An Engineered Bacterial Display System for the Development of a High‐Throughput Screen for Compounds that Block Amyloid Fiber Formation

Abstract

The molecular basis for a number of neuronal diseases, such as Alzheimer's and Parkinson's, is aberrant peptide or protein aggregation that result in the formation of amyloid fibers. For example, the Aβ-42 peptide is a molecular component of the plaques found in the brains of Alzheimer's patients, and the protein α-synuclein is a constituent of Lewy bodies, a hallmark of Parkinson's disease. The exact role these proteins play in disease is currently not completely clear but it is generally believed that aberrant self-association contributes in large part to the disease state. For Aβ-42 it is not currently known whether it is low-order complexes (e.g., trimers, tetramers, etc.) or higher-order aggregates that lead to cell death and neuronal degradation. With Parkinson's disease, genetic variations in the gene for α-synuclein give rise to overexpression or aggregate prone variants of the protein. Regardless of the exact disease mechanism, or what level of self-association causes disease, it is generally recognized that blocking the aberrant self-association of these protein-based elements is of high scientific merit. To this end we have engineered a fluorescence-based Bacterial Surface Display (BSD) system that enables a relatively high expression level of a protein-of-interest (POI) on the outer surface of E. coli. Upon expression the POI is fused to an N-terminal periplasmic leader sequence, and to its C-terminus the red fluorescent protein mCherry, and finally a transmembrane protein that functions to anchor the entire fusion construct in the bacterial outer membrane. When the BSD system is used to display Aβ-42, or α-synuclein, the bacterial cells are driven to strongly self-associate. Thus far we have successfully demonstrated the utility of the system using antibodies against Aβ-42. Drug-like compounds that have been proven to block amyloid fiber formation in vitro will also be tested using the Aβ-42 and α-synuclein BSD systems. Immediate and longer-range goals entail the development of a high-throughput screen for additional compounds that bind Aβ-42 and α-synuclein and block their ability to self-associate into amyloid fibers. In addition, the BSD system will also be used for the directed evolution of a small, stable test protein that will be engineered to bind immobilized Aβ-42 and α-synuclein. The test protein, the β1 domain of streptococcal protein-G (Gβ1), consists of an α-helix overlaying a β-sheet made up of two β-hairpins. Codons for select residue positions, located at the edge of one side of the β-sheet, will be randomized and the resulting library displayed using the BSD system. The bacteria displaying the library will be incubated against immobilized Aβ-42 and α-synuclein where successful complex formation (binding) will function as the selection process. The β-hairpin, which contains the amino acids that drive binding, will be expressed/produced separately and assessed for its ability to bind the Aβ-42 and α-synuclein and block their ability to form amyloid fibers. A long-term goal is to convert this β-hairpin peptide into a more drug-like molecule using chemical biology methods such as converting the α amino acids into β forms and using unnatural D stereoisomers of amino acids. The ultimate goal is to develop drugs that effectively combat the devastating neuronal diseases caused by aberrant protein aggregation. Support or Funding InformationThis material is based upon work supported in part by the U.S. Army Research Laboratory and the U.S. Army Research Office under grant number W911NF-13-1-0155. Bacterial Surface Display of Aggregation-Prone Proteins. (A) Aβ-42 peptide (B) α-synuclein