract

The advent of ligand based receptor capture methodologies, allows the identification of unknown receptor candidates for orphan extracellular ligands. However, further target validation can be tedious, laborious and time consuming. Here, we present a methodology that provides a fast and costefficient alternative for candidate target verification on living cells for a peptide, a protein and an antibody.

Author

Cardiac Targeting Peptide, a Novel Cardiac Vector: Studies in Bio-Distribution, Imaging Application, and Mechanism of Transduction

Refernce

Maliha Zahid, Kyle S. Feldman, Gabriel Garcia-Borrero, Timothy N. Feinstein, Nicholas Pogodzinski, Xinxiu Xu, Raymond Yurko, Michael Czachowski , Yijen L. Wu, Neale S. Mason and CeciliaW. Lo Biomolecules 2018, 8, 147; doi:10.3390/biom8040147 Received: 24 September 2018 / Accepted: 8 November 2018 / Published: 14 November 2018

Abstract

Our previous work identified a 12-amino acid peptide that targets the heart, termed cardiac targeting peptide (CTP).We now quantitatively assess the bio-distribution of CTP, show a clinical application with the imaging of the murine heart, and study its mechanisms of transduction. Bio-distribution studies of cyanine5.5-N-Hydroxysuccinimide (Cy5.5) labeled CTP were undertaken in wild-type mice. Cardiac targeting peptide was labeled with Tc. 99m (99mTc) using the chelator hydrazino-nicotinamide (HYNIC), and imaging performed using micro-single photon emission computerized tomography/computerized tomography (SPECT/CT). Human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMCs) were incubated with dual-labeled CTP, and imaged using confocal microscopy. TriCEPs technology was utilized to study the mechanism of transduction. Bio-distribution studies showed peak uptake of CTP at 15 min. 99mTc-HYNIC-CTP showed heart-specific uptake. Robust transduction of beating human iPSC-derived CMCs was seen. TriCEPs experiments revealed five candidate binding partners for CTP, with Kcnh5 being felt to be the most likely candidate as it showed a trend towards being competed out by siRNA knockdown. Transduction efficiency was enhanced by increasing extracellular potassium concentration, and with Quin., a Kcnh5 inhibitor, that blocks the channel in an open position. We demonstrate that CTP transduces the normal heart as early as 15 min. 99mTc-HYNIC-CTP targets the normal murine heart with substantially improved targeting compared with 99mTc Sestamibi. Cardiac targeting peptide’s transduction ability is not species limited and has human applicability. Cardiac targeting peptide appears to utilize Kcnh5 to gain cell entry, a phenomenon that is affected by pre-treatment with Quin. and changes in potassium levels.

Author

Maliha Zahid 1,*, Kyle S. Feldman 1, Gabriel Garcia-Borrero 1, Timothy N. Feinstein 1, Nicholas Pogodzinski 1, Xinxiu Xu 1, Raymond Yurko 2, Michael Czachowski 3, Yijen L. Wu 1, Neale S. Mason 3 and CeciliaW. Lo 1 1 Department of Developmental Biology, University of Pittsburgh, Pittsburgh, PA 15201, USA; ksf23@pitt.edu (K.S.F.); gag44@pitt.edu (G.G.-B.); tnf8@pitt.edu (T.N.F.); nrp30@pitt.edu (N.P.);xux@pitt.edu (X.X.); yijenwu@pitt.edu (Y.L.W.); cel36@pitt.edu (C.W.L.) 2 Peptide Synthesis Facility, University of Pittsburgh, Pittsburgh, PA 15201, USA; yurko@pitt.edu 3 Department of Radiology, University of Pittsburgh, Pittsburgh, PA 15201, USA; michael.czachowski@chp.edu (M.C.); masonns@upmc.edu (N.S.M.)

Leukocyte differentiation by histidine-rich glycoprotein/stanniocalcin-2 complex regulates murine glioma growth through modulation of anti-tumor immunity

Reference

Francis P Roche, Ilkka Pietilä, Hiroshi Kaito, Elisabet O Sjöström, Nadine Sobotzki, Oriol Noguer, Tor Persson Skare, Magnus Essand, Bernd Wollscheid, Michael Welsh and Lena Claesson-Welsh DOI: 10.1158/1535-7163.MCT-18-0097 Received January 27, 2018, Revision received April 21, 2018, Accepted June 19, 2018, Copyright ©2018, American Association for Cancer Research. PDF

Abstract

The plasma-protein histidine-rich glycoprotein (HRG) is implicated in phenotypic switching of tumor-associated macrophages, regulating cytokine production and phagocytotic activity, thereby promoting vessel normalization and anti-tumor immune responses. To assess the therapeutic effect of HRG gene delivery on CNS tumors, we used adenovirus-encoded HRG to treat mouse intracranial GL261 glioma. Delivery of Ad5-HRG to the tumor site resulted in a significant reduction in glioma growth, associated with increased vessel perfusion and increased CD45+ leukocyte and CD8+ T cell accumulation in the tumor. Antibody-mediated neutralization of colony-stimulating factor-1 suppressed the effects of HRG on CD45+ and CD8+ infiltration. Using a novel protein interaction-decoding technology, TRICEPS-based ligand receptor capture (LRC), we identified Stanniocalcin-2 (STC2) as an interacting partner of HRG on the surface of inflammatory cells in vitro and co-localization of HRG and STC2 in gliomas. HRG reduced the suppressive effects of STC2 on monocyte CD14+ differentiation and STC2-regulated immune response pathways. In consequence, Ad5-HRG treated gliomas displayed decreased numbers of Interleukin-35+ Treg cells, providing a mechanistic rationale for the reduction in GL261 growth in response to Ad5-HRG delivery. We conclude that HRG suppresses glioma growth by modulating tumor inflammation through monocyte infiltration and differentiation. Moreover, HRG acts to balance the regulatory effects of its partner, STC2, on inflammation and innate and/or acquired immunity. HRG gene delivery therefore offers a potential therapeutic strategy to control anti-tumor immunity.

Author

Francis P Roche1, Ilkka Pietilä2, Hiroshi Kaito3, Elisabet O Sjöström1, Nadine Sobotzki4, Oriol Noguer1, Tor Persson Skare1, Magnus Essand1, Bernd Wollscheid5, Michael Welsh2, and Lena Claesson-Welsh1,*

¹Department of Immunology, Uppsala University

²Department of Medical Cell Biology, Uppsala University

³Department of Immunology, Uppsala university

⁴Department of Health Sciences, ETH Zurich

⁵Department of Health Sciences, ETH Zürich

↵* Corresponding Author:

Glycomics and Proteomics Approaches to Investigate Early Adenovirus–Host Cell Interactions

Reference

Lisa Lasswitz, Naresh Chandra, Niklas Arnberg, Gisa Gerold jmb Journal of Molecular Biology, doi.org/10.1016/j.jmb.2018.04.039 Received 15 February 2018, Revised 24 April 2018, Accepted 30 April 2018, Available online 7 May 2018.

Abstract

Adenoviruses as most viruses rely on glycan and protein interactions to attach to and enter susceptible host cells. The Adenoviridae family comprises more than 80 human types and they differ in their attachment factor and receptor usage, which likely contributes to the diverse tropism of the different types. In the past years, methods to systematically identify glycan and protein interactions have advanced. In particular sensitivity, speed and coverage of mass spectrometric analyses allow for high-throughput identification of glycans and peptides separated by liquid chromatography. Also, developments in glycan microarray technologies have led to targeted, high-throughput screening and identification of glycan-based receptors. The mapping of cell surface interactions of the diverse adenovirus types has implications for cell, tissue, and species tropism as well as drug development. Here we review known adenovirus interactions with glycan- and protein-based receptors, as well as glycomics and proteomics strategies to identify yet elusive virus receptors and attachment factors. We finally discuss challenges, bottlenecks, and future research directions in the field of non-enveloped virus entry into host cells.

Author

Lisa Lasswitz¹Naresh Chandra^2,3Niklas Arnberg^2,3Gisa Gerold^1,2,4
¹Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, 30625 Hannover, Germany

²Department of Clinical Microbiology, Virology, Umeå University, SE-90185 Umeå, Sweden

³Molecular Infection Medicine Sweden (MIMS), Umeå University, SE-90185 Umea, Sweden

⁴Wallenberg Centre for Molecular Medicine (WCMM), Umeå University, SE-90185 Umea, Sweden

HATRIC-based identification of receptors for orphan ligands

Reference

Nadine Sobotzki, Michael A. Schafroth, Alina Rudnicka, Anika Koetemann, Florian Marty, Sandra Goetze, Yohei Yamauchi, Erick M. Carreira & Bernd Wollscheid Nature Communications, volume 9, Article number: 1519 (2018) doi:10.1038/s41467-018-03936-z Published online: 17 April 2018

Abstract

Cellular responses depend on the interactions of extracellular ligands, such as nutrients, growth factors, or drugs, with specific cell-surface receptors. The sensitivity of these interactions to non-physiological conditions, however, makes them challenging to study using in vitro assays. Here we present HATRIC-based ligand receptor capture (HATRIC-LRC), a chemoproteomic technology that successfully identifies target receptors for orphan ligands on living cells ranging from small molecules to intact viruses. HATRIC-LRC combines a click chemistry-based, protein-centric workflow with a water-soluble catalyst to capture ligand-receptor interactions at physiological pH from as few as 1 million cells. We show HATRIC-LRC utility for general antibody target validation within the native nanoscale organization of the surfaceome, as well as receptor identification for a small molecule ligand. HATRIC-LRC further enables the identification of complex extracellular interactomes, such as the host receptor panel for influenza A virus (IAV), the causative agent of the common flu.

Author

Staphylococcal Superantigens Use LAMA2 as a Coreceptor GPCT signaling To Activate T Cells

Reference

Zhigang Li, Joseph J. Zeppa, Mark A. Hancock, John K. McCormick, Terence M. Doherty, Geoffrey N. Hendy and Joaquín Madrenas J Immunol January 15, 2018, ji1701212; DOI: https://doi.org/10.4049/jimmunol.1701212 (Published online February 5, 2018) This work was supported by the Canadian Institutes for Health Research. J.M. holds a tier I Canada Research Chair in Human Immunology. The Department of Microbiology and Immunology Flow Cytometry and Cell Sorting Facility and McGill Surface Plasmon Resonance–Mass Spectrometry Facility are supported by the Canada Foundation for Innovation.

Abstract

Canonical Ag-dependent TCR signaling relies on activation of the src-family tyrosine kinase LCK. However, staphylococcal superantigens can trigger TCR signaling by activating an alternative pathway that is independent of LCK and utilizes a Gα11-containing G protein–coupled receptor (GPCR) leading to PLCβ activation. The molecules linking the superantigen to GPCR signaling are unknown. Using the ligand-receptor capture technology LRC-TriCEPS, we identified LAMA2, the α2 subunit of the extracellular matrix protein laminin, as the coreceptor for staphylococcal superantigens. Complementary binding assays (ELISA, pull-downs, and surface plasmon resonance) provided direct evidence of the interaction between staphylococcal enterotoxin E and LAMA2. Through its G4 domain, LAMA2 mediated the LCK-independent T cell activation by these toxins. Such a coreceptor role of LAMA2 involved a GPCR of the calcium-sensing receptor type because the selective antagonist NPS 2143 inhibited superantigen-induced T cell activation in vitro and delayed the effects of toxic shock syndrome in vivo. Collectively, our data identify LAMA2 as a target of antagonists of staphylococcal superantigens to treat toxic shock syndrome.

Author

Toll like receptors TLR1/2, TLR6 and MUC5B as binding interaction partners with cytostatic proline rich polypeptide 1 in human chondrosarcoma

Reference

International Journal of Oncology, published online on: November 9, 2017 doi.org/10.3892/ijo.2017.4199 Authors: Karina Galoian, Silva Abrahamyan, Gor Chailyan, Amir Qureshi, Parthik Patel, Gil Metser, Alexandra Moran, Inesa Sahakyan, Narine Tumasyan, Albert Lee, Tigran Davtyan, Samvel Chailyan and Armen Galoyan Metastatic chondrosarcoma is a bone malignancy not responsive to conventional therapies; new approaches and therapies are urgently needed.

Abstract

Metastatic chondrosarcoma is a bone malignancy not responsive to conventional therapies; new approaches and therapies are urgently needed. We have previously reported that mTORC1 inhibitor, antitumorigenic cytostatic proline rich polypeptide 1 (PRP-1), galarmin caused a significant upregulation of tumor suppressors including TET1/2 and SOCS3 (known to be involved in inflammatory processes), downregulation of oncoproteins and embryonic stem cell marker miR-302C and its targets Nanog, c-Myc and Bmi-1 in human chondrosarcoma. To understand better the mechanism of PRP-1 action it was very important to identify the receptor it binds to. Nuclear pathway receptor and GPCR assays indicated that PRP-1 receptors are not G protein coupled, neither do they belong to family of nuclear or orphan receptors. In the present study, we have demonstrated that PRP-1 binding interacting partners belong to innate immunity pattern recognition toll like receptors TLR1/2 and TLR6 and gel forming secreted mucin MUC5B. MUC5B was identified as PRP-1 receptor in human chondrosarcoma JJ012 cell line using Ligand-receptor capture technology. Toll like receptors TLR1/2 and TLR6 were identified as binding interaction partners with PRP-1 by western blot analysis in human chondrosarcoma JJ012 cell line lysates. Immunocytochemistry experiments confirmed the finding and indicated the localization of PRP-1 receptors in the tumor nucleus predominantly. TLR1/2, TLR6 and MUC5B were downregulated in human chondrosarcoma and upregulated in dose-response manner upon PRP-1 treatment. Experimental data indicated that in this cellular context the mentioned receptors had tumor suppressive function.

Author

Phenotypic screening—the fast track to novel antibody discovery

Reference

ScienceDirekt, doi.org/10.1016/j.ddtec.2017.03.004

Ralph R. Minter,
Alan M. Sandercock,
Steven J. Rust^,

Department of Antibody Discovery and Protein Engineering, MedImmune, Milstein Building, Granta Park, Cambridge CB21 6GH, UK Available online 25 April 2017

Abstract

The majority of antibody therapeutics have been isolated from target-led drug discovery, where many years of target research preceded drug program initiation. However, as the search for validated targets becomes more challenging and target space becomes increasingly competitive, alternative strategies, such as phenotypic drug discovery, are gaining favour. This review highlights successful examples of antibody phenotypic screens that have led to clinical drug candidates. We also review the requirements for performing an effective antibody phenotypic screen, including antibody enrichment and target identification strategies. Finally, the future impact of phenotypic drug discovery on antibody drug pipelines will be discussed.

Author

Identification of Putative Receptors for the Novel Adipokine CTRP3 Using Ligand-Receptor Capture Technology

Reference

PLoS One. 2016 Oct 11;11(10):e0164593. doi: 10.1371/journal.pone.0164593. eCollection 2016. Li Y¹, Ozment T², Wright GL¹, Peterson JM^1,³. We used Ligand-receptor glycocapture technology with TriCEPS-based ligand-receptor capture (LRC-TriCEPS; Dualsystems Biotech AG). The LRC-TriCEPS experiment with CTRP3-FLAG protein as ligand and insulin as a control ligand was performed on the H4IIE rat hepatoma cell line.

Abstract

Initial analysis demonstrated efficient coupling of TriCEPS to CTRP3. Further, flow cytometry analysis (FACS) demonstrated successful oxidation and crosslinking of CTRP3-TriCEPS and INS-TriCEPS complexes to cell surface glycans. Demonstrating the utility of TriCEPS under these conditions, the>INS receptor was identified in the control dataset. In the CTRP3 treated cells a total enrichment of 261 peptides was observed. From these experiments 5 putative receptors for CTRP3 were identified with two reaching statistically significance: Lysosomal-associated membrane protein 1 (LAMP-1) and Lysosome membrane protein 2 (LIMP II). Follow-up Co-immunoprecipitation analysis confirmed the association between LAMP1 and CTRP3 and further testing using a polyclonal antibody to block potential binding sites of LAMP1 prevented CTRP3 binding to the cells. Conclusion The LRC-TriCEPS methodology was successful in identifying potential novel receptors for CTRP3. Relevance The identification of the receptors for CTRP3 are important prerequisites for the development of small molecule drug candidates, of which none currently exist, for the treatment NAFLD.

Author

Li Y¹, Ozment T², Wright GL¹, Peterson JM^1,³.

¹Quillen College of Medicine, Department of Biomedical Sciences, East Tennessee State University, Johnson City, Tennessee, United States of America.
²Quillen College of Medicine, Department of Internal Medicine, East Tennessee State University, Johnson City, Tennessee, United States of America.
³College of Public Health, Department of Health Sciences, East Tennessee State University, Johnson City, Tennessee, United States of America.

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