Crystallization and preliminary X-ray diffraction analysis of a specific VHH domain against mouse prion protein

Abskharon, Romany; Soror, Sameh H.; Pardon, Els; Hassan, Hassan El; Legname, Giuseppe; Steyaert, Jan; Wohlkonig, A.

doi:10.1107/s1744309110042168

Cited by 9 publications

(11 citation statements)

References 17 publications

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“…The second complex crystal was grown at concentration 68 mg/mL complex in MD-proplex screen in G1 (0.1 M Tris pH 8, 1.5 M ammonium sulfate) within one year. Nb484 has crystallized and diffracted to 1.2 Å resolution as described previously[49]. All crystals were grown at 20 °C and cryoprotected using 15% glycerol.…”

Section: Methodsmentioning

confidence: 99%

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Structural evidence for the critical role of the prion protein hydrophobic region in forming an infectious prion

et al. 2019

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Prion or PrPSc is the proteinaceous infectious agent causing prion diseases in various mammalian species. Despite decades of research, the structural basis for PrPSc formation and prion infectivity remains elusive. To understand the role of the hydrophobic region in forming infectious prion at the molecular level, we report X-ray crystal structures of mouse (Mo) prion protein (PrP) (residues 89–230) in complex with a nanobody (Nb484). Using the recombinant prion propagation system, we show that the binding of Nb484 to the hydrophobic region of MoPrP efficiently inhibits the propagation of proteinase K resistant PrPSc and prion infectivity. In addition, when added to cultured mouse brain slices in high concentrations, Nb484 exhibits no neurotoxicity, which is drastically different from other neurotoxic anti-PrP antibodies, suggesting that the Nb484 can be a potential therapeutic agent against prion disease. In summary, our data provides the first structure-function evidence supporting a crucial role of the hydrophobic region of PrP in forming an infectious prion.

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Section: Methodsmentioning

confidence: 99%

“…The structures of the different MoPrP•Nb484 complexes were determined by molecular replacement (PHASER)[51] using the HuPrP•Nb484 crystal structure (PDB entry 4KML)[12]. Nb484 was processed using iMOSFLM as described previously[49]. The Nb484 structure was solved by molecular replacement using PDB entry 1OL0 as search model.…”

Section: Methodsmentioning

confidence: 99%

Structural evidence for the critical role of the prion protein hydrophobic region in forming an infectious prion

et al. 2019

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“…VHHs also proved as being particularly useful for studying the intermediates of aggregation-prone polypeptides such as in the case of prion protein [ 173 ] and of the amyloidogenic fragments [ 174 ]. Single domain antibodies of different origin enabled the elucidation of the variety of morphologically distinct Aβ aggregates, blocking their polymerization development into non-toxic intermediates, and the clarification of the plausible mechanism of amyloidogenic protein self-association [ 175 - 180 ].…”

Section: Introductionmentioning

confidence: 99%

Biotechnological applications of recombinant single-domain antibody fragments

Marco

2011

Microb Cell Fact

146

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BackgroundSingle-domain antibody fragments possess structural features, such as a small dimension, an elevated stability, and the singularity of recognizing epitopes non-accessible for conventional antibodies that make them interesting for several research and biotechnological applications.ResultsThe discovery of the single-domain antibody's potentials has stimulated their use in an increasing variety of fields. The rapid accumulation of articles describing new applications and further developments of established approaches has made it, therefore, necessary to update the previous reviews with a new and more complete summary of the topic.ConclusionsBeside the necessary task of updating, this work analyses in detail some applicative aspects of the single-domain antibodies that have been overseen in the past, such as their efficacy in affinity chromatography, as co-crystallization chaperones, protein aggregation controllers, enzyme activity tuners, and the specificities of the unconventional single-domain fragments.

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“…In vivo studies also showed that anti-PrP KDEL-fused intrabodies can prevent scrapie infectivity in mice [44]. On the other hand, crystallographic studies based on anti-PrP scFvs and nanobodies also helped characterize the bovine PrP unstructured domain [45,46,96].…”

Section: Targets For Prion Disordersmentioning

confidence: 99%

“…In vitro in crystallography studies [45,46] Cellular PrP AA123-125, 164-170, and 174-185 Camelid nanobody (VHH) Nb484…”

Section: Introductionmentioning

confidence: 99%

Engineered Extracellular Vesicles/Exosomes as a New Tool against Neurodegenerative Diseases

et al. 2020

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Neurodegenerative diseases are commonly generated by intracellular accumulation of misfolded/aggregated mutated proteins. These abnormal protein aggregates impair the functions of mitochondria and induce oxidative stress, thereby resulting in neuronal cell death. In turn, neuronal damage induces chronic inflammation and neurodegeneration. Thus, reducing/eliminating these abnormal protein aggregates is a priority for any anti-neurodegenerative therapeutic approach. Although several antibodies against mutated neuronal proteins have been already developed, how to efficiently deliver them inside the target cells remains an unmet issue. Extracellular vesicles/exosomes incorporating intrabodies against the pathogenic products would be a tool for innovative therapeutic approaches. In this review/perspective article, we identify and describe the major molecular targets associated with neurodegenerative diseases, as well as the antibodies already developed against them. Finally, we propose a novel targeting strategy based on the endogenous engineering of extracellular vesicles/exosomes constitutively released by cells of the central nervous system.

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Crystallization and preliminary X-ray diffraction analysis of a specific VHH domain against mouse prion protein

Cited by 9 publications

References 17 publications

Structural evidence for the critical role of the prion protein hydrophobic region in forming an infectious prion

Structural evidence for the critical role of the prion protein hydrophobic region in forming an infectious prion

Biotechnological applications of recombinant single-domain antibody fragments

Engineered Extracellular Vesicles/Exosomes as a New Tool against Neurodegenerative Diseases

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