Camelid single-domain antibody fragments: Uses and prospects to investigate protein misfolding and aggregation, and to treat diseases associated with these phenomena

Abstract

Keywords:Variable domain of heavy-chain antibody Inhibition of protein misfolding and aggregation Protein misfolding diseases Amyloidoses V H H Nanobody a b s t r a c tThe deposition of misfolded peptides and proteins in the form of amyloid fibrils is the hallmark of nearly fifty medical disorders, including Alzheimer's disease, Parkinson's disease, prion diseases and type II diabetes. These disorders, referred to as amyloidoses, generally become apparent late in life. Their psycho-sociological and economic incidence in western societies will be therefore considerable in the coming decades due to the ageing of the population. Neither preventing nor curative treatments are available yet. These disorders constitute therefore a medical challenge of great importance. Thus, an extensive research is being carried out to understand, at the molecular level, (i) how amyloidogenic proteins misfold and convert from their soluble form into amyloid fibrils, and (ii) how these aggregates or some of their oligomeric precursor species are toxic. The formation of amyloid fibrils proceeds through a complex nucleation/polymerisation mechanism with the formation of various species, including small oligomers. In this review, we focus on how V H Hs or nanobodies, the antigen-binding domains of camelid heavy-chain antibodies, are being increasingly used to characterise each of the species formed on the pathway of fibril formation in terms of structure, stability, kinetics of formation and toxicity. We first introduce the characteristic features of nanobodies compared to those of conventional antibody fragments. Thereafter, we discuss how nanobodies, due to their unique properties, are used as probes to dissect the molecular mechanisms of misfolding and aggregation of six proteins associated with diseases, i.e. human lysozyme, b2-microglobulin, a-synuclein, prion, polyadenylate binding protein nuclear 1 and amyloid b-peptide. A brief general presentation of each disease and the associated peptide/protein is also provided. In addition, we discuss how nanobodies could be used as early diagnostic tools and as novel strategies to treat diseases associated with protein misfolding and aggregation.© 2015 Elsevier B.V. and Societe Française de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.Abbreviations: Ab, antibody; Ab, amyloid b-peptide; AD, Alzheimer's disease; ANS, anilino naphthalene-sulfonic acid; AP, alkaline phosphatase; APP, amyloid precursor protein; aSyn, a-synuclein; b2m, b2-microglobulin; BBB, bloodebrain barrier; CDR, complementarity determining region; C m , concentration of mid-denaturation; CR, Congo red; CSF, cerebrospinal fluid; DN6b2m, a truncated form of b2m lacking the six N-terminal amino acids; DLS, dynamic light scattering; DRA, dialysis-related amyloidosis; FR, framework; FTIR, Fourier transform infrared spectroscopy; Fv, variable fragment made of the VH and VL domains of conventional antibodies; GFP, green fluorescent protein; HCAb, heavy-chain antibody; H/D, hydrogen/deuterium; HSQC, heteronuclear s...

Biophysical characterization of antibodies with isothermal titration calorimetry

“…Display technologies are used to screen libraries by affinity selection, yielding clones that are specific and high-affinity for their cognate targets. One such scaffold classification is "nanobodies," which are single-domain fragments of camelid antibodies [54]. Nanobodies are emerging as versatile tools in biotechnology.…”

A Nanobody‐Based Approach to Amyloid Diseases, the Gelsolin Case Study

“…Nanobodies are also capable of stabilizing proteins in a particular conformation and of minimizing structural flexibility. This has been nicely demonstrated in the case of human lysozyme amyloidosis, a hereditary systemic disease associated with at least seven amyloidogenic lysozyme variants [39,40]. Aggregation of these lysozyme variants leads to the accumulation of amyloid in the extracellular matrix of several tissues and organs.…”

Use, Applications and Mechanisms of Intracellular Actions of Camelid VHHs

“…These therapeutic approaches are feasible when using nanobodies as a tool: increasing the stability of the correctly folded proteins, neutralization of toxic protein/peptide species, and inhibiting or reversing the aggregation of misfolded proteins into oligomers or fibrils [56]. Several research groups have already exploited the use of nanobodies for targeting protein misfolding diseases [57][58][59]; however, most of the time, they aim at extracellular targets.…”

“…PABPN1 is a multifunctional protein and is involved in pre-mRNA polyadenylation, transcription regulation, and mRNA nucleocytoplasmic transport [56].…”