Dual roles of tau R peptides on Cu(II)/(I)-mediated reactive oxygen species formation.

Metal dyshomeostasis plays a critical role in the reactive oxygen species (ROS) formation and protein misfolding and aggregation; hence, contributing to neurodegeneration. Tau protein plays a key role in normal cellular function by maintaining microtubule formation in brain. The role of metal ions on tau protein biochemistry has not been systematically evaluated, but earlier reports indicated that metal ions modulate the complex biochemistry of this protein and its peptides. Herein, we evaluated interactions of biologically-relevant Cu(II) ions with the four repeat peptides of tau protein (R1 through R4) and their role on the formation of ROS, Cu(II) to Cu(I) reduction, and ultimately, peptide aggregation. The role of R peptides on ROS formation was characterized in the absence and presence of biological reducing agent, ascorbate by using UV-Vis and fluorescence spectroscopy. In the presence of the reducing agent, all Cu(II)-peptide complexes reduced hydroxyl radical (OH·), while only Cu(II)-R3 complex depleted the hydrogen peroxide (H2O2). In the absence of a reducing agent, only Cu(II)-R2 and Cu(II)-R3 complexes, which contain Cys and His residues, produced OH· and H2O2. Only R2 and R3 peptides, but not R1 and R4, reduced Cu(II) to Cu(I). The aggregation propensities of R peptides were modulated by Cu(II) and ascorbate, and were imaged by transmission electron microscopy. All metallo-peptides were characterized predominantly as singly charged mononuclear complexes by mass spectrometry. The data indicate that Cu(II)-peptide complexes may act as pro-oxidants or antioxidants and exhibit unique aggregation propensities under specific environmental conditions, with implications in the biological setting.

Aggregation of gelsolin wild-type and G167K/R, N184K, and D187N/Y mutant peptides and inhibition.

Gelsolin, an actin-binding protein, is localized intra- and extracellularly in the bloodstream and throughout the body. Gelsolin amyloidosis is a disease characterized by several point mutations that lead to cleavage and fibrillization of gelsolin. The D187 mutation to N or Y leads to aggregation of peptide fragments with shortest aggregating peptide identified as 182SFNNGDCFILD192. Recently, G167 has also been identified as relevant gelsolin mutation, which leads to gelsolin deposits in kidneys, but its aggregation is much less understood. Hence, we systematically investigated in vitro the aggregation propensities of the following gelsolin peptides: 167GRRVV171 (1), 161RLFQVKG167 (2), 184NNGDCFILDL193 (3), 188CFILDL193 (4), 187DCFILDL193 (5), and their respective mutants (G167K, G167R, N184K, D187Y, D187N), by using spectroscopic methods [fluorescence Proteostat, Thioflavin T (ThT), turbidity assay, and Dynamic Light Scattering (DLS)], and Transmission Electron Microscopy (TEM). The (non) mutant peptides containing CFILDL sequence aggregated into fibrillar networks, while G167R mutation promoted aggregation compared to the wild-type sequence. In the presence of inhibitors, Methylene Blue (MB) and epigallocatechin gallate (EGCG), the gelsolin peptide (3-5) aggregation was reduced with the IC50 values in the 2-13 µM range. We discovered that inhibitors have dual functionality, as aggregation inhibitors and disaggregation promoters, potentially allowing for the prevention and reversal of gelsolin amyloidosis. Such therapeutic strategies may improve outcomes related to other amyloidogenic diseases of the heart, brain, and eye.