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https://www.readbyqxmd.com/read/29674997/development-by-genetic-immunization-of-monovalent-antibodies-against-human-vasoactive-intestinal-peptide-receptor-1-vpac1-new-innovative-and-versatile-tools-to-study-vpac1-receptor-function
#1
Xavier Peyrassol, Toon Laeremans, Vannessa Lahura, Maja Debulpaep, Hassan El Hassan, Jan Steyaert, Marc Parmentier, Ingrid Langer
Multi-membrane spanning proteins, such as G protein-coupled receptors (GPCRs) and ion channels, are extremely difficult to purify as native proteins. Consequently, the generation of antibodies that recognize the native conformation can be challenging. By combining genetic immunization, phage display, and biopanning, we identified a panel of monovalent antibodies (nanobodies) targeting the vasoactive intestinal peptide receptor 1 (VPAC1) receptor. The nine unique nanobodies that were classified into four different families based on their CDR3 amino acid sequence and length, were highly specific for the human receptor and bind VPAC1 with moderate affinity...
2018: Frontiers in Endocrinology
https://www.readbyqxmd.com/read/29670611/advances-in-biomarker-guided-therapy-for-pediatric-and-adult-onset-neuroinflammatory-disorders-targeting-chemokines-cytokines
#2
REVIEW
Michael R Pranzatelli
The concept and recognized components of "neuroinflammation" are expanding at the intersection of neurobiology and immunobiology. Chemokines (CKs), no longer merely necessary for immune cell trafficking and positioning, have multiple physiologic, developmental, and modulatory functionalities in the central nervous system (CNS) through neuron-glia interactions and other mechanisms affecting neurotransmission. They issue the "help me" cry of neurons and astrocytes in response to CNS injury, engaging invading lymphoid cells (T cells and B cells) and myeloid cells (dendritic cells, monocytes, and neutrophils) (adaptive immunity), as well as microglia and macrophages (innate immunity), in a cascade of events, some beneficial (reparative), others destructive (excitotoxic)...
2018: Frontiers in Immunology
https://www.readbyqxmd.com/read/29658806/nanobodies-and-cancer-current-status-and-new-perspectives
#3
Alessandro Allegra, Vanessa Innao, Demetrio Gerace, Doriana Vaddinelli, Andrea Gaetano Allegra, Caterina Musolino
Reducing the dimension of antigen-binding proteins to an only immunoglobulin domain has been one of the objectives of antibody manufacturing. Heavy chain antibodies were encountered while attempting to separate the blood serum proteins of dromedaries. Later the term "nanobodies" (Nbs) was introduced. The advantageous features of Nbs comprise little immunogenicity, stability at low/high pH, capacity to target antigens that are less antigenic, and, lastly, easy capability to be used for therapy against tumor cells...
April 16, 2018: Cancer Investigation
https://www.readbyqxmd.com/read/29621251/ultrastructural-localisation-of-protein-interactions-using-conditionally-stable-nanobodies
#4
Nicholas Ariotti, James Rae, Nichole Giles, Nick Martel, Emma Sierecki, Yann Gambin, Thomas E Hall, Robert G Parton
We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome...
April 5, 2018: PLoS Biology
https://www.readbyqxmd.com/read/29603451/exploring-the-role-of-post-translational-modifications-in-regulating-%C3%AE-synuclein-interactions-by-studying-the-effects-of-phosphorylation-on-nanobody-binding
#5
Farah El Turk, Erwin De Genst, Tim Guilliams, Bruno Fauvet, Mirva Hejjaoui, Justin Di Trani, Anass Chiki, Anthony Mittermaier, Michele Vendruscolo, Hilal A Lashuel, Christopher M Dobson
Intracellular deposits of α-synuclein in the form of Lewy bodies are major hallmarks of Parkinson's disease (PD) and a range of related neurodegenerative disorders. Post-translational modifications (PTMs) of α-synuclein are increasingly thought to be major modulators of its structure, function, degradation and toxicity. Among these PTMs, phosphorylation near the C-terminus at S129 has emerged as a dominant pathogenic modification as it is consistently observed to occur within the brain and cerebrospinal fluid (CSF) of post-mortem PD patients, and its level appears to correlate with disease progression...
March 30, 2018: Protein Science: a Publication of the Protein Society
https://www.readbyqxmd.com/read/29602806/oversized-galactosides-as-a-probe-for-conformational-dynamics-in-lacy
#6
Irina Smirnova, Vladimir Kasho, Xiaoxu Jiang, Hong-Ming Chen, Stephen G Withers, H Ronald Kaback
Binding kinetics of α-galactopyranoside homologs with fluorescent aglycones of different sizes and shapes were determined with the lactose permease (LacY) of Escherichia coli by FRET from Trp151 in the binding site of LacY to the fluorophores. Fast binding was observed with LacY stabilized in an outward-open conformation ( k on = 4-20 μM-1 ·s-1 ), indicating unobstructed access to the binding site even for ligands that are much larger than lactose. Dissociation rate constants ( k off ) increase with the size of the aglycone so that K d values also increase but remain in the micromolar range for each homolog...
March 30, 2018: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29579624/modulating-ion-channel-function-with-antibodies-and-nanobodies
#7
REVIEW
Catelijne Stortelers, Carolina Pinto-Espinoza, Diane Van Hoorick, Friedrich Koch-Nolte
Immune cells express various voltage-gated and ligand-gated ion channels that mediate the influx and efflux of charged ions across the plasma membrane, thereby controlling the membrane potential and mediating intracellular signal transduction pathways. These channels thus present potential targets for experimental modulation of immune responses and for therapeutic interventions in immune disease. Small molecule drugs and natural toxins acting on ion channels have illustrated the potential therapeutic benefit of targeting ion channels on immune cells...
March 23, 2018: Current Opinion in Immunology
https://www.readbyqxmd.com/read/29577475/selectable-high-yield-recombinant-protein-production-in-human-cells-using-a-gfp-yfp-nanobody-affinity-support
#8
Matthew J Schellenberg, Robert M Petrovich, Christine C Malone, R Scott Williams
Recombinant protein expression systems that produce high yields of pure proteins and multi-protein complexes are essential to meet the needs of biologists, biochemists, and structural biologists using X-ray crystallography and cryo-electron microscopy. An ideal expression system for recombinant human proteins is cultured human cells where the correct translation and chaperone machinery are present. However, compared to bacterial expression systems, human cell cultures present several technical challenges to their use as an expression system...
March 25, 2018: Protein Science: a Publication of the Protein Society
https://www.readbyqxmd.com/read/29569425/comparative-analysis-of-nanobody-sequence-and-structure-data
#9
Laura S Mitchell, Lucy J Colwell
Nanobodies are a class of antigen-binding protein derived from camelids that achieve comparable binding affinities and specificities to classical antibodies, despite comprising only a single 15 kDa variable domain. Their reduced size makes them an exciting target molecule with which we can explore the molecular code that underpins binding specificity - how is such high specificity achieved? Here we use a novel dataset of 90 non-redundant, protein-binding nanobodies with antigen-bound crystal structures to address this question...
March 22, 2018: Proteins
https://www.readbyqxmd.com/read/29567535/nanobodies-targeting-cortactin-proline-rich-helical-and-actin-binding-regions-downregulate-invadopodium-formation-and-matrix-degradation-in-scc-61-cancer-cells
#10
Laurence Bertier, Tim Hebbrecht, Elien Mettepenningen, Natasja De Wit, Olivier Zwaenepoel, Adriaan Verhelle, Jan Gettemans
Cortactin is a multidomain actin binding protein that activates Arp2/3 mediated branched actin polymerization. This is essential for the formation of protrusive structures during cancer cell invasion. Invadopodia are cancer cell-specific membrane protrusions, specialized at extracellular matrix degradation and essential for invasion and tumor metastasis. Given the unequivocal role of cortactin at every stage of invadopodium formation, it is considered an invadopodium marker and potential drug target. We used cortactin nanobodies to examine the role of cortactin domain-specific function at endogenous protein level...
March 19, 2018: Biomedicine & Pharmacotherapy, Biomédecine & Pharmacothérapie
https://www.readbyqxmd.com/read/29562137/binding-specificities-of-nanobody%C3%A2-membrane-protein-complexes-obtained-from-chemical-cross-linking-and-high-mass-maldi-mass-spectrometry
#11
Martin Köhler, Christoph Neff, Camilo Perez, Cyrill Brunner, Els Pardon, Jan Steyaert, Gisbert Schneider, Kaspar P Locher, Renato Zenobi
The application of nanobodies as binding partners for structure stabilization in protein x-ray crystallography is taking an increasingly important role in structural biology. However, the addition of nanobodies to the crystallization matrices might complicate the optimization of the crystallization process, which is why analytical techniques to screen and characterize suitable nanobodies are useful. Here, we show how chemical cross-linking combined with high-mass matrix-assisted laser/desorption ionization mass spectrometry can be employed as a fast screening technique to determine binding specificities of intact nanobody•membrane protein complexes...
March 21, 2018: Analytical Chemistry
https://www.readbyqxmd.com/read/29545609/single-and-two-photon-imaging-of-human-micrometastases-and-disseminated-tumour-cells-with-conjugates-of-nanobodies-and-quantum-dots
#12
Fernanda Ramos-Gomes, Julia Bode, Alyona Sukhanova, Svetlana V Bozrova, Mara Saccomano, Miso Mitkovski, Julia Eva Krueger, Anja K Wege, Walter Stuehmer, Pavel S Samokhvalov, Daniel Baty, Patrick Chames, Igor Nabiev, Frauke Alves
Early detection of malignant tumours and, especially, micrometastases and disseminated tumour cells is still a challenge. In order to implement highly sensitive diagnostic tools we demonstrate the use of nanoprobes engineered from nanobodies (single-domain antibodies, sdAbs) and fluorescent quantum dots (QDs) for single- and two-photon detection and imaging of human micrometastases and disseminated tumour cells in ex vivo biological samples of breast and pancreatic metastatic tumour mouse models expressing human epidermal growth factor receptor 2 (HER2) or carcinoembryonic antigen (CEA)...
March 15, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29543347/development-and-characterization-of-a-camelid-single-domain-antibody-directed-to-human-cd22-biomarker
#13
Fatemeh Faraji, Nader Tajik, Mahdi Behdani, Mohammad Ali Shokrgozar, Amir Hassan Zarnani, Fatemeh Shahhosseini, Mahdi Habibi-Anbouhi
CD22 is a B-cell-specific trans-membrane glycoprotein which is found on the surface of the most B-cells and modulates their function, survival, and apoptosis. Recently, targeting this cell surface biomarker in B-cell malignancies and disorders has attracted a lot of attention. The variable domain of camelid single chain antibodies (VHH, Nanobody) is a form of antibodies with novel properties including small size (15-17 kDa), thermal and chemical stability, high affinity and homology to human antibody sequences...
March 15, 2018: Biotechnology and Applied Biochemistry
https://www.readbyqxmd.com/read/29527688/delivery-of-an-anti-ttr-nanobody-to-the-brain-through-intranasal-administration-reveals-ttr-expression-and-secretion-by-motor-neurons
#14
João R Gomes, Inês Cabrito, Hugo R Soares, Susete Costelha, Anabela Teixeira, Angela Wittelsberger, Catelijne Stortelers, Peter Vanlandschoot, Maria J Saraiva
Transthyretin (TTR) is a transport protein of retinol and thyroxine in serum and cerebrospinal fluid (CSF), which is mainly secreted in liver and choroid plexus, and in smaller amounts in other cells throughout the body. The exact role of TTR and its specific expression in Central Nervous System (CNS) remains understudied. We investigated TTR expression and metabolism in CNS, through the intranasal and intracerebroventricular delivery of a specific anti-TTR Nanobody to the brain, unveiling Nanobody pharmacokinetics to the CNS...
March 12, 2018: Journal of Neurochemistry
https://www.readbyqxmd.com/read/29526739/insights-into-maleimide-thiol-conjugation-chemistry-conditions-for-efficient-surface-functionalization-of-nanoparticles-for-receptor-targeting
#15
Lucía Martínez-Jothar, Sofia Doulkeridou, Raymond M Schiffelers, Javier Sastre Torano, Sabrina Oliveira, Cornelus F van Nostrum, Wim E Hennink
Maleimide-thiol chemistry is widely used for the design and preparation of ligand-decorated drug delivery systems such as poly(lactide-co-glycolide) (PLGA) based nanoparticles (NPs). While many publications on nanocarriers functionalized exploiting this strategy are available in the literature, the conditions at which this reaction takes place vary among publications. This paper presents a comprehensive study on the conjugation of the peptide cRGDfK and the nanobody 11A4 (both containing a free thiol group) to maleimide functionalized PLGA NPs by means of the maleimide-thiol click reaction...
March 8, 2018: Journal of Controlled Release: Official Journal of the Controlled Release Society
https://www.readbyqxmd.com/read/29511492/design-of-a-humanized-anti-vascular-endothelial-growth-factor-nanobody-and-evaluation-of-its-in-vitro-function
#16
Fatemeh Kazemi-Lomedasht, Serge Muyldermans, Mahdi Habibi-Anbouhi, Mahdi Behdani
Objectives: Nanobodies, the single domain antigen binding fragments of heavy chain-only antibodies occurring naturally in camelid sera, are the smallest intact antigen binding entities. Their minimal size assists in reaching otherwise largely inaccessible regions of antigens. However, their camelid origin raises a possible concern of immunogenicity when used for human therapy. Humanization is a promising approach to overcome the problem. Materials and Methods: Here, we designed a humanized version of previously developed nanobody (anti vascular endothelial growth factor nanobody), evaluated and compared its predicted 3D structure, affinity and biological activity with its original wild type nanobody...
March 2018: Iranian Journal of Basic Medical Sciences
https://www.readbyqxmd.com/read/29507400/speed-up-to-find-the-right-ones-rapid-discovery-of-functional-nanobodies
#17
Ulrich Rothbauer
No abstract text is available yet for this article.
March 2018: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/29507218/mechanism-of-the-g-protein-mimetic-nanobody-binding-to-a-muscarinic-g-protein-coupled-receptor
#18
Yinglong Miao, J Andrew McCammon
Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method...
March 5, 2018: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29502411/evaluation-of-an-anti-her2-nanobody-labeled-with-225-ac-for-targeted-%C3%AE-particle-therapy-of-cancer
#19
Marek Pruszynski, Matthias D'Huyvetter, Frank Bruchertseifer, Alfred Morgenstern, Tony Lahoutte
Human epidermal growth factor receptor type 2 (HER2) is overexpressed in numerous carcinomas. Nanobodies (Nbs) are the smallest antibody-derived fragments with beneficial characteristics for molecular imaging and radionuclide therapy. Therefore, HER2-targeting nanobodies could offer a valuable platform for radioimmunotherapy, especially when labeled with α-particle emitters, which provide highly lethal and localized radiation to targeted cells with minimal exposure to surrounding healthy tissues. In this study, the anti-HER2 2Rs15d-nanobody was conjugated with 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid ( p-SCN-Bn-DOTA) and radiolabeled with an α-emitter225 Ac with a high yield (>90%) and a radiochemical purity above 95%...
March 7, 2018: Molecular Pharmaceutics
https://www.readbyqxmd.com/read/29500346/a-peptide-tag-specific-nanobody-enables-high-quality-labeling-for-dstorm-imaging
#20
David Virant, Bjoern Traenkle, Julia Maier, Philipp D Kaiser, Mona Bodenhöfer, Christian Schmees, Ilijana Vojnovic, Borbála Pisak-Lukáts, Ulrike Endesfelder, Ulrich Rothbauer
Dense fluorophore labeling without compromising the biological target is crucial for genuine super-resolution microscopy. Here we introduce a broadly applicable labeling strategy for fixed and living cells utilizing a short peptide tag-specific nanobody (BC2-tag/bivBC2-Nb). BC2-tagging of ectopically introduced or endogenous proteins does not interfere with the examined structures and bivBC2-Nb staining results in a close-grained fluorophore labeling with minimal linkage errors. This allowed us to perform high-quality dSTORM imaging of various targets in mammalian and yeast cells...
March 2, 2018: Nature Communications
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