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Investigation on the Physiological and Pathological Aspects of the Proline-Rich Region of the Microtubule-Associated Protein Tau

dc.contributor.advisorZweckstetter, Markus Prof. Dr.
dc.contributor.authorSavastano, Adriana
dc.titleInvestigation on the Physiological and Pathological Aspects of the Proline-Rich Region of the Microtubule-Associated Protein Taude
dc.contributor.refereeZweckstetter, Markus Prof. Dr.
dc.description.abstractengThe microtubule-associated protein tau is involved in several neurodegenerative diseases including Alzheimer’s disease (AD), Pick’s disease (PiD), Progressive Supranuclear Palsy (PSP) and others. The aggregation and fibrillization of hyperphosphorylated tau are considered disease-causing agents in these diseases, which are therefore termed tauopathies.  A major physiological activity of tau is its interaction with microtubules and the regulation of their dynamic rearrangement. Tau phosphorylation regulates its affinity to microtubules and is linked to pathological conditions when aberrant. Phosphorylation is mediated by several kinases and occurs in the proline-rich region as well as in the pseudo-repeat domain of the tau sequence. In cryo-electron microscopy studies of tau fibrils purified from patients with AD and PiD, as well as in vitro studies of tau bound to microtubules, structural information regarding the pseudo-repeats was obtained. In contrast, little is known about the structural properties of the proline-rich region when tau is bound to microtubules or aggregated into amyloid fibrils. In this work both physiological and pathological aspects of the structure of tau have been addressed, with a specific focus on the proline-rich region sequence of tau. In the first project reported in this work, solid-state nuclear magnetic resonance (ssNMR) was used to investigate the contribution of the proline-rich region to the structure of tau fibrils. In vitro fibrils obtained from the tau construct K32, comprising the P2 domain and the R1, R2, R3, R4 and R’ domains of the pseudo-repeat region, and from two simplistic models, the peptides P2R2 and P2R3, were studied using a combination of 13C-13C correlation experiments and INEPT-based transfer experiments, which probe the flexible regions of the fibrils. The use of simplistic models, such as the peptides P2R2 and P2R3, improved the quality of the ssNMR spectra and facilitated the identification of residues partially recruited into the core of the fibrils. The analysis of the ssNMR spectra indicated a partial recruitment of the P2 domain within the fibrils, especially of the most hydrophobic patch of the domain, i.e. the225KVAVVRT231 sequence. In the INEPT spectra,the absence of cross peaks from this region suggested a loss of flexibility, most likely due to the formation of hydrophobic interactions between the 225KVAVVRT231 sequence and the hexapeptides in the R2 and the R3 domains. Liquid-liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs) into compartments without surrounding membranes is implicated in the regulation of biological processes. However, little is known regarding the molecular mechanisms that influence biological processes in condensed phases. In the second project, I therefore studied LLPS of tau and its connection to the polymerization of tubulin into microtubules. The studies showed that LLPS of tau, its phosphorylation, and conformational changes upon binding to microtubules are functionally linked. Tau phosphorylated at disease-associated epitopes condensed into liquid-like compartments, tubulin partitioned into these drops, but it was unable to grow into microtubules. The functional link between LLPS and tubulin polymerization was provided by a conformational change in the proline-rich region of tau upon binding to tubulin. Phosphorylation blocks the functionally required conformational change through formation of salt bridges between phosphate groups and conserved arginine residues in the proline-rich region of tau. The data established a mechanistic framework in which LLPS and conformational changes in tau cooperate to drive formation of cytoskeletal tracks. de
dc.contributor.coRefereeOuteiro, Tiago Fleming Prof. Dr.
dc.subject.gerPathological rolede
dc.subject.engAlzheimer´s Disease,de
dc.subject.engStructural biologyde
dc.subject.engNuclear Magnetic resonancede
dc.affiliation.instituteBiologische Fakultät für Biologie und Psychologiede
dc.subject.gokfullBiologie (PPN619462639)de

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