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https://www.readbyqxmd.com/read/28065273/screening-and-characterization-strategies-for-nanobodies-targeting-membrane-proteins
#1
S Veugelen, M Dewilde, B De Strooper, L Chávez-Gutiérrez
The study of membrane protein function and structure requires their successful detection, expression, solubilization, and/or reconstitution, which poses a challenging task and relies on the availability of suitable tools. Several research groups have successfully applied Nanobodies in the purification, as well as the functional and structural characterization of membrane proteins. Nanobodies are small, single-chain antibody fragments originating from camelids presenting on average a longer CDR3 which enables them to bind in cavities and clefts (such as active and allosteric sites)...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28055308/binding-to-ocular-albumin-as-a-half-life-extension-principle-for-intravitreally-injected-drugs-evidence-from-mechanistic-rat-and-rabbit-studies
#2
Holger Fuchs, Frederik Igney
PURPOSE: The ocular half-life of intravitreally (IVT) injected drugs is of major relevance for the suitability of a drug intended for chronic intraocular treatment, as the half-life determines the dosing frequency. Thus, half-life extension principles are very attractive as they can reduce the IVT dosing frequency. In this study, we investigated the ocular pharmacokinetics (PK) of the IVT injected Nanobody(®) BI-X and whether the noncovalent binding of BI-X to vitreous albumin could increase its ocular half-life...
January 5, 2017: Journal of Ocular Pharmacology and Therapeutics
https://www.readbyqxmd.com/read/27982020/intrabody-targeting-vascular-endothelial-growth-factor-receptor-2-mediates-downregulation-of-surface-localization
#3
E Alirahimi, A Ashkiyan, F Kazemi-Lomedasht, K Azadmanesh, M Hosseininejad-Chafi, M Habibi-Anbouhi, R Moazami, M Behdani
Angiogenesis is among the most important mechanisms that helps cancer cells to survive, grow and undergo metastasis. Therefore, inhibiting angiogenesis will suppress tumor growth. Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) are believed to be important players of angiogenesis. The goal of this study was to evaluate the success of a novel nanobody against VEGFR2 in tethering its target inside the endoplasmic reticulum and preventing its transport to the cell membrane. Nanobody sequence was cloned in a mammalian vector in fusion with green fluorescent protein and a KDEL retention signal...
December 16, 2016: Cancer Gene Therapy
https://www.readbyqxmd.com/read/27959623/nanobodies-to-study-g-protein-coupled-receptor-structure-and-function
#4
Aashish Manglik, Brian K Kobilka, Jan Steyaert
Ligand-induced activation of G protein-coupled receptors (GPCRs) is a key mechanism permitting communication between cells and organs. Enormous progress has recently elucidated the structural and dynamic features of GPCR transmembrane signaling. Nanobodies, the recombinant antigen-binding fragments of camelid heavy-chain-only antibodies, have emerged as important research tools to lock GPCRs in particular conformational states. Active-state stabilizing nanobodies have elucidated several agonist-bound structures of hormone-activated GPCRs and have provided insight into the dynamic character of receptors...
January 6, 2017: Annual Review of Pharmacology and Toxicology
https://www.readbyqxmd.com/read/27940234/construction-of-iron-polymer-graphene-nanocomposites-with-low-nonspecific-adsorption-and-strong-quenching-ability-for-competitive-immunofluorescent-detection-of-biomarkers-in-gm-crops
#5
Kaifei Yin, Anran Liu, Li Shangguan, Li Mi, Xu Liu, Yuanjian Liu, Yuewu Zhao, Ying Li, Wei Wei, Yuanjian Zhang, Songqin Liu
We developed a new immunofluorescent biosensor by utilizing a novel nanobody (Nb) and iron-polymer-graphene nanocomposites for sensitive detection of 5-enolpyruvylshikimate-3-phosphate synthase from Agrobacdterium tumefaciens strain CP4 (CP4-EPSPS), which considered as biomarkers of genetically modified (GM) crops. Specifically, we prepared iron doped polyacrylic hydrazide modified reduced graphene nanocomposites (Fe@RGO/PAH) by in-situ polymerization approach and subsequent a one-pot reaction with hydrazine...
April 15, 2017: Biosensors & Bioelectronics
https://www.readbyqxmd.com/read/27917276/preparation-and-characterization-of-a-novel-nanobody-against-t-cell-immunoglobulin-and-mucin-3-tim-3
#6
Vida Homayouni, Mazdak Ganjalikhani-Hakemi, Abbas Rezaei, Hossein Khanahmad, Mahdi Behdani, Fatemeh Kazemi Lomedasht
OBJECTIVES: As T-cell immunoglobulin and mucin domain 3 (TIM-3) is an immune regulatory molecule; its blocking or stimulating could alter the pattern of immune response towards a desired condition. Based on the unique features of nanobodies, we aimed to construct an anti-TIM-3 nanobody as an appropriate tool for manipulating immune responses for future therapeutic purposes. MATERIALS AND METHODS: We immunized a camel with TIM-3 antigen and then, synthesized a VHH phage: mid library from its B cell's transcriptome using nested PCR...
November 2016: Iranian Journal of Basic Medical Sciences
https://www.readbyqxmd.com/read/27895170/prevention-of-v%C3%AE-9v%C3%AE-2-t-cell-activation-by-a-v%C3%AE-9v%C3%AE-2-tcr-nanobody
#7
Renée C G de Bruin, Anita G M Stam, Anna Vangone, Paul M P van Bergen En Henegouwen, Henk M W Verheul, Zsolt Sebestyén, Jürgen Kuball, Alexandre M J J Bonvin, Tanja D de Gruijl, Hans J van der Vliet
Vγ9Vδ2 T cell activation plays an important role in antitumor and antimicrobial immune responses. However, there are conditions in which Vγ9Vδ2 T cell activation can be considered inappropriate for the host. Patients treated with aminobisphosphonates for hypercalcemia or metastatic bone disease often present with a debilitating acute phase response as a result of Vγ9Vδ2 T cell activation. To date, no agents are available that can clinically inhibit Vγ9Vδ2 T cell activation. In this study, we describe the identification of a single domain Ab fragment directed to the TCR of Vγ9Vδ2 T cells with neutralizing properties...
January 1, 2017: Journal of Immunology: Official Journal of the American Association of Immunologists
https://www.readbyqxmd.com/read/27889311/structural-evaluation-of-a-nanobody-targeting-complement-receptor-vsig4-and-its-cross-reactivity
#8
Yurong Wen, Zhenlin Ouyang, Steve Schoonooghe, Siyu Luo, Patrick De Baetselier, Wuyuan Lu, Serge Muyldermans, Geert Raes, Fang Zheng
Vsig4 is a recently identified immune regulatory protein related to the B7 family with dual functionality: a negative regulator of T cell activation and a receptor for the complement components C3b and C3c. Here we present a structural evaluation of a nanobody, Nb119, against the extracellular IgV domain protein of both mouse and human recombinant Vsig4, which have a high degree of sequence identity. Although mouse and human Vsig4 bind to Nb119 with a 250 times difference in dissociation constants, the interaction results in a highly identical assembly with a RMSD of 0...
November 18, 2016: Immunobiology
https://www.readbyqxmd.com/read/27881823/nanobodies-that-block-gating-of-the-p2x7-ion-channel-ameliorate-inflammation
#9
Welbeck Danquah, Catherine Meyer-Schwesinger, Björn Rissiek, Carolina Pinto, Arnau Serracant-Prat, Miriam Amadi, Domenica Iacenda, Jan-Hendrik Knop, Anna Hammel, Philine Bergmann, Nicole Schwarz, Joana Assunção, Wendy Rotthier, Friedrich Haag, Eva Tolosa, Peter Bannas, Eric Boué-Grabot, Tim Magnus, Toon Laeremans, Catelijne Stortelers, Friedrich Koch-Nolte
Ion channels are desirable therapeutic targets, yet ion channel-directed drugs with high selectivity and few side effects are still needed. Unlike small-molecule inhibitors, antibodies are highly selective for target antigens but mostly fail to antagonize ion channel functions. Nanobodies-small, single-domain antibody fragments-may overcome these problems. P2X7 is a ligand-gated ion channel that, upon sensing adenosine 5'-triphosphate released by damaged cells, initiates a proinflammatory signaling cascade, including release of cytokines, such as interleukin-1β (IL-1β)...
November 23, 2016: Science Translational Medicine
https://www.readbyqxmd.com/read/27852863/cryoem-structures-of-expanded-poliovirus-with-vhhs-sample-the-conformational-repertoire-of-the-expanded-state
#10
Mike Strauss, Lise Schotte, Krishanthi S Karunatilaka, David J Filman, James M Hogle
: Using cryoelectron microscopy, expanded 80S-like poliovirions were visualized in complexes with four 80S-specific camelid VHHs (Nanobodies®). In all four complexes, the VHHs bind to a site on the top surface of capsid protein VP3, which is hidden in native virus. Interestingly, although the four VHHs bind to the same site, the structures of the expanded virus differ in detail in each complex, suggesting that each of the nanobodies has sampled a range of low energy structures available to the expanded virion...
November 16, 2016: Journal of Virology
https://www.readbyqxmd.com/read/27832973/neutralization-of-tnf%C3%AE-in-tumor-with-a-novel-nanobody-potentiates-paclitaxel-therapy-and-inhibits-metastasis-in-breast-cancer
#11
Xuemei Ji, Zhengxin Peng, Xiaorui Li, Zhonghui Yan, Yue Yang, Zheng Qiao, Yu Liu
Metastatic disease is the major cause of death from cancer, and immunotherapy and chemotherapy have had limited success in reversing its progression. Researchers have suggested that inflammatory factors in the tumor environment can promote cancer invasion and metastasis, stimulating cancer progression. Thus, novel strategies that target cytokines and modulate the tumor microenvironment may emerge as important approaches for treating metastatic breast cancer. Specific neutralization of pathogenic TNF signaling using a TNFα antibody has gained increasing attention...
February 1, 2017: Cancer Letters
https://www.readbyqxmd.com/read/27825023/two-dimensional-crystallization-of-the-mouse-serotonin-5-ht3a-receptor
#12
Jan Rheinberger, Ghérici Hassaine, Mohamed Chami, Henning Stahlberg, Horst Vogel, Xiaodan Li
The mouse serotonin 5-HT3A receptor is a homo-pentameric ligand-gated ion channel (pLGIC) mediating fast excitatory neurotransmission in the central nervous system. The molecular mechanism of ion permeation of 5-HT3A receptors triggered by the neurotransmitter serotonin is not yet fully understood. The recent X-ray structure of the mouse serotonin 5-HT3A receptor in complex with a stabilizing nanobody revealed for the first time the entire structure of a mammalian pLGIC in detergent. Structural information of the receptor in a lipid bilayer however is still limited primarily due to the lack of 2D crystals of the receptor in a lipid bilayer...
January 2017: Micron: the International Research and Review Journal for Microscopy
https://www.readbyqxmd.com/read/27824888/bifunctional-anti-non-amyloid-component-%C3%AE-synuclein-nanobodies-are-protective-in-situ
#13
David C Butler, Shubhada N Joshi, Erwin De Genst, Ankit S Baghel, Christopher M Dobson, Anne Messer
Misfolding, abnormal accumulation, and secretion of α-Synuclein (α-Syn) are closely associated with synucleinopathies, including Parkinson's disease (PD). VH14 is a human single domain intrabody selected against the non-amyloid component (NAC) hydrophobic interaction region of α-Syn, which is critical for initial aggregation. Using neuronal cell lines, we show that as a bifunctional nanobody fused to a proteasome targeting signal, VH14PEST can counteract heterologous proteostatic effects of mutant α-Syn on mutant huntingtin Exon1 and protect against α-Syn toxicity using propidium iodide or Annexin V readouts...
2016: PloS One
https://www.readbyqxmd.com/read/27810917/subdiffractional-tracking-of-internalized-molecules-reveals-heterogeneous-motion-states-of-synaptic-vesicles
#14
Merja Joensuu, Pranesh Padmanabhan, Nela Durisic, Adekunle T D Bademosi, Elizabeth Cooper-Williams, Isabel C Morrow, Callista B Harper, WooRam Jung, Robert G Parton, Geoffrey J Goodhill, Andreas Papadopulos, Frédéric A Meunier
Our understanding of endocytic pathway dynamics is severely restricted by the diffraction limit of light microscopy. To address this, we implemented a novel technique based on the subdiffractional tracking of internalized molecules (sdTIM). This allowed us to image anti-green fluorescent protein Atto647N-tagged nanobodies trapped in synaptic vesicles (SVs) from live hippocampal nerve terminals expressing vesicle-associated membrane protein 2 (VAMP2)-pHluorin with 36-nm localization precision. Our results showed that, once internalized, VAMP2-pHluorin/Atto647N-tagged nanobodies exhibited a markedly lower mobility than on the plasma membrane, an effect that was reversed upon restimulation in presynapses but not in neighboring axons...
October 24, 2016: Journal of Cell Biology
https://www.readbyqxmd.com/read/27800572/intracellular-delivery-of-nanobodies-for-imaging-of-target-proteins-in-live-cells
#15
Ruth Röder, Jonas Helma, Tobias Preiß, Joachim O Rädler, Heinrich Leonhardt, Ernst Wagner
PURPOSE: Cytosolic delivery of nanobodies for molecular target binding and fluorescent labeling in living cells. METHODS: Fluorescently labeled nanobodies were formulated with sixteen different sequence-defined oligoaminoamides. The delivery of formulated anti-GFP nanobodies into different target protein-containing HeLa cell lines was investigated by flow cytometry and fluorescence microscopy. Nanoparticle formation was analyzed by fluorescence correlation spectroscopy...
January 2017: Pharmaceutical Research
https://www.readbyqxmd.com/read/27791182/crystal-structure-of-a-lacy-nanobody-complex-in-a-periplasmic-open-conformation
#16
Xin Jiang, Irina Smirnova, Vladimir Kasho, Jianping Wu, Kunio Hirata, Meng Ke, Els Pardon, Jan Steyaert, Nieng Yan, H Ronald Kaback
The lactose permease of Escherichia coli (LacY), a dynamic polytopic membrane protein, catalyzes galactoside-H(+) symport and operates by an alternating access mechanism that exhibits multiple conformations, the distribution of which is altered by sugar binding. We have developed single-domain camelid nanobodies (Nbs) against a mutant in an outward (periplasmic)-open conformation to stabilize this state of the protein. Here we describe an X-ray crystal structure of a complex between a double-Trp mutant (Gly46→Trp/Gly262→Trp) and an Nb in which free access to the sugar-binding site from the periplasmic cavity is observed...
November 1, 2016: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/27791003/graded-activation-and-free-energy-landscapes-of-a-muscarinic-g-protein-coupled-receptor
#17
Yinglong Miao, J Andrew McCammon
G-protein-coupled receptors (GPCRs) recognize ligands of widely different efficacies, from inverse to partial and full agonists, which transduce cellular signals at differentiated levels. However, the mechanism of such graded activation remains unclear. Using the Gaussian accelerated molecular dynamics (GaMD) method that enables both unconstrained enhanced sampling and free energy calculation, we have performed extensive GaMD simulations (∼19 μs in total) to investigate structural dynamics of the M2 muscarinic GPCR that is bound by the full agonist iperoxo (IXO), the partial agonist arecoline (ARC), and the inverse agonist 3-quinuclidinyl-benzilate (QNB), in the presence or absence of the G-protein mimetic nanobody...
October 25, 2016: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/27784791/an-affinity-directed-protein-missile-system-for-targeted-proteolysis
#18
Luke J Fulcher, Thomas Macartney, Polyxeni Bozatzi, Annika Hornberger, Alejandro Rojas-Fernandez, Gopal P Sapkota
The von Hippel-Lindau (VHL) protein serves to recruit the hypoxia-inducible factor alpha (HIF1α) protein under normoxia to the CUL2 E3 ubiquitin ligase for its ubiquitylation and degradation through the proteasome. In this report, we modify VHL to engineer an affinity-directed protein missile (AdPROM) system to direct specific endogenous target proteins for proteolysis in mammalian cells. The proteolytic AdPROM construct harbours a cameloid anti-green fluorescence protein (aGFP) nanobody that is fused to VHL for either constitutive or tetracycline-inducible expression...
October 2016: Open Biology
https://www.readbyqxmd.com/read/27779240/specificity-evaluation-and-disease-monitoring-in-arthritis-imaging-with-complement-receptor-of-the-ig-superfamily-targeting-nanobodies
#19
Fang Zheng, Harris Perlman, Patrick Matthys, Yurong Wen, Tony Lahoutte, Serge Muyldermans, Shemin Lu, Patrick De Baetselier, Steve Schoonooghe, Nick Devoogdt, Geert Raes
Single-photon emission computed tomography combined with micro-CT (SPECT/μCT) imaging using Nanobodies against complement receptor of the Ig superfamily (CRIg), found on tissue macrophages such as synovial macrophages, has promising potential to visualize joint inflammation in experimental arthritis. Here, we further addressed the specificity and assessed the potential for arthritis monitoring. Signals obtained with (99m)Tc-labelled NbV4m119 Nanobody were compared in joints of wild type (WT) versus CRIg(-/-) mice with collagen-induced arthritis (CIA) or K/BxN serum transfer-induced arthritis (STIA)...
October 25, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27776189/the-tax-inducible-actin-bundling-protein-fascin-is-crucial-for-release-and-cell-to-cell-transmission-of-human-t-cell-leukemia-virus-type-1-htlv-1
#20
Christine Gross, Veit Wiesmann, Sebastian Millen, Martina Kalmer, Thomas Wittenberg, Jan Gettemans, Andrea K Thoma-Kress
The delta-retrovirus Human T-cell leukemia virus type 1 (HTLV-1) preferentially infects CD4+ T-cells via cell-to-cell transmission. Viruses are transmitted by polarized budding and by transfer of viral biofilms at the virological synapse (VS). Formation of the VS requires the viral Tax protein and polarization of the host cytoskeleton, however, molecular mechanisms of HTLV-1 cell-to-cell transmission remain incompletely understood. Recently, we could show Tax-dependent upregulation of the actin-bundling protein Fascin (FSCN-1) in HTLV-1-infected T-cells...
October 2016: PLoS Pathogens
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