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Amyloid Beta – Biotherapy Target and Biotechnological Tool

Time: Fri 2020-05-29 10.00

Location:, Stockholm (English)

Subject area: Biotechnology

Doctoral student: Sebastian W. Meister , Proteinvetenskap, Stefan Ståhl

Opponent: Prof. Maarten Merkx, Department of Biomedical Engineering, Eindhoven University of Technology (TU/e), Eindhoven, The Netherlands

Supervisor: Professor Stefan Ståhl, Proteinvetenskap, Bioteknologi, Albanova VinnExcellence Center for Protein Technology, ProNova; Associate professor John Löfblom, Skolan för teknikvetenskap (SCI), Protein Engineering

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Alzheimer’s disease is a progressive neurodegenerative disease characterized primarily by memory impairment and cognitive decline. As of 2020, an estimated 50 million people suffer from Alzheimer’s disease or related dementia and no disease-modifying treatment options are yet approved for clinical practice. A major pathological feature of Alzheimer’s disease is the presence of cerebral senile plaques with aggregated amyloid beta (Aβ) peptides as the main constituent. In this thesis, Aβ is used in five separate studies either as a target for the development of potential biotherapeutical interventions or as a tool in biotechnological research.

In the first study, a high-throughput screening method was developed that enables functiondriven selection of protein-based aggregation inhibitors from combinatorial libraries. The method employs a reporter protein consisting of Aβ42 fused to the N-terminus of green fluorescent protein (GFP). The reporter protein misfolds due to the aggregating nature of Aβ42. Following protein expression in Escherichia coli a low whole-cell GFP fluorescence signal was detected using flow cytometry. However, when co-expressed with an affibodybased aggregation inhibitor, the reporter protein was rescued from aggregation and an increased whole-cell GFP fluorescence signal was detected in flow cytometry. By combining the screening method with flow cytometric cell sorting, the aggregation-inhibiting affibody molecule could successfully be enriched from a large background of non-inhibiting affibody molecules. The results thus demonstrated that the developed method enables highthroughput screening and sorting of combinatorial protein libraries based on the Aβ aggregation inhibiting ability.

The second study explored a strategy to increase the uptake of a biotherapeutical candidate protein into the central nervous system (CNS) via receptor-mediated transcytosis across the blood-brain barrier (BBB). The affibody-based candidate ZSYM73 binds monomeric Aβ and inhibits Aβ aggregation. Here, ZSYM73 was fused to the C-terminus of a single-chain variable fragment (scFv8D3) binding the transferrin receptor (TfR); a receptor expressed on the BBB. An engineered albumin-binding domain (ABD) was fused to ZSYM73 to extend the circulatory half-life of the fusion protein. In a mouse study, the tri-specific fusion protein scFv8D3-ZSYM73-ABD exhibited increased cerebrospinal fluid (CSF) bioavailability compared to the control protein ZSYM73-ABD, indicating an active transport mechanism into the CNS.

In the third study, a novel method for combinatorial protease engineering was developed and applied to generate highly proteolytic active variants of the coxsackievirus 3C protease. The method is based on the findings form the first study and employs a reporter protein consisting of Aβ42 fused to the N-terminus of GFP via a peptide linker containing a protease substrate sequence. The reporter protein misfolds upon expression in E. coli, which resulted in a low whole-cell GFP fluorescence signal detected in flow cytometry. Co-expression of a protease with activity on the substrate sequence led to proteolytic separation of the aggregation-prone Aβ42 peptide from GFP and restored whole-cell fluorescence. This method was used in combination with flow cytometric cell sorting to isolate highly proteolytic active variants from a randomly mutated 3C protease library. The aim of the fourth study was to evaluate the potential of the newly developed method from the third study to engineer the substrate specificity of proteases. A semi-rational tobacco etch virus (TEV) protease library was screened for variants with proteolytic activity on a novel target substrate. The target substrate differed substantially from the wild-type TEV consensus substrate and exhibited high sequence similarity to the aggregation-inducing hydrophobic core region of Aβ. After three rounds of flow cytometric cell sorting, a set of TEV protease variants was enriched that exhibited proteolytic activity on the novel substrate.

In the fifth study, a methodology employing flow cytometric sorting of multiprotein aggregates was developed to investigate the protein interactome related to Aβ plaques. It was demonstrated that in human serum or human CSF, Aβ aggregates bound to a fluorescent probe can be detected and isolated using flow cytometry. Quantitative mass spectrometry analysis was performed on Aβ aggregates isolated from human CSF. The abundances and functional features of proteins found in the isolated aggregates were investigated, and a hypothetical model of the layered architecture of Aβ aggregates was proposed. 

In conclusion, this thesis describes the development of new concepts and methods that will hopefully contribute to increasing the understanding and improving the therapy of Alzheimer’s disease and other diseases associated with protein aggregation.