Aggregate-selective removal of pathological tau by clustering-activated degraders.

Selective degradation of pathological protein aggregates while sparing monomeric forms is of major therapeutic interest. The E3 ligase tripartite motif-containing protein 21 (TRIM21) degrades antibody-bound proteins in an assembly state-specific manner due to the requirement of TRIM21 RING domain clustering for activation, yet effective targeting of intracellular assemblies remains challenging. Here, we fused the RING domain of TRIM21 to a target-specific nanobody to create intracellularly expressed constructs capable of selectively degrading assembled proteins.

Recombinant Production and Characterization of VHHs/Nanobodies Targeting Tau to Block Fibrillar Assembly.

Tau protein was extensively studied using nuclear magnetic resonance spectroscopy, providing a powerful way to determine interaction sites between Tau and partner proteins. Here we used this analytical tool to describe the epitopes of Tau-specific VHHs (variable domain of the heavy chain of the heavy chain-only antibodies, aka nanobodies) selected from a synthetic library. An in vitro Tau aggregation assay was subsequently used as a functional screen to check VHH efficacy as aggregation inhibitors.

A selection and optimization strategy for single-domain antibodies targeting the PHF6 linear peptide within the tau intrinsically disordered protein.

The use of variable domain of the heavy-chain of the heavy-chain-only antibodies (VHHs) as disease-modifying biomolecules in neurodegenerative disorders holds promises, including targeting of aggregation-sensitive proteins. Exploitation of their clinical values depends however on the capacity to deliver VHHs with optimal physico-chemical properties for their specific context of use. We described previously a VHH with high therapeutic potential in a family of neurodegenerative diseases called tauopathies.

Inhibition of Tau seeding by targeting Tau nucleation core within neurons with a single domain antibody fragment.

Tau proteins aggregate into filaments in brain cells in Alzheimer's disease and related disorders referred to as tauopathies. Here, we used fragments of camelid heavy-chain-only antibodies (VHHs or single domain antibody fragments) targeting Tau as immuno-modulators of its pathologic seeding. A VHH issued from the screen against Tau of a synthetic phage-display library of humanized VHHs was selected for its capacity to bind Tau microtubule-binding domain, composing the core of Tau fibrils.

Extracellular vesicles: Major actors of heterogeneity in tau spreading among human tauopathies.

Tauopathies are neurodegenerative diseases characterized by tau inclusions in brain cells. Seed-competent tau species have been suggested to spread from cell to cell in a stereotypical manner, indicating that this may involve a prion-like mechanism. Although the intercellular mechanisms of transfer are unclear, extracellular vesicles (EVs) could be potential shuttles.

Nuclear Magnetic Resonance Spectroscopy Insights into Tau Structure in Solution: Impact of Post-translational Modifications.

Although Tau is an intrinsically disordered protein, some level of structure can still be defined, corresponding to short stretches of dynamic secondary structures and a preferential global fold described as an ensemble of conformations. These structures can be modified by Tau phosphorylation, and potentially other post-translational modifications. The analytical capacity of Nuclear Magnetic Resonance (NMR) spectroscopy provides the advantage of offering a residue-specific view of these modifications, allowing to link specific sites to a particular structure.

Exacerbation of C1q dysregulation, synaptic loss and memory deficits in tau pathology linked to neuronal adenosine A2A receptor.

Accumulating data support the role of tau pathology in cognitive decline in ageing and Alzheimer's disease, but underlying mechanisms remain ill-defined. Interestingly, ageing and Alzheimer's disease have been associated with an abnormal upregulation of adenosine A2A receptor (A2AR), a fine tuner of synaptic plasticity. However, the link between A2AR signalling and tau pathology has remained largely unexplored.

Single Domain Antibody Fragments as New Tools for the Detection of Neuronal Tau Protein in Cells and in Mice Studies.

Tau is a neuronal protein linked to pathologies called tauopathies, including Alzheimer's disease. In Alzheimer's disease, tau aggregates into filaments, leading to the observation of intraneuronal fibrillary tangles. Molecular mechanisms resulting in tau aggregation and in tau pathology spreading through the brain regions are still not fully understood.

Prevention of tau seeding and propagation by immunotherapy with a central tau epitope antibody.

Tauopathies are neurodegenerative diseases characterized by the intraneuronal accumulation of aggregated tau. The staging of this neurodegenerative process is well established for Alzheimer's disease as well as for other tauopathies. The stereotypical pattern of tau pathology in these diseases is consistent with the hypothesis that the tau protein can spread in a 'prion-like' manner.

Gradient reconstitution of membrane proteins for solid-state NMR studies.

We here adapted the GRecon method used in electron microscopy studies for membrane protein reconstitution to the needs of solid-state NMR sample preparation. We followed in detail the reconstitution of the ABC transporter BmrA by dialysis as a reference, and established optimal reconstitution conditions using the combined sucrose/cyclodextrin/lipid gradient characterizing GRecon. We established conditions under which quantitative reconstitution of active protein at low lipid-to-protein ratios can be obtained, and also how to upscale these conditions in order to produce adequate amounts for NMR.

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins.

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau.

Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus.

We describe the expression of the hepatitis C virus nonstructural protein 4B (NS4B), which is an integral membrane protein, in a wheat germ cell-free system, the subsequent purification and characterization of NS4B and its insertion into proteoliposomes in amounts sufficient for multidimensional solid-state NMR spectroscopy. First spectra of the isotopically [(2)H,(13)C,(15)N]-labeled protein are shown to yield narrow (13)C resonance lines and a proper, predominantly α-helical fold. Clean residue-selective leucine, isoleucine and threonine-labeling is demonstrated.