Development of a pan-tau multivalent nanobody that binds tau aggregation motifs and recognizes pathological tau aggregates.

Alzheimer's disease and other tauopathies are characterized by the misfolding and aggregation of the tau protein into oligomeric and fibrillar structures. Antibodies against tau play an increasingly important role in studying these neurodegenerative diseases and the generation of tools to diagnose and treat them. The development of antibodies that recognize tau protein aggregates, however, is hindered by complex immunization and antibody selection strategies and limitations to antigen presentation. Here, we have taken a facile approach to identify single-domain antibodies, or nanobodies, that bind to many forms of tau by screening a synthetic yeast surface display nanobody library against monomeric tau and creating multivalent versions of our lead nanobody, MT3.1, to increase its avidity for tau aggregates. We demonstrate that MT3.1 binds to tau monomer, oligomers, and fibrils, as well as pathogenic tau from a tauopathy mouse model, despite being identified through screens against monomeric tau. Through epitope mapping, we discovered binding epitopes of MT3.1 contain the key motif VQIXXK which drives tau aggregation. We show that our bivalent and tetravalent versions of MT3.1 have greatly improved binding ability to tau oligomers and fibrils compared to monovalent MT3.1. Our results demonstrate the utility of our nanobody screening and multivalent design approach in developing nanobodies that bind amyloidogenic protein aggregates. This approach can be extended to the generation of multivalent nanobodies that target other amyloid proteins and has the potential to advance the research and treatment of neurodegenerative diseases.

Quantitative flow cytometric selection of tau conformational nanobodies specific for pathological aggregates.

Single-domain antibodies, also known as nanobodies, are broadly important for studying the structure and conformational states of several classes of proteins, including membrane proteins, enzymes, and amyloidogenic proteins. Conformational nanobodies specific for aggregated conformations of amyloidogenic proteins are particularly needed to better target and study aggregates associated with a growing class of associated diseases, especially neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. However, there are few reported nanobodies with both conformational and sequence specificity for amyloid aggregates, especially for large and complex proteins such as the tau protein associated with Alzheimer's disease, due to difficulties in selecting nanobodies that bind to complex aggregated proteins. Here, we report the selection of conformational nanobodies that selectively recognize aggregated (fibrillar) tau relative to soluble (monomeric) tau. Notably, we demonstrate that these nanobodies can be directly isolated from immune libraries using quantitative flow cytometric sorting of yeast-displayed libraries against tau aggregates conjugated to quantum dots, and this process eliminates the need for secondary nanobody screening. The isolated nanobodies demonstrate conformational specificity for tau aggregates in brain samples from both a transgenic mouse model and human tauopathies. We expect that our facile approach will be broadly useful for isolating conformational nanobodies against diverse amyloid aggregates and other complex antigens.

Flow cytometric isolation of drug-like conformational antibodies specific for amyloid fibrils.

Antibodies that recognize specific protein conformational states are broadly important for research, diagnostic and therapeutic applications, yet they are difficult to generate in a predictable and systematic manner using either immunization or in vitro antibody display methods. This problem is particularly severe for conformational antibodies that recognize insoluble antigens such as amyloid fibrils associated with many neurodegenerative disorders. Here we report a quantitative fluorescence-activated cell sorting (FACS) method for directly selecting high-quality conformational antibodies against different types of insoluble (amyloid fibril) antigens using a single, off-the-shelf human library. Our approach uses quantum dots functionalized with antibodies to capture insoluble antigens, and the resulting quantum dot conjugates are used in a similar manner as conventional soluble antigens for multi-parameter FACS selections. Notably, we find that this approach is robust for isolating high-quality conformational antibodies against tau and α-synuclein fibrils from the same human library with combinations of high affinity, high conformational specificity and, in some cases, low off-target binding that rival or exceed those of clinical-stage antibodies specific for tau (zagotenemab) and α-synuclein (cinpanemab). This approach is expected to enable conformational antibody selection and engineering against diverse types of protein aggregates and other insoluble antigens (e.g., membrane proteins) that are compatible with presentation on the surface of antibody-functionalized quantum dots.

Experimental and Analytical Framework for "Mix-and-Read" Assays Based on Split Luciferase.

The use of immunodetection assays including the widely used enzyme-linked immunosorbent assay (ELISA) in applications such as point-of-care detection is often limited by the need for protein immobilization and multiple binding and washing steps. Here, we describe an experimental and analytical framework for the development of simple and modular "mix-and-read" enzymatic complementation assays based on split luciferase that enable sensitive detection and quantification of analytes in solution. In this assay, two engineered protein binders targeting nonoverlapping epitopes on the target analyte were each fused to nonactive fragments of luciferase to create biosensor probes. Binding proteins to two model targets, lysozyme and Sso6904, were isolated from a combinatorial library of Sso7d mutants using yeast surface display. In the presence of the analyte, probes were brought into close proximity, reconstituting enzymatic activity of luciferase and enabling detection of low picomolar concentrations of the analyte by chemiluminescence. Subsequently, we constructed an equilibrium binding model that relates binding affinities of the binding proteins for the target, assay parameters such as the concentrations of probes used, and assay performance (limit of detection and concentration range over which the target can be quantified). Overall, our experimental and analytical framework provides the foundation for the development of split luciferase assays for detection and quantification of various targets.

Using experimental reintroductions to resolve the roles of habitat quality and metapopulation dynamics on patch occupancy in fragmented landscapes.

Declines of species in fragmented landscapes can potentially be reversed either by restoring connectivity or restoring local habitat quality. Models fitted to snapshot occupancy data can be used to predict the effectiveness of these actions. However, such inferences can be misleading if the reliability of the habitat and landscape metrics used is unknown. The only way to unambiguously resolve the roles of habitat quality and metapopulation dynamics is to conduct experimental reintroductions to unoccupied patches so that habitat quality can be measured directly from data on vital rates. We, therefore, conducted a 15-year study that involved reintroducing a threatened New Zealand bird to unoccupied forest fragments to obtain reliable data on their habitat quality and reassess initial inferences made by modeling occupancy against habitat and landscape metrics. Although reproductive rates were similar among fragments, subtle differences in adult survival rates resulted in λ (finite rate of increase) estimations of <0.9 for 9 of the 12 fragments that were previously unoccupied. This was the case for only 1 of 14 naturally occupied fragments. This variation in λ largely explained the original occupancy pattern, reversing our original conclusion from occupancy modeling that this occupancy pattern was isolation driven and suggesting that it would be detrimental to increase connectivity without improving local habitat quality. These results illustrate that inferences from snapshot occupancy should be treated with caution and subjected to testing through experimental reintroductions in selected model systems.

Uso de Reintroducciones Experimentales para Determinar las Funciones de la Calidad delHábitat y las Dinámicas Metapoblacionales en la Ocupación de Paisajes Fragmentados Resumen La declinación de las especies en paisajes fragmentados tiene el potencial de ser revertida mediante la restauración de la conectividad o de la calidad del hábitat. Se pueden utilizar los modelos ajustados a los datos de ocupación instantánea para predecir la efectividad de estas acciones. Sin embargo, estas inferencias pueden ser engañosas si se desconoce la confiabilidad de las medidas usadas para el hábitat y el paisaje. La única manera de determinar inequívocamente las funciones de la calidad del hábitat y de las dinámicas metapoblacionales es mediante la realización de reintroducciones experimentales en los fragmentos no ocupados, de tal manera que se puede medir directamente la calidad del hábitat a partir de los datos de las tasas vitales. Por lo tanto, realizamos un estudio de 15 años que involucró la reintroducción de un ave neozelandesa amenazada en fragmentos no ocupados de bosque para así obtener datos confiables de la calidad del hábitat y reevaluar las inferencias iniciales hechas por los modelos de ocupación en relación con las medidas de hábitat y paisaje. Aunque las tasas de reproducción fueron similares entre los fragmentos, algunas diferencias sutiles en las tasas de supervivencia de los adultos resultaron en estimaciones λ (una tasa finita de incremento) <0.9 en nueve de los doce fragmentos que no estaban ocupados previamente. Este fue el caso para uno solo de los 14 fragmentos ocupados naturalmente. Esta variación λ explicó en su mayoría el patrón original de ocupación, revirtiendo nuestra conclusión original obtenida del modelo de ocupación de que este patrón estuvo causado por el aislamiento y sugiriendo que sería perjudicial incrementar la conectividad sin mejorar la calidad del hábitat local. Estos resultados muestran que las inferencias a partir de la ocupación instantánea deberían abordarse con cautela y estar sujetas al análisis mediante reintroducciones experimentales en sistemas modelados selectos.

Misinformation tactics protect rare birds from problem predators.

Efficient decision-making integrates previous experience with new information. Tactical use of misinformation can alter choice in humans. Whether misinformation affects decision-making in other free-living species, including problem species, is unknown. Here, we show that sensory misinformation tactics can reduce the impacts of predators on vulnerable bird populations as effectively as lethal control. We repeatedly exposed invasive mammalian predators to unprofitable bird odors for 5 weeks before native shorebirds arrived for nesting and for 8 weeks thereafter. Chick production increased 1.7-fold at odor-treated sites over 25 to 35 days, with doubled or tripled odds of successful hatching, resulting in a 127% increase in modeled population size in 25 years. We demonstrate that decision-making processes that respond to changes in information reliability are vulnerable to tactical manipulation by misinformation. Altering perceptions of prey availability offers an innovative, nonlethal approach to managing problem predators and improving conservation outcomes for threatened species.

Quantitative Yeast-Yeast Two Hybrid for the Discovery and Binding Affinity Estimation of Protein-Protein Interactions.

Quantifying the binding affinity of protein-protein interactions is important for elucidating connections within biochemical signaling pathways, as well as characterization of binding proteins isolated from combinatorial libraries. We describe a quantitative yeast-yeast two-hybrid (qYY2H) system that not only enables the discovery of specific protein-protein interactions but also efficient, quantitative estimation of their binding affinities (KD). In qYY2H, the bait and prey proteins are expressed as yeast cell surface fusions using yeast surface display. We developed a semiempirical framework for estimating the KD of monovalent bait-prey interactions, using measurements of bait-prey yeast-yeast binding, which is mediated by multivalent interactions between yeast-displayed bait and prey. Using qYY2H, we identified interaction partners of SMAD3 and the tandem WW domains of YAP from a cDNA library and characterized their binding affinities. Finally, we showed that qYY2H could also quantitatively evaluate binding interactions mediated by post-translational modifications on the bait protein.

Isolation of Chemically Cyclized Peptide Binders Using Yeast Surface Display.

Cyclic peptides with engineered protein-binding activity have gained increasing attention for use in therapeutic and biotechnology applications. We describe the efficient isolation and characterization of cyclic peptide binders from genetically encoded combinatorial libraries using yeast surface display. Here, peptide cyclization is achieved by disuccinimidyl glutarate-mediated cross-linking of amine groups within a linear peptide sequence that is expressed as a yeast cell surface fusion. Using this approach, we first screened a library of cyclic heptapeptides using magnetic selection, followed by fluorescence activated cell sorting (FACS) to isolate binders for a model target (lysozyme) with low micromolar binding affinity (KD ∼ 1.2-3.7 µM). The isolated peptides bind lysozyme selectively and only when cyclized. Importantly, we showed that yeast surface displayed cyclic peptides can be used to efficiently obtain quantitative estimates of binding affinity, circumventing the need for chemical synthesis of the selected peptides. Subsequently, to demonstrate broader applicability of our approach, we isolated cyclic heptapeptides that bind human interleukin-17 (IL-17) using yeast-displayed IL-17 as a target for magnetic selection, followed by FACS using recombinant IL-17. Molecular docking simulations and follow-up experimental analyses identified a candidate cyclic peptide that likely binds IL-17 in its receptor binding region with moderate apparent affinity (KD ∼ 300 nM). Taken together, our results show that yeast surface display can be used to efficiently isolate and characterize cyclic peptides generated by chemical modification from combinatorial libraries.

An Engineered Sso7d Variant Enables Efficient Magnetization of Yeast Cells.

Magnetization using cheap and minimally toxic materials, such as iron oxide nanoparticles can enable easy separation of cells from culture medium and is relevant to several industrial applications. Here, we show that cell surface expression of a mutant protein that binds iron oxide can enable efficient magnetization of yeast cells. We screened a combinatorial library of mutants derived from the Sso7d protein scaffold to isolate proteins that exhibit preferential binding to iron oxide. One of the isolated mutants, SsoFe2, was chosen for further characterization. Yeast cells expressing SsoFe2 as fusions to a cell wall protein-but not other Sso7d mutants with similar overall protein charge or amino acid composition-preferentially bind iron oxide when present in a solution with high protein concentration and in the presence of 1000-fold excess of competitor yeast cells. Moreover, coexpression of cell surface SsoFe2 enables efficient magnetic capture and separation of yeast cells expressing an enzyme (glucose oxidase) on the cell surface from yeast culture medium, and solutions with high protein concentration or containing other metal oxides. Therefore, SsoFe2-enabled magnetization can enable a range of industrial and biotechnology applications, where easy separation of cells or organelles from complex media is desirable.

Strategic rat control for restoring populations of native species in forest fragments.

Forest fragments have biodiversity value that may be enhanced through management such as control of non-native predators. However, such efforts may be ineffective, and research is needed to ensure that predator control is done strategically. We used Bayesian hierarchical modeling to estimate fragment-specific effects of experimental rat control on a native species targeted for recovery in a New Zealand pastoral landscape. The experiment was a modified BACI (before-after-control-impact) design conducted over 6 years in 19 forest fragments with low-density subpopulations of North Island Robins (Petroica longipes). The aim was to identify individual fragments that not only showed clear benefits of rat control, but also would have a high probability of subpopulation growth even if they were the only fragment managed. We collected data on fecundity, adult and juvenile survival, and juvenile emigration, and modeled the data in an integrated framework to estimate the expected annual growth rate (λ) of each subpopulation with and without rat control. Without emigration, subpopulation growth was estimated as marginal (λ = 0.95-1.05) or negative (λ = 0.74-0.90) without rat control, but it was estimated as positive in all fragments (λ = 1.4-2.1) if rats were controlled. This reflected a 150% average increase in fecundity and 45% average increase in adult female survival. The probability of a juvenile remaining in its natal fragment was 0.37 on average, but varied with fragment connectivity. With juvenile emigration added, 6 fragments were estimated to have a high (>0.8) probability of being self-sustaining (λ > 1) with rat control. The key factors affecting subpopulation growth rates under rat control were low connectivity and stock fencing because these factors were associated with lower juvenile emigration and higher fecundity, respectively. However, there was also substantial random variation in adult survival among fragments, illustrating the importance of hierarchical modeling for fragmentation studies.