Investigating the Effect of Physiological Factors on Amyloid Formation and Developing Its Targeted Inhibitors

Amyloid fibrils are highly insoluble protein aggregates with fibrillar morphology. They include functional amyloids and pathological amyloids, which are strongly implicated in a range of diseases. Functional amyloids serve as the structural scaffold within the bacterial biofilm matrix, facilitating bacterial surface adhesion for colonization and consolidation, and conferring resistance to antibiotics and immune systems, thereby contributing to biofilm-related infections. In contrast, the presence of pathological amyloids is a characteristic feature of various neurodegenerative diseases, such as tauopathies and Parkinson’s disease. These aggregates, deposited in the brain, lead to synaptic dysfunction, neuronal loss, and brain atrophy. Given the serious health risks posed by amyloid fibrils, it is critical to understand how physiological factors influence protein aggregation and to explore effective strategies to inhibit their formation and propagation. This dissertation first investigates the role of shear rate, one of the critical physiological factors within blood vessels, on amyloid formation from phenol-soluble modulins (PSMs). PSM amyloids serve as a crucial component fortifying the Staphylococcus aureus (S. aureus) biofilm matrix. In this work, using a combination of thioflavin T fluorescence kinetic studies, circular dichroism spectrometry, and electron microscopy, we demonstrate that shear rates accelerate the fibrillation of PSMa1, a3, and a4 into amyloids, resulting in elongated amyloid structures. Furthermore, PSMa1, a3, and a4 predominantly self-assembled into amyloid fibers with a cross-a structure under shear conditions, deviating from the typical ß-sheet configuration of PSM amyloids. These findings imply that shear rates within the bloodstream enhance PSM self-assembly, which is associated with S. aureus biofilm formation. Following this, we explored strategies to inhibit the aggregation of tau proteins for the treatment of tauopathies. Pathological aggregation of tau proteins into neurofibrillary tangles is a hallmark of tauopathies, which are a group of neurodegenerative disorders. Moreover, the propagation of tau aggregates between neurons leads to disease progression. Although numerous small molecules are known to inhibit tau aggregation and block tau transmission, their therapeutic application remains challenging due to poor specificity and low blood-brain barrier (BBB) penetration. Graphene quantum dots (GQDs) have been demonstrated to penetrate the BBB and are amenable to functionalization for targeted delivery. Moreover, these nanoscale biomimetic particles can self-assemble or assemble with various proteins. In this work, we show that GQDs and cysteine functionalized GQDs (Cys-GQDs) block the seeding activity of tau fibrils by inhibiting the fibrillization of monomeric tau and triggering the disaggregation of tau filaments, which is attributed to electrostatic and p-p stacking interactions of GQDs with tau. These findings suggest the potential of GQDs as therapeutic agents for tauopathies. However, the non-specific binding of GQDs to various proteins in the physiological environment limits their clinical translation. To further enhance the specificity of GQDs toward tau proteins, we incorporated D-cysteine functionalized GQDs (D-GQDs) with a tau-targeting N-amino peptide, mxyl-NAP2. The mxyl-NAP2/D-GQD complex demonstrated improved selectivity for tau protein over serum albumin, effectively enhancing the inhibition of tau aggregation. To further minimize off-target effects and optimize therapeutic delivery, the complex was loaded into small extracellular vesicles (sEVs), followed by functionalization of sEVs with a neuron targeting ligand, rabies viral glycoprotein peptides. This strategy not only reduced off-target effects, but also enhanced uptake by neuron cells, further improving the inhibition of tau transmission between neurons. These findings indicate that this targeted and efficient therapeutic platform holds great promise for overcoming the off-target limitations of GQDs and advancing the development of precision therapeutics for tauopathies.<p></p>