RATIONAL AND COMBINATORIAL DESIGN OF MOLECULES THAT TARGET AMYLOID β 42 AND SUPEROXIDE DISMUTASE1 MUTANT

Protein misfolding and aggregation has been linked to many neurodegenerative disorders. In the process of aggregation, proteins adopt ensemble of heterogeneous conformations and these intermediates are found to be more toxic than the mature fibrils. Among the many proteins that adopt fibrillar conformation upon cellular stress, Aβ42 and mutants of SOD1 have gained attention due to their involvement in Alzheimer`s disease and familiar amyotrophic lateral sclerosis (ALS) diseases, respectively. Thus, the prevention of aggregation of these proteins is a goal for many therapeutic efforts as well as being a possible route to trapping intermediates for understanding the role aggregation plays in disease pathology. In our research, we used HTB1, the hypotherphilic variant of B1 domain of protein G, as a molecular scaffold to engineer protein variants that bind to Aβ42 and SOD1^G93A with high specificity and that may modulate and inhibit the aggregation pathway of these two proteins. Yeast displayed mutated HTB1 library was created by selecting twelve residue positions based on steered molecular dynamics simulation (SMD) calculations and further screened by high-throughput flow cytometric sorting to identify high affinity clones. Several unique clones were identified which bind specifically to Aβ42 and SOD1^G93A. The next steps in our research are to express the selected clones as soluble proteins and test their affinity to the target proteins and their in vitro aggregation prevention capacity. We will also test the potential of these proteins to reduce cellular toxicity level during amyloid aggregation process. Our research will identify structurally well-defined β-sheet epitopes that target Aβ42 and SOD1^G93A, that are not based upon the Aβ42 and SOD1^G93A protein sequence and do not possess the complexity of an antibody.