Biophysics

Amyloid fibrils are fibrous protein aggregates that arise from misfolding and self-assembly processes, collectively referred to as amyloidosis. These aggregates are strongly associated with incurable neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease, and Amyotrophic Lateral Sclerosis (ALS). Elevated levels of Reactive Oxygen Species (ROS) and dysregulated metal-ion homeostasis often impaired by environmental and lifestyle factors can induce oxidative stress that undermines cellular antioxidant defenses, which cause the amyloid formation and toxicity. This thesis investigates multiple amyloidosis models, emphasizing the contribution of metal ions and ROS to aggregation pathways, and evaluates the potential inhibitory or protective roles of cytokinin (CK) plant hormone.Chapter 2 focuses on Gelsolin amyloidosis, a hereditary condition driven by point mutations that promote aberrant amyloid formation. Using microscopic and spectroscopic approaches, this work characterizes the aggregation behavior of peptides derived from domain 2 of plasma gelsolin and secreted by muscle cells. Three peptides were studied: the wild-type(WT) sequence and two clinically relevant mutants, K184N and N187Y. Each variant exhibited distinct aggregation rates, reflecting mutation-dependent effects on self-assembly. Furthermore, two CKs Kinetin (Kin) and trans-Zeatin (tZ) were shown to modulate gelsolin aggregation, suggesting their potential as anti-aggregation molecules. Chapter 3 revolves on the aggregation properties of TDP-43 peptides associated with ALS pathology. Within the RRM I domain, two cysteine residues serve as key redox-active sites susceptible to oxidation. ESI-MS and spectroscopic methods were used to analyze three peptide variants: WT, a mutant (MT) in which cysteine were substituted with alanine, and WT-S, a disulfide-linked dimer. All variants displayed higher aggregation under mildly acidic conditions. CKs, Kin and isopentenyl-adenine (iP) showed antioxidant capacity and their influence on peptide stability. Chapter 4 investigates the effects of copper(II)-induced oxidative stress in C2C12 muscle cells and evaluates cellular responses to various CK forms. ESI-MS profiling identified 20 CKs in copper-treated samples and revealed 24 untargeted metabolites with significant level changes, indicating their possible involvement in metal-induced oxidative pathways. In conclusion, this thesis highlights the multifaceted roles of CKs in biological systems, particularly their potential to mitigate ROS overproduction, counteract metal-driven amyloidgenesis, promote fibril destabilization, and lessen oxidative stress.

Author Keywords: Amyloid, Anti aggregation, cytokinins, inhibition, Peptide aggregation, Protein aggregation

Transactive response (TAR) DNA-binding protein of 43 kDa (TDP-43) pathology, including fibrillar aggregates and mutations, develops in amyotrophic lateral sclerosis (ALS) and is characterized by hyperphosphorylation and aggregation patterns, a mechanism largely understudied. In addition, ALS remains without a cure. Herein, in vitro aggregation of phosphorylated TDP-43 was explored, and the anti-TDP-43 antibodies tested for their inhibitor efficacies. Additionally, in vitro phosphorylation of TDP-43 by protein kinases was conducted to identify which protein kinases catalyze phosphorylation. The aggregation of phosphorylated and unphosphorylated full-length TDP-43 protein (pS410) was monitored by transmission electron microscopy (TEM), turbidity absorbance, and thioflavin (ThT) fluorescence spectroscopy. The protein aggregates were largely insoluble, ThT-positive and characterized with heterogeneous morphologies. Antibodies specific to epitopes within the RNA-recognition motifs and the C-terminal domains reduced the formation of β-sheets and insoluble aggregates, with outcomes highly dependent on the type of antibodies, indicating dual functionality. The only protein kinase able to phosphorylate TDP-43 at S410 was MARK4, indicating its role in the onset of PTMs in the protein. Thus, targeting TDP-43 epitopes for inhibition of aggregation and in vitro phosphorylation represent viable biochemical assays for screening protein kinase inhibitors as potential drugs against ALS.

Author Keywords: aggregation, ALS, antibody-based inhibition, phosphorylation, protein kinase, TDP-43