Interactions between biological macromolecules: Prion protein and its ligands
Cavaliere, Paola Maria Giovanna (2011) Interactions between biological macromolecules: Prion protein and its ligands. [Tesi di dottorato] (Inedito)
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A family of rare but all fatal neurodegenerative diseases which affect not only humans but also various animal species is related to the prion protein (PrP). The unique feature of these diseases is that, in addition to sporadic and inherited forms, they may be acquired by transmission of an infectious agent, which is represented by a misfolded form of prion protein, called PrPSc. Despite great efforts in the prion field, many questions remain unresolved related to the pathogenic mechanisms underlying prion diseases. The understanding of the mechanisms that lead to oligomerization and aggregation of PrP is necessary to find effective therapeutic strategies to antagonize prionopathies. In addition to the role of PrP on prionopathies, the physiological function of this protein in the cell is still a mystery. Many biological roles have been assigned to PrP, leading to presume that this protein can act in multicellular process, interacting with many biological macromolecules. Taking in consideration the above main issues on prion field, this thesis work was focused on the study of the interaction between PrP and three different molecules: a heterocyclic aromatic molecule, methylene blue (MB); small DNA and RNA aptamers with a sequence (GGA)4; and a glycolytic enzyme, Aldolase C. These molecules were chosen for different aims, in order to obtain further information about: i) the possibility to use a compound, such as MB, for therapy against prionopathies; ii) the affinity of the interaction between PrP and DNA and RNA aptamers with specific features, that could be used for diagnosis of prion diseases and could give information about the biological meaning of these interactions; and finally iii) a possible physiological function of PrP regulated by the interaction with aldolases. In searching for a compound that could interfere with the PrPC->PrPSc conversion, and be potentially used as drug against prion diseases, we chose methylene blue for its many properties: first, MB fulfills the safety features required for drugs delivery to humans and animals, and secondly, it is able to cross the blood brain barrier and, thus, suitable to target the toxic species formed in the brain, leading to prion diseases. In this thesis work, MB was tested to evaluate its potential inhibition action on both oligomerization and fibrillization processes that are thought to be on the pathway to the PrP disease occurrence. To this aim, several methodologies were used, such as fluorescence, surface plasmon resonance (SPR), nuclear magnetic resonance, differential scanning calorimetry, static light scattering and transmission electron microscopy. We demonstrate that MB can slow down the formation of PrP oligomers and can limit the amount of oligomers. Finally we demonstrate that MB is able to completely suppress the formation of PrP fibrils. These findings deserve the evaluation of MB for in vivo studies and preclinical testing for prion disease. The nucleic acids (NA) correlation with prion protein has ever been an issue of debate since the “protein only” hypothesis brought a new biological paradigm. Small DNA and RNA aptamers have shown to interact with PrP with high affinity. In an attempt to find a common shared sequence among the aptamers studied, it was found that some of them contain contiguous GG, and moreover, they can adopt a quadruplex structure. On this basis, we have studied the interaction of PrP with two reported DNA and RNA aptamers of sequence (GGAGGAGGAGGA), called D12 and R12. These aptamers, that fold in a G-quadruplex structure and are dimers, have shown a specific interaction with the native form of PrP. In this thesis work, a complete thermodynamic and kinetic investigation was performed using SPR, isothermal titration calorimetry and circular dichroism, in order to gain further information about the forces that drive this interaction. Using various PrP domains, it was possible to investigate also the nucleic acids-binding regions of PrP. The results obtained demonstrate that the interaction between the full-length PrP and both D12 and R12 is driven by different type of forces. The binding between full-length PrP and R12 occurs through the formation of a complex in which one molecule of PrP interacts with one R12 dimer. Differently, both aptamers bind to a deleted PrP form with a diverse stoichiometry. Finally, three putative binding sites on PrP are presumably involved in the interaction with these aptamers. This study has also allowed to evaluate the conformational changes induced by the PrP-nucleic acids complex formation, highlighting that PrP undergoes structural changes upon D12 and R12 interaction, whereas structural variations were also observed for R12, but not for D12. The binding study of DNA and RNA aptamers is an useful tool for the development of diagnostic strategies, since aptamers can be used to concentrate PrP from biological fluids to remove normal prions from a sample, and consequently enrich PrPSc. The lack of a deep understanding of PrP role in the complex machinery of living cells has led to an intensive study of the biological prion partners. In fact, PrP has a considerable number of interactors. One of these is the glycolytic enzyme Aldolase C, belonging to the fructose-bisphosphate aldolase family and mainly expressed in the brain. In our work, taking into account that, so far, no data regarding the binding strength of PrP-AldoC interaction have been reported, a detailed thermodynamic and kinetic analysis of the binding of PrP to AldoC by SPR was conducted. Our interaction study was extended to the other two aldolase isoenzymes, aldolase A and B, that share a high sequence identity, to investigate whether the binding was sequence specific or not. Our results showed that all three aldolases interact with PrP with a binding constant within the micromolar range. We surmise that the binding process between PrP and aldolase enzymes occurs through a 3D-structural recognition. Further research is required to determine the relationship between PrPC and its biological ligands, how the absence of the interaction is compensated, and whether the loss-of-function of PrP and its interactors is related to prion diseases.
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