CEI - DBMyB - Tesis

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https://rephip.unr.edu.ar/handle/2133/8017

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Examinando CEI - DBMyB - Tesis por Autor "Fernández, Claudio"

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  • Miniatura
    ÍtemAcceso Abierto
    Alpha-synuclein amyloid aggregation: From basic to translational research
    (2024-05-06) Buratti, Fiamma Ayelén; Fernández, Claudio; Zweckstetter, Markus
    Neurodegenerative diseases are a heterogeneous group of disorders affecting the lives of millions of people worldwide. A shared pathologic hallmark is the appearance of insoluble aggregates in the brain. The components of these are different, given a wide spectrum of neurodegenerative diseases. The presence of amyloid fibrils of specific proteins in the deposits allowed the identification of proteins involved in the development of the disease, and their study in vitro. Parkinson’s disease is considered a neurological disorder where spherical intraneuronal inclusions known as Lewy bodies are found in brain regions. The determination of inherited mutations has aided the research of alpha-synuclein, as one protein involved in the misfolding and deposition into Lewy bodies. Alpha-synuclein is an intrinsically disordered protein, found free or bound to membranes under physiological conditions. However, in pathological conditions adopt b-sheet conformations, resulting in amyloid fibrils. Many factors are modulating the pathways mentioned before, such as some regions of the protein, point mutations, and post-translational modifications. The physiological conditions could be addressed following the interaction of monomers with membranes, as one model for gaining knowledge in parameters such as membrane affinity, membrane-bound conformations, and even residues involved in the binding. Amyloid fibrils of alpha-synuclein can be generated in vitro, and be able to uncover fibril core, specific residues implicated in the aggregation process, stability and structure of fibrils. These tools permit the study of how different factors affect the physiological and pathological condition. Our study aimed to unravel the aggregation properties of alpha-synuclein through the characterization of conformational assemblies, as well as in terms of binding properties. Key factors in this process have been studied. A combination of kinetics assays, nuclear magnetic resonance, circular dichroism, dynamic light scattering, cell culture, fluorescent probes, electron and atomic force microscopy, was chosen to gain insight into all the features investigated. Structural changes induced for point mutations or post-translational modifications gave relevance to the implication of residue-specific in the pathological and functional state of the protein. We observed how particular amino acids can impair the in vitro amyloid assembly and even impact the binding to membranes. These factors are likely modulating structural conformations, that may be attributed then to the variety of alpha-synucleinopathies. The work presented here is a step forward towards understanding the role of alpha-synuclein in the pathology of Parkinson’s disease.
  • Miniatura
    ÍtemAcceso Abierto
    Characterization of the phosphomimetic mutant Y39E of α-Synuclein
    (2024-04-10) Böffinger, Nicola Martina; Fernández, Claudio; Griesinger, Christian
    α-Synuclein (aSyn) has long been identified as a key factor in the pathogenesis of Parkinson's Disease (PD), with familial mutations in the SNCA gene contributing to the aberrant aggregation of aSyn. Its aggregation results in the formation of Lewy Bodies, a characteristic hallmark of PD, ultimately leading to neurodegeneration and cell loss. While mutations have been extensively studied, post-translational modifications (PTMs), particularly phosphorylation, have emerged as crucial players in aSyn's physiological function and pathological aggregation. The kinase c-abl is known to phosphorylate aSyn predominantly at Tyrosine 39 (Y39), a modification often observed in advanced PD stages associated with elevated c-abl levels. Given the challenges in purifying phosphorylated proteins, phosphomimetic mutants, in this case Y39E, serves to simulate phosphorylation effects. This study extensively characterizes the Y39E aSyn mutant across various aspects, including monomeric structure and dynamics, membrane binding, in vitro and in vivo aggregation properties, dopaminergic neurodegeneration, and toxicity. Employing a multidisciplinary approach, biophysical methods such as nuclear magnet resonance (NMR), circular dichroism (CD) spectroscopy, Thioflavin-T (ThT) fluorescence measurements, and size exclusion chromatography (SEC) were combined with biological methods including molecular cloning, protein expression, and in vivo investigations using cell-based assays and the animal model C. elegans. Structural analysis revealed that the Y39E monomer closely resembled the wild-type (WT) monomeric structure, with no significant differences in backbone dynamics nor in the hydrodynamic radius. However, slightly higher R2 relaxation rates and minor changes in transient long-range interactions were observed in the Y39E variant. The overall membrane affinity remained equal between WT and Y39E, although the NAC region of Y39E exhibited reduced interaction. Notably, during the aggregation process, Y39E incorporated fewer monomers and displayed a prolonged lag phase with concentration-dependent kinetics. In cellular and animal models, the Y39E mutant demonstrated fewer cellular inclusions and smaller aggregates, respectively. Compared to the WT species, dopaminergic neurodegeneration was significantly elevated when expressing Y39E aSyn in C. elegans, impacting the nematode's behavior, while mitochondrial pathways were ruled out as a cause of toxicity. Additionally, data acquired on pY39 aSyn by other research groups aligned closely with results obtained in this study, indicating the Y39E mutant as a valuable tool for probing phosphorylation at this specific site, and confirming that pY39 can be mimicked by the Y39E mutant. Furthermore, the significance of pY39 is underscored by evidence of various research groups, showing that inhibiting phosphorylation effectively hinders disease progression and diminishes pathology in animal models. In summary, this work characterizes de Y39E mutant of aSyn. The structure and dynamics of the Y39E aSyn monomer closely resembled those of the WT species. However, differences were observed in the aggregation profiles between WT and Y39E aSyn, and dopaminergic neurodegeneration was found to be elevated for the Y39E mutant. Furthermore, this work highlights the crucial role of PTMs in aSyn pathology, showing that mutations, such as Y39E emerge as valuable instruments for mimicking PTMs, thereby simplifying the research process.