The global population is not only increasing but also rapidly aging, while neurodegenerative disorders such as Alzheimer’s disease represent a true silent epidemic of modern times. Understanding the propensity of proteins to fibrillate and the mechanisms underlying their toxicity is therefore crucial for the development of effective therapeutic approaches and new drugs that could prevent or slow the progression of these diseases.
We investigated the fibrillization of the model protein hen egg white lysozyme (HEWL) and the influence of various factors on this process. The results confirmed a fibril formation mechanism, proceeding through nucleation, followed by the protofibril growth phase, and finally reaching the plateau phase. HEWL proved to be a suitable model protein due to its small size, well characterized structure, and sensitivity to denaturing conditions, which enable relatively rapid fibrillization. Circular dichroism spectroscopy was used to monitor the transition of the secondary structure from α helices to β structures, while Thioflavin T fluorescence emission spectroscopy and Congo red UV Vis spectroscopy confirmed the formation of the cross β structure characteristic of amyloid fibrils. We found that aging fibrils at different temperatures affects their stability. At room temperature, the process is faster and fibrils precipitate more quickly from the solution, whereas aging in the refrigerator proceeds more slowly, allowing for prolonged monitoring. Furthermore, we examined the effect of the nucleotide derivative thymine 1 acetate and the macromolecular crowding agent PEG 10,000. The nucleotide did not show a strong influence on fibrillization, and its kinetics could not be reliably monitored due to its poor solubility in glycine buffer. In contrast, PEG 10,000 strongly inhibited fibrillization, likely by stabilizing the native protein structure and introducing steric hindrance. These results indicate that appropriate molecules can modulate the fibrillization process already in its early stages.
Understanding fibrillization mechanisms is essential for the development of therapeutic strategies against neurodegenerative diseases such as Alzheimer’s disease. Future studies should focus on less toxic compounds with interactions similar to PEG that could inhibit fibrillization under physiological conditions.
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