Effect of DNAJB6 on α-synuclein amyloid fibril aggregation and dis-aggregation

Abstract: Amyloid proteins are a group of proteins with the ability to form large fibrillar aggregates, identifiable by their cross-β core structure. These aggregates are linked to several different neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease and due to the growing rate of people diagnosed, there is a need to combat these diseases. Aggregation of amyloid proteins is caused by the misfolding of nascent protein. The human body, however, has a machinery to prevent this, through a protein class known as chaperones. They are defined as protein which can prevent misfolding or aggregation of other protein. One chaperone from the Hsp40 class, DNAJB6, has been shown to prevent aggregation of amyloid protein. An attempt to describe the thermodynamic background behind this phenomena has been done by Linse et al. (2021), with the ”unhappy chaperone” hypothesis [1]. Through the formation of co-aggregates between chaperones and amyloid protein, there is a lowering of the free energy of the system, leading to an increase in solubility for the amyloid protein. An increase in solubility is only possible through an increase in free energy for the amyloid protein, however this is offset by a larger decrease for the chaperone. The decrease in free energy for the chaperone when forming co-aggregates explains the ”unhappiness” of chaperones to be free in solution. The aim of this project was to exhibit this increase in solubility for the amyloid protein α-synuclein, whose aggregation is the cause of Parkinson’s disease. DNAJB6 was the chosen chaperone, and the goal was to determine if an increase in solubility is a true thermodynamic equilibrium, which means this effect should lead to the same solubility both from a system consisting of 100% monomeric α-synuclein or 100% fibrillar α-synuclein. Also, there was prior method development for how to best measure the monomer concentration. α-synuclein aggregation at 37◦ C and pH 5.5, was measured both without DNAJB6 and with 1% DNAJB6, this was done through a developed HPLC absorbance protocol and OPA fluorescence. Possible α-synuclein disaggregation was also investigated on samples with aggregated α-synuclein, where 1% and 10% DNAJB6 was added, these also measured with HPLC and OPA. α-synuclein solubility were furthermore also measured with NMR, and DNAJB6 was also added here, at the end of fibrillization to detect possible dis-aggregation. From the results, the following conclusions could be determined. α-synuclein solubility was determined to be 0.39 ±0.016 µM measured with OPA, and 0.11 ±0.013 µM, measured with HPLC. Aggregation in the presence of 1% DNAJB6 lead to delayed aggregation and possible increase in solubility. With SDS-PAGE and MALDI, the formation of aggregates consisting of both α-synuclein and DNAJB6 was identified. Dis-aggregation was not observed and could be attributed to the low solubility of DNAJB6 at pH 5.5. HPLC was determined to be a sensitive and accurate system for measuring α-synuclein monomer concentration, but further development should be done to improve robustness and reproducibility. Lastly, NMR was determined as a viable method for measuring α-synuclein solubility at pH 5.5, although it is not yet dependable for discontinuous measurements.