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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
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
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
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
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
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
Ulrich Rothbauer
No abstract text is available yet for this article.
March 2018: Nature Structural & Molecular Biology
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
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 5, 2018: Molecular Pharmaceutics
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
Rasmus K Jensen, Rasmus Pihl, Trine A F Gadeberg, Jan K Jensen, Kasper R Andersen, Steffen Thiel, Nick S Laursen, Gregers Rom Andersen
The complement system is a complex, carefully regulated proteolytic cascade for which suppression of aberrant activation is of increasing clinical relevance and inhibition of the complement alternative pathway is a subject of intense research. Here, we describe the nanobody hC3Nb1 that binds to multiple functional states of C3 with sub-nanomolar affinity. The nanobody causes a complete shutdown of alternative pathway activity in human and murine serum when present in concentrations comparable to C3, and hC3Nb1 is shown to prevent both proconvertase assembly as well as binding of the C3 substrate to C3 convertases...
March 1, 2018: Journal of Biological Chemistry
Robert Alvin Bernedo-Navarro, Ema Romão, Tomomasa Yano, Joar Pinto, Henri De Greve, Yann G-J Sterckx, Serge Muyldermans
BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) are a subset of pathogens leading to illnesses such as diarrhea, hemolytic uremic syndrome and even death. The Shiga toxins are the main virulence factors and divided in two groups: Stx1 and Stx2, of which the latter is more frequently associated with severe pathologies in humans. RESULTS: An immune library of nanobodies (Nbs) was constructed after immunizing an alpaca with recombinant Shiga toxin-2a B subunit (rStx2aB), to retrieve multiple rStx2aB-specific Nbs...
March 1, 2018: Toxins
Jessica R Ingram, Florian I Schmidt, Hidde L Ploegh
The unique class of heavy chain-only antibodies, present in Camelidae, can be shrunk to just the variable region of the heavy chain to yield VHHs, also called nanobodies. About one-tenth the size of their full-size counterparts, nanobodies can serve in applications similar to those for conventional antibodies, but they come with a number of signature advantages that find increasing application in biology. They not only function as crystallization chaperones but also can be expressed inside cells as such, or fused to other proteins to perturb the function of their targets, for example, by enforcing their localization or degradation...
February 28, 2018: Annual Review of Immunology
Hyeon-Yeol Cho, Taek Lee, Jinho Yoon, Zhenlin Han, Hudifah Rabie, Ki Bum Lee, Wei Wen Su, Jeong-Woo Choi
In the present study, we fabricated magnetic oleosomes functionalized with recombinant proteins as a new carrier for oil-based lipophilic drugs for cancer treatment. The bioengineered oleosome is composed of neutral lipids surrounded by a phospholipid monolayer with embedded oleosin fusion proteins. The oleosin was genetically fused to a nanobody of a green fluorescent protein (GFP). A recombinant protein consisting of immunoglobulin-binding protein LG fused to GFP was used to couple the oleosome to an antibody for targeted delivery to breast cancer cells...
February 28, 2018: ACS Applied Materials & Interfaces
Ágnes Klein, Mátyás Kovács, Adél Muskotál, Hajnalka Jankovics, Balázs Tóth, Mihály Pósfai, Ferenc Vonderviszt
In this work we addressed the problem how to fabricate self-assembling tubular nanostructures displaying target recognition functionalities. Bacterial flagellar filaments, composed of thousands of flagellin subunits, were used as scaffolds to display single-domain antibodies (nanobodies) on their surface. As a representative example, an anti-GFP nanobody was successfully inserted into the middle part of flagellin replacing the hypervariable surface-exposed D3 domain. A novel procedure was developed to select appropriate linkers required for functional internal insertion...
February 26, 2018: Scientific Reports
Bernadette Lee, Shuang Sun, Ester Jiménez-Moreno, André A Neves, Gonçalo J L Bernardes
Site-selective protein modification strategies can be used to insert non-natural functional groups into protein structures. Herein, we report on the use of the bis-electrophile 3-bromo-2-bromomethyl-1-propene as a reagent to introduce an electrophilic handle at cysteine residues under mild conditions. This method is demonstrated on a variety of proteins containing a solvent-exposed cysteine residue, including an anti-HER2 nanobody. Chemically distinct protein conjugates are then efficiently formed through further reaction of the electrophilic site with various nucleophiles, including thiols and amines...
February 17, 2018: Bioorganic & Medicinal Chemistry
Bianca Baudisch, Ingrid Pfort, Eberhard Sorge, Udo Conrad
Here, we present data showing the directed degradation of target proteins recognized by a specific nanobody in transgenic plants. Green fluorescent protein was depleted by a chimeric nanobody fused to a distinct F-box domain, which enables protein degradation via the ubiquitin proteasome pathway. This technique could thus be used to knock out other proteins of interest in planta using specific, high-affinity binding proteins.
2018: Frontiers in Plant Science
Wenshuai Liu, Haipeng Song, Quan Chen, Jianli Yu, Mo Xian, Rui Nian, Dongxiao Feng
Nanobodies represent the next-generation antibody-derived biologics with significant advances over conventional antibodies. Several rapid and robust techniques for isolating highly specific nanobodies have been developed. Antigen specific nanobodies are selected from constructed nanobody libraries, which can be classified into 3 main types: immune library, naïve library, and semisynthetic/synthetic library. The immune library is the most widely used strategy for nanobody screening. Target specific nanobodies are highly enriched in immune libraries than in non-immune libraries; however, it is largely limited by the natural antigenicity of antigens...
February 22, 2018: Molecular Immunology
Els Pardon, Cecilia Betti, Toon Laeremans, Florent Chevillard, Karel Guillemyn, Peter Kolb, Steven Ballet, Jan Steyaert
The conformational complexity of G protein-coupled receptor (GPCR) transmembrane signaling is a central hurdle for the design of screens for receptor agonists. In their basal states, GPCRs have lower affinities for agonists compared to their G protein-bound active state conformations. Moreover, different agonists can stabilize distinct active receptor conformations and do not uniformly activate all cellular signaling pathways linked to a given receptor (agonist bias). Herein, comparative fragment screens were performed on a β2 adrenoreceptor-Nanobody fusion locked in its active state conformation by a G protein mimicking Nanobody, and the same receptor in its basal state conformation...
February 22, 2018: Angewandte Chemie
Ned Van Eps, Christian Altenbach, Lydia N Caro, Naomi R Latorraca, Scott A Hollingsworth, Ron O Dror, Oliver P Ernst, Wayne L Hubbell
More than two decades ago, the activation mechanism for the membrane-bound photoreceptor and prototypical G protein-coupled receptor (GPCR) rhodopsin was uncovered. Upon light-induced changes in ligand-receptor interaction, movement of specific transmembrane helices within the receptor opens a crevice at the cytoplasmic surface, allowing for coupling of heterotrimeric guanine nucleotide-binding proteins (G proteins). The general features of this activation mechanism are conserved across the GPCR superfamily...
February 20, 2018: Proceedings of the National Academy of Sciences of the United States of America
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